NEMATOLOGY TRAINING MANUAL

Transcription

1 NEMATOLOGY TRAINING MANUAL FUNDED BY NIESA and UNIVERSITY OF NAIROBI, CROP PROTECTION DEPARTMENT CONTRIBUTORS: J. Kimenju, Z. Sibanda, H. Talwana and W. Wanjohi 1

2 CHAPTER 1 TECHNIQUES FOR NEMATODE DIAGNOSIS AND HANDLING Herbert A. L. Talwana Department of Crop Science, Makerere University P. O. Box 7062, Kampala Uganda Section Objectives Going through this section will enrich you with skill to be able to: diagnose nematode problems in the field considering all aspects involved in sampling, extraction and counting of nematodes from soil and plant parts, make permanent mounts, set up and maintain nematode cultures, design experimental set-ups for tests with nematodes Section Content sampling and quantification of nematodes extraction methods for plant-parasitic nematodes, free-living nematodes from soil and plant parts mounting of nematodes, drawing and measuring of nematodes, preparation of nematode inoculum and culturing nematodes, set-up of tests for research with plant-parasitic nematodes, A. Nematode sampling Unlike some pests and diseases, nematodes cannot be monitored by observation in the field. Nematodes must be extracted for microscopic examination in the laboratory. Nematodes can be collected by sampling soil and plant materials. There is no problem in finding nematodes, but getting the species and numbers you want may be trickier. In general, natural and undisturbed habitats will yield greater diversity and more slow-growing nematode species, while temporary and/or disturbed habitats will yield fewer and fastmultiplying species. Sampling considerations Getting nematodes in a sample that truly represent the underlying population at a given time requires due attention to sample size and depth, time and pattern of sampling, and handling and storage of samples. Since plant parasitic nematodes feed on plant tissue, their distribution is influenced by the distribution of their hosts, soil types, nematode species involved and season. In most situations, nematodes are clustered or aggregated. When the host crop is present, depth and lateral distribution of nematodes in soil generally mirrors the 2

3 degree of proliferation of the root system (particularly the fine roots on which nematodes feed). When the field is fallowed, the existing distribution will remain except that the bulk of the population may be slightly deeper in soil (largely due to desiccation of nematodes near the surface). Nematodes are rarely distributed evenly across a field. Some species are favoured by certain soils and their distribution may change with subtle changes in soil texture. Besides, nematode populations fluctuate over time; numbers increase in the presence of a host crop, the rate of increase being greatest when the environmental conditions are favourable. When the field is under fallow or a non-host crop is present, nematodes die of starvation and populations decline. The number of nematodes in a sample will therefore be affected by sampling time. Additionally, many plant parasitic nematodes have the capacity to survive periods of dryness through a behavioural adaptation in which their surface area is reduced by a process known as coiling. Since nematodes are very susceptible to mechanical damage in this desiccated state, the process of collecting samples from dry soils may damage nematodes. Nematode population densities are therefore likely to be underestimated in dry soil and it is preferable to wait until soil moisture is adequate before collecting samples. This uneven distribution makes collection of truly representative samples difficult as it introduces sampling errors. Other sources of sampling error are low population densities, latent infections, sub-sampling and counting of nematodes. Therefore, absence of nematodes in a sample may indicate their absence in the sampled field but may also indicate that the populations are too low to be detected by sampling. In order to increase the probability of detecting nematode infestations, roots of weeds and volunteer crops should be collected when soil is sampled. Sampling equipment For collecting soil samples to a depth of 20 cm or more, a soil sampling auger can be used. Garden trowels, narrow-bladed shovels or spades are also useful especially when sampling cropped areas or areas with rocky soil (Figure 1). Remember to clean all equipment and footwear of adhering soil before leaving a sampling site to reduce the risk of spreading nematodes to uninfected areas. Sample size The goal of a nematode sampling would be to capture information about the presence of nematode species occurring in one or more landscapes, ecosystems or geographic regions. The number of samples to be taken will depend on the breadth and depth of the sampling; where sampling breadth denotes the relative number of sites from which samples are collected, and sampling depth denotes the extent of taxonomic characterization per sampling site. Apportioning resources between sampling breadth and depth entails a choice between extensive or intensive nematode sampling. An extensive sampling maximizes the number of sampling sites and characterizes a limited number of taxa per site. 3

4 . Figure 1. Examples of tools for soil sampling to diagnose nematode infestations A B C D Figure 2. Recommended soil sampling patterns; A. and B: Patterns for perennial plants (When sampling tree crops, collect sub-samples from around the drip-line, towards and on alternate sides of the trunk) C: Pattern for annual crop or fallow field; D: Sampling pattern 4

5 for diagnosing nematode problems (Take soil cores from the margin of affected areas and only from the roots of affected plants that are still alive). The size of the sampling unit will vary from crop to crop and is largely determined by the value of the crop. Thus, sampling can be more intensive on high value horticultural and ornamental crops than on low value field crops. A sample should consist of 10 or more sub-samples representative of the area being sampled. Any number of sub-samples can be combined to form a composite sample. If an entire composite sample can not be processed, sub-sample by mixing the soil or plant tissue sample very gently but thoroughly by hand, avoiding excessive handling to prevent mechanical damage to the nematodes. The size of the sub sample depends on the extraction procedure used but should be at least 100ml of soil and 50g of plant material. Sampling pattern The sampling pattern will depend on the purpose of sampling: sampling to predict a problem or sampling to diagnose a problem. When sampling for predictive nematode assays (Figure 2), take samples before the desired crop is in the ground. For annual crops, this is usually soon after (or just before) harvest of the existing crop and several months before planting the next crop. Sampling close to harvest ensures that nematode populations are at their peak and that the assay will be a good indicator of any potential problems An intensive sampling limits the total number of sampled sites but maximizes the number of characterizations per site. The rationale for sampling will determine whether an intensive or extensive approach is more appropriate. For perennial crops, plan to collect samples well before planting so that there will be time to treat the fields if necessary. It is very difficult to manage nematodes on an established crop. In high-value established landscapes like golf courses, however, it can be prudent to sample for nematodes on a regular basis so that management can be scheduled for off-peak seasons. Additional information required for predictive sampling includes: Current crop and cultivar (or the crop and cultivar to be planted in the case of pre-plant samples) Details of the site (e.g. area, soil type, variability of soil, previous cropping history). Standard of crop management, particularly with regard to irrigation and nutrition. Likely importance of other pests and pathogens of roots. Details of previous nematicide use and the responses that were obtained. When sampling to diagnose a problem, use a combination of agronomic tests when trying to diagnose whether an existing plant growth problem might be due to nematodes. Collect a soil sample with roots for nematode problem diagnosis as well as a comparison sample from a nearby area where growth is more nearly normal. Because most problems have more than one cause, submit samples for soil nutrient and plant tissue analyses as well. Package each different kind of sample separately. You can send matching samples from the same area for soil testing or plant analysis. You can collect samples for nematode problem 5

6 diagnosis any time plants are actively growing and the soil is in good working condition. Take soil from the root zone of plants that are affected but still alive. Never sample beneath dead plants. Diagnoses are improved if adequate background information is provided. The following details are particularly important: Crop and cultivar Previous crop Area involved Description of symptoms and their distribution Soil texture, soil depth and variability of soil Frequency of irrigation and/or rainfall Previous nematode control methods used and details of responses obtained For larger cropped areas, divide the field into 1 hectare units and make a grid pattern that covers the entire 1 ha unit. The length of intervals between sampling points on the grid will depend on the sampling precision that you require. Very close intervals, e.g. 2m x 2m will reflect the nematode distribution more precisely than, for example, 10m x 10m. Collect a sub-sample from at least 20 points throughout the fields. Collect separate samples for areas with different soil types, different cropping histories, or different management objectives. Sampling depth The depth of sampling has to consider the depth of rooting of the crop being sampled. For most crops, a sampling depth of 20 cm is adequate; for deep rooted perennial crops, different depths can be sampled, for example, 15, 30, 60, 100 cm. Sampling time Soil and root samples can be taken and reliably processed as needed, whenever the soil is not dry. For best results, collect samples before planting a field but fairly close to planting time, as the nematodes present at this time can generally be related to yield in annual crops. Samples can also be collected between mid season and harvest as nematode numbers increase towards harvest. For established perennial crops, collect samples during growth flushes. Care of samples Caution! Collect samples in sturdy plastic bags and close the bags firmly. Place above ground plant materials and soil samples in separate bags but place the roots together with soil in the same bag; the roots should be covered with soil. Do not moisten soil samples that were collected from dry soil. Transport samples in a cool-box or other insulated containers, preferably with ice packs. Put samples in a cool place or refrigerate until submission. If you do not want to extract straightaway, most nematodes in the samples will survive storage at 4 o C for several weeks. Do not allow samples to dry as the nematodes will die before the sample arrives in the laboratory. Temperatures above 35 o C will also kill many nematodes. Submit samples to the nematode laboratory promptly and if possible process within one week from collection! Direct examination of plant material 6

7 Nematodes can usually be seen by examining small amounts of gently washed plant tissue such as roots, leaves, stems or seeds with a stereomicroscope. The plant tissue is examined in water in an open Petri dish or large watch glass and teased apart with strong mounted needles. Nematodes released from the tissue will float out and can be collected with a handling needle or fine pipette. Since nematodes are translucent and difficult to see in plant tissues, staining helps to make them more visible. The plant tissue may be cleared in diluted sodium hypochlorite bleach before examination. The strength and time of bleaching will depend on the plant material and type of bleach. Rinse out the bleach before transferring the plant material to a glass vial or beaker with acid fuschsin solution (875ml of lactic acid, 63ml of glycerol, 62 ml of water and 0.1g of acid fuschsin). Boil the plant material in the solution for about 30 seconds in a microwave or on a hotplate (should be in a ventilated area to avoid lactic acid fumes). Several small samples can be stained in one operation by wrapping each in a piece of muslin cloth. The plant material is allowed to cool before washing off the excess stain in running tap water. Thereafter, the plant material is transferred to a solution of equal volumes of glycerol and water acidified with a few drops of lactic acid. After several hours to a few days, depending upon the type of material, the differentially stained nematodes can be examined in the largely unstained plant tissue. B. Extracting live nematodes Extracting nematodes from Soil An endless range of extraction tools and techniques have been developed mostly because no single technique will efficiently extract all sizes and kinds of nematodes. Some are rarely used, others have been adapted to suit local conditions and requirements and availability of equipment. Modified Baermann Technique - Extraction tray method (Whitehead and Hemming, 1965) This is the simplest method which was adapted from the Baermann Funnel Technique. This method is not quantitatively reliable as it selects against slow and non-moving nematodes, but it is extremely cheap, relatively fast, very good at yielding large numbers of live active worms and it extracts live, active nematodes from soil and plant material in a clear suspension. The three components required are: (1) a large plastic tray, (2) a wide mesh and (3) reasonably strong kitchen paper (Figure 3). The larger the tray, the more material can be extracted in one run. A handy size and type is e.g. a dissection tray or a photographic developing tray. The mesh can be a single layer of thick plastic or rubber netting, or alternatively it can consist of a plastic-coated metal grid covered with thin plastic gauze. It should NOT contain any bare metal, because this may release toxic ions. Place the mesh in the tray, cover its bottom and sides with a single sheet of kitchen paper (one or two layers) and spread a thin, uniform layer of soil out over the paper. Gently pour clean water (tap water may contain nematodes!) into the tray until the soil is wet but not submerged. Avoid spilling soil particles along the sides or through the filter paper - they 7

8 will cloud up the extract and make it difficult to see the nematodes. Leave the tray until the next day. Actively moving nematodes will sooner or later crawl down through the kitchen paper and sink to the bottom of the tray. A large proportion of the sampled nematodes will thus collect on the bottom of the tray overnight, and the next morning you should have plenty of nematodes ready for further treatment. Concentrate the suspended nematodes by pouring the water out of the tray over a fine sieve (mesh 25 m or less) and washing the nematodes off into a small beaker with about ml water. If you do not have a sieve, pour the extract water into a sufficiently large beaker and allow the nematodes to settle (takes about 30 minutes). Then pour off most of the top water and transfer the remaining water to a smaller beaker. Repeat until you have ml suspension left. If you have sampled on a good spot, you should now have hundreds or even thousands of nematodes ready for further processing. This method is called Active method and it is not effective for inactive nematodes such as Trichodorids, Longidorids and Criconematids. Therefore, to be consistent, it is advisable to supplement it with other techniques the Passive methods. There are two major methods Elutriation and Sieving. Elutriation involves separating nematodes from soil by an upcurrent of water, the strength of which is such that the nematodes are held in suspension whilst the heavier soil particles sink. With these techniques, both active and sluggish nematodes are extracted from soil, results are quickly available but they involve fairly complicated apparatus, require considerable expertise, and depend upon a plentiful supply of clean water at high pressure, thus limiting their usefulness. Sieving Technique The sieving technique is also known as the bucket-sieving method. Although crude, it is widely used as it enables the extraction of large numbers of both active and inactive nematodes in a relatively short time. Equipment required includes two plastic buckets (about 5L), sieves of cm diameter made with wire mesh, preferably stainless steel of aperture size of 2mm, 720, 250, 125, 90, 63, 45,25 m, respectively and tall 100 ml beakers for the residue from sieves (Figure 4). Usually only three or four sets of sieves will be used for a particular sample, with the sieves selected to match the size of nematode it is hoped to extract, and to suit the type of soil involved. In general, sieve openings should be no greater than 1/10 of the nematode length. Most adults of large nematodes (e.g. Anguina, Belonolaimus, Hirshmanniella, Longidorus and Xiphinema) are caught on a 250 m aperture sieve, adults of average sized nematodes (e.g. Aphelenchoides, Ditylenchus and Hemicycliophora) on a 90 m aperture sieve, and many juveniles and small adults (e.g. Criconemoides, Paratrichodorus, Paratylenchus, Pratylenchus and Radopgolus) on a 63 m aperture. A 45 m or even 25 m aperture sieve is used to recover small juveniles (e.g. Meloidogyne, Heterodera, and most others). Ready made sieves are expensive. Caution! Use sieves singly, never stack them and never attempt to work samples through them all simultaneously, as this may reduce the efficiency of recovery. Fine sieves are easily clogged, but this can partially be avoided by pouring the suspension on a sieve inclined at an angle of about 30 o to the horizontal. However the number of nematodes recovered on the sieve will be reduced. Gently patting the underside 8

9 of the sieve into the water bucket below and lifting it in and out a few times will help to clear it. Procedure 1. Mix the soil sample thoroughly and place a known volume of soil in a bucket and fill to about ¾ with water. Dry soils should be soaked for a few hours. The mixture is stirred to free nematodes from the soil and suspend them in the water. Flocculating agents such as Separan NP10 (12.5 g/ml) might be used to help break up soil aggregates in heavy clay soils. 2. Let the mixture settle for seconds and decant over a 2mm aperture sieve into another bucket. Avoid pouring the sediment. Add less water to the sediment in the first bucket and repeat this step 2 3 times to increase nematode recovery. Any sediment left in the first bucket is discarded and the bucket washed out. The sieve is rinsed over the second bucket. 3. The contents in the second bucket are stirred, allowed to settle for about 10 seconds and then poured through a 710 m aperture sieve into the clean bucket, leaving behind heavy soil particles to which more water is added and the process repeated, if desired. Collect the residue nematodes on the sieve by directing a gentle stream of water on the upper surface of the sieve so as to wash out small nematodes and eggs. Transfer the nematodes on the sieve into a beaker using a gentle stream of water leaving behind any heavy particles. 4. Repeat the process using 250, 125 and 90 m aperture sieves and collecting the residues as described above. The residues of each sieve can be pooled in one beaker or kept separate in different beakers. If the contents of the beakers appear cloudy, it is because the residue on the sieve was inadequately rinsed. If necessary, the contents should be poured back onto the sieve and rinsed again over the bucket containing the remaining suspension before proceeding to the next sieve in the series. The contents of the collecting beakers are allowed to settle for 1-2 hours and the supernatant liquid is carefully decanted or siphoned off leaving about 20ml in the bottom (some nematodes tend not to settle and may require refrigerating the supernatant liquid). The material can be transferred to a viewing dish and examined. If the suspension still contains a significant amount of debris, further processing by centrifugal floatation or modified Baermann techniques will result in an almost clean nematode suspension. However, sluggish and inactive nematodes can be lost. If you use 3 same sieves in series, you can be sure that you will recover about 100% of your nematodes. For example if the soil sample had 10 nematodes, 1 st Sieve will retain 7, 3 will escape through. The 2 nd sieve will retain 70% of the 3 nematodes ~ 9 nematodes recovered and 30% will escape through (~ 1 nematode). The 3 rd sieve will retain 70% of the 1 nematode ~ 100% recovery. 1. Sieving/Centrifugation method This is an extension of the sieving method. 1. After decanting some of the supernatant liquid in step 4 above, transfer the supernatant to four 50ml centrifuge tubes. 2. Centrifuge for 7 minutes at 1750rpm 3. Decant supernatant from tubes and discard 9

10 4. Add sugar solution (450g/l water) to tubes 5. Shake tubes, centrifuge for 3 minutes at 1750 rpm 6. Pour suspension through the 45 m sieve Rinse the residue from sieve for examination Extraction of Nematodes from plant materials Baermann Funnel Technique and its modifications The Baermann funnel technique uses a funnel of cm with rubber tubing attached to the funnel stem and closed with a spring or screw clip. The funnel is placed in a suitable support and almost filled with tap water. Plant material containing nematodes is chopped into small pieces (< 1cm length), placed in a muslin cloth or nylon gauze, which is folded to enclose the material and then gently submerged into the water in the funnel. Soil kitchen paper Wide mesh Plastic tray Figure 3: - Extraction tray method Figure 4: The sieving technique (bucket-sieving method) Nematodes emerge from the tissues and sink to the bottom of the funnel stem. After hours, fully open the clamp and rapidly withdraw 5 10 ml of water containing the nematodes and transfer it to a shallow viewing dish for examination. When this technique 10

11 is used in its original form, nematode recovery is low (about 20% of other methods) because of anaerobic conditions that develop due to bacterial decay of submerged organic matter and lack of oxygen at the base of the funnel stem. This technique is handy and, in order to improve its efficiency, it has been modified in several ways to become the standard method for extraction of nematodes from plant tissues and soil. For example, 1. Modified Baermann funnel technique: This technique uses a supporting mesh to hold the plant material partly submerged in water to avoid anaerobic decomposition. Supporting gauze-mesh is fixed at the bottom of a plastic ring (cut from Perspex, polythelene or vinyl tubes). Wet-strength facial tissue or milk filter is placed on the supporting mesh and materials from which active nematodes can be extracted, such as chopped plant material, thin layers of soil or residues obtained by sieving or maceration, are then placed onto the facial tissue. Water is added to the funnel so that the facial tissue is partly submerged in water. After hours, collect nematodes as described above. 2. The other modification is to use a shallow tray, dish or bowl to further improve aeration and reduce the number of nematodes remaining on the wall of the funnel. Similar to the above, a facial tissue is placed on a support and the chopped plant material or soil is placed on it. The support and the material for nematode extraction is placed in a tray (or dish, bowl, etc) filled with tap water. Small feet (for example cut pieces of polythelene) can be attached to the support ring to give a space of about 2mm between the base of the support and the collecting tray. The material on the sieve should be moist (but not flooded) and it may be kept moist by placing another facial tissue on top or using a relatively large tissue and folding it over the material (Figure 5). Do not pour water over the sample to avoid washing debris through the tissue. After hours (sometimes less), the support with the sample is gently removed and the contents on the tray transferred to a beaker. Any nematodes remaining on the tray can be rinsed into the beaker using a spray bottle. The sample can be re-extracted if necessary. Oxygenation (and hence nematode recovery) can be improved by adding 1 3 % H 2 O 2. Maceration techniques Maceration can be used for extracting active nematodes as well as immobile stages of the sedentary nematodes from plant tissues (e.g. corms, bulbs, cloves, storage roots, crowns, leaves and small plants). The plant material is chopped into lengths of < 1 cm and then placed in about 100 ml of water and macerated in an electric blender. The maceration time required depends on the type of blender and the type of plant material. Maceration needs to be continued long enough to give nematodes easy way out from the tissues but not to damage or render them immobile. The resulting suspension can be extracted using the modified Baermann techniques described above. 11

12 Figure 5: Baermann Funnel Technique (top) and one of its modifications Bioassays for detecting and quantifying nematodes Because of the aggregated distribution pattern of nematodes, the chance of detecting a small population of nematodes (for example 1 juvenile per 2 L soil) is relatively low when small volumes of soil are processed. Difficulties in detecting low but economically important populations of some nematodes can be overcome to a certain extent with bioassay procedures. For example, to detect and quantify root-knot nematodes, samples are collected as described previously but at least 1L of soil must be retained. Samples of 1-5 L of soil are added to pots, a susceptible tomato seedling is planted and the plants are grown in a warm environment (ideally C). If information on the presence or absence of the nematode is all that is required, plants are carefully washed from soil after 4-8 weeks and roots observed for signs of galling. High gall ratings indicate a high population of rootknot nematode. Bioassays can also be used to quantify the root-knot nematode population, provided plants are harvested before a second generation of juveniles invades roots. Since reproduction can begin in 4-5 weeks under ideal conditions (i.e. temperatures of C), plants are best removed from soil 3-4 weeks after planting. Provided nematode populations 12

13 are low, (i.e. less than about 100 root-knot nematodes/litre), a single gall will generally represent a single nematode. Thus, counting the number of galls on the root system will give an indication of the number of root-knot nematodes in the potted soil volume. When counting galls, roots should be floated in water in a tray with a dark coloured base, as this enables small galls to be seen more easily with the naked eye. C. HANDLING NEMATODES Extracted nematodes can be examined directly under a microscope to the genus level using viewing dishes or counting slides or can be processed further to slides. All or part of extracted nematode suspension (depending on nematode density) can be transferred to an open counting slide 1 and examined directly under a microscope to genus level (at times up to species level) and the nematode populations can be counted using hand held tally counters or a bank of counters. However, for nematode identification to the species level, it is advisable that temporary or permanent slides are made. The nematodes must first be killed, fixed and properly mounted. Killing and fixing nematodes A few specimens can be killed by transferring them to a drop of water on a glass slide, which is then heated over a small flame for a few seconds or by placing the slide on a hot plate at O C so that the nematodes assume their heat relaxed shape. The specimen can be examined directly under the microscope or can be transferred to a fixative or fixed on the slide by adding an equal amount of double strength fixative. A number of fixatives are commonly used for preserving nematodes. Most of these contain formalin and should be handled with due care (rumour has it that a substantial percentage of Nematologists died from cancers induced by a lifetime of inhaling formalin vapours!). After fixation, nematodes are usually transferred to glycerine via a dehydration procedure, because this will stop further denaturising and decay, while also resulting in highly transparent specimen ideal for observation through high-powered light microscopes. Stains are not commonly used, because these will not only highlight certain features but also mask other structures. Below, two easy and quick methods for fixing and processing nematodes are presented. Prior to this, however, it is important to stress that you must at all costs avoid contraction of the nematodes prior to death. For example, never fix live nematodes with cold fixative. They will not be killed instantaneously, but (apart from suffering a gruesome death) will contract and coil to a degree that often makes them useless for detailed study. To make sure nematodes are nicely stretched upon fixation, you must kill them instantaneously, either by using hot fixative or by heat-killing them prior to adding fixative. 1 there are various types of counting slides developed by various nematologists with varying capacities which range from a petri dish or watch glass with a grid, which can be marked on the inside of its base to guide searching of nematodes, to multi-chambered counting slides which allow examination of several samples on one slide 13

14 Both procedures result in what is known euphemistically as "heat relaxation", relying on a knock-out heat shock to instantly relax the muscles of the nematodes. Note also that it may sometimes be advisable to starve the nematodes for a few days prior to killing and fixing, because well-fed nematodes can contain so many intestinal granules that other organs remain obscured even after transfer to glycerine. The best way to kill nematodes that are collected in a small volume of water (e.g. from an extraction tray or from cultures) is to transfer them to a glass vial and put this in a O C water bath. Stir the vial for seconds and check under a stereomicroscope that the nematodes are all motionless and stretched out. Make sure they are not boiled - this disrupts cellular structure. After heat-killing, fix the nematodes with hot fixative under a flow hood (hot fixative will be more chemically active and you should avoid inhaling it at all times). Fixatives 1. Formalin: 8ml Formalin (40% formaldehyde) topped with distilled water up to 100ml. CaCO 4 powder can be added to neutralize the free formic acid that can cause darkening and granulation of tissue 2. Formal acetic (FA) or formal propionic (FP) 4:1: 10 ml Formalin (40% formaldehyde), 1ml glacial acetic acid (or propionic acid), 2 ml glycerol, topped with distilled water up to 100ml. Addition of glycerol means that the nematodes can be brought directly from fixative to glycerol by slow evaporation. Also it means that the nematodes stored in vials will eventually end up in glycerol should the fixative evaporate (see processing and mounting nematodes below). 3. TAF: 7ml Formalin (2 4% formaldehyde) + 2% glycerol, 2 ml tri-ethanol-amine (to neutralize the free formic acid that can cause darkening and granulation of tissue), 91 ml water. FA 4:1 and FP 4:1 are probably the most widely used fixatives that also allow long term preservation. TAF is a commonly used fixative, as nematodes fixed in TAF retain their lifelike appearance in it for several hours, but it is not good for long term preservation as some degeneration of the nematode cuticle can occur. Processing nematodes further to glycerol improves preservation. Fixation of plant tissue and soil In some (if not most cases) plant tissues and soil samples will be processed for nematodes within a few days after sampling. In order to prevent population changes during extended storage (and also avoid quarantine restrictions applicable to live material), roots, shoots and soil can be fixed for storage and subsequent examination by adding to them an equal volume of hot (65-70 O C) double-strength fixative, in a sufficiently large plastic bottle with screw-cap, which is closed and shaken thoroughly. Alternatively, fresh plant material can be put directly into hot lactoglycerol; this softens tissues and is particularly helpful in the recovery of root-knot nematode females from roots. 14

15 Note that dead nematodes don't wriggle, so you cannot extract them with an extraction tray. If you fix a plant tissue/soil sample, you will therefore need to use more complicated extraction devices such as Cobb sieves, centrifuge, etc. Processing and Mounting Nematodes In fixed nematodes, much of the internal body contents, especially the gonad structure, may be obscured by the granular appearance of the intestines. Specimens can be cleared by processing with lactophenol (Caution: phenol fumes are dangerous to health!). Lactoglycerol (equal amounts of lactic acid, glycerol and distilled water) or glycerol, to which a stain (0.05% acid fuchsin or 0.05% methyl blue) is preferred. Fixing with formalin-glycerine and transferring to glycerine through ethanol (Seinhorst, 1962) Prepare double-strength Formalin Glycerine fixative containing 8% formalin and 2% glycerine in distilled water. Transfer live nematodes to a small glass vial and allow them to settle to the bottom. Draw off surplus water until they are left in about 2 ml water. Kill the nematodes by stirring the vial seconds in a O C water bath, check they are all dead and stretched, and then add an equal volume of O C fixative. Stir, and then leave the vial for a day to allow the fixative to penetrate and act on all tissues. Take the vial with Formalin Glycerine fixed nematodes and draw off as much fixative as possible without losing nematodes (if the vial has a narrow opening transfer the nematodes to a cavity block). Fill the vial or block with a solution made from 20 ml of 96% ethanol, 1 ml glycerol and 79 ml distilled water (to the brim if in a cavity block, to about 5 mm high if in a vial). Place the block or vial on a platform inside a closed glass jar containing an excess of 96% ethanol (1/10 volume of the glass jar). Leave overnight in an incubator at C. This will allow all water in the suspension with the nematodes to be replaced with ethanol. The next day, take the vial or block out of the glass vessel and leave it open in the O C incubator for 2 3 hours, to evaporate about half of the ethanol (if necessary cover partly to prevent complete evaporation). Refill with 5% glycerin-95% ethanol solution, leave for another 2 3 hours, and refill one last time before leaving the vial or block overnight in the incubator at O C. By the next day, the nematodes will be impregnated in pure glycerine and ready for mounting in slides, or for storing without fear of desiccation. Note that the nematodes processed to glycerol are very soft and should be handled carefully. The entire Formalin Glycerine ethanol procedure takes only three days and usually results in well-fixed nematodes that will not decay for decades. Transferring through ethanol dissolves cuticular lipids, however, and may result in a finely wrinkled cuticle that will show up as such under the scanning electron microscope. If you want to avoid this, try the slightly slower method below. Fixing with TAF and transferring to glycerine through evaporation 15

16 Prepare double-strength TAF fixative containing 8% formalin and 2% triethanolamine in distilled water. Transfer live nematodes to a small glass vial and allow them to settle to the bottom. Draw off surplus water until they are left in about 2 ml water. Kill the nematodes by stirring the vial seconds in a O C water bath, check they are all dead and stretched, and then add an equal volume of O C fixative. Stir, and then leave the vial alone for a day to allow the fixative to penetrate and act on all tissues. Take the vial with TAF fixed nematodes; transfer the nematodes to a cavity block (this could be easier for subsequent manipulation under a stereo microscope). Draw off as much fixative as possible without losing nematodes, and then fill the vial or block with a solution of 5% glycerine in distilled water (to the brim if in a cavity block, to about 5 mm high if in a vial). Place the block or vial in an incubator at O C and cover it nearly completely, leaving a narrow slit for slow evaporation. Leave until a substantial amount of water has evaporated. Refill with 5% glycerine, and then leave at least two more days in the incubator until all water has evaporated. Check the degree of dehydration by transferring two or three specimens to a drop of pure glycerine, if their cuticle collapses, they are not yet completely dehydrated - return everything to the incubator for another day or two. The entire TAF/evaporation procedure takes four to six days and often results in perfectly fixed nematodes. In comparison with Seinhorst slow method, this method is recommended for better instantaneous preservation and for Scanning Electron Microscope (SEM) material, but less suitable for long-term preservation. Mounting Nematodes Temporary Slides Temporary slides with freshly killed/fixed specimens mounted in TAF can be made to view some important features of nematodes. Place the specimens and supports (e.g. glass fibre or beads) in a small drop of fixative and put a coverslip. Blot off excess fixative with tissue paper, seal the cover slip with Vaseline or nail varnish and observe under a microscope. Observation of detailed morphology of live nematodes can also be done by making a temporary slide. Add 1 2 drops of hot 4 5% agar on a glass slide, and immediately flatten this agar with another glass slide having spacer strips of thick plastic tape. Carefully remove the top slide when the agar has set. Add a drop of water on the agar, transfer the nematode to it and place a coverslip on top. The pressure between the coverslip and the hard agar will slow down the worm sufficiently to observe it with oil immersion magnification. If you want to immobilize it almost completely, smear some Vaseline on the rims of the coverslip, place it on top of the agar and nematode, and very carefully press down the rims of the coverslip until the nematode is trapped but not squashed. A complete Vaseline seal will also prevent desiccation. It is usually possible to recover the live nematode from such a slide after studying it. Permanent Slides 16

17 Once nematodes have been fixed and transferred to glycerine, permanent mounts can be made. Step by step procedure 1. Preparing a glass slide: Fill a Petri dish with paraffin granules, melt them at about 60 O C and allow the paraffin to set into a solid layer. Take a 10 cm long cross-cut metal tube with smooth, thin rim and slightly smaller diameter than the coverslips you use (e.g. a 16 mm diameter tube for 18 mm diameter coverslips) and heat one end in a flame. When the other end of the tube is beginning to get hot in your hand, push the heated end down vertically in the paraffin so that it gets covered by melting paraffin, and then press this end down vertically on the middle of a glass slide. Lift the tube, and you should leave behind a complete 3-4 mm thick ring of setting paraffin. Transfer a very small drop (can be applied using a syringe fixed with a small needle) of anhydrous glycerol (heated for 4 hours at O C in an oven) to the centre of this wax ring on a slide, leaving a spot of 4-5 mm on the slide. Repeat this for as many slides as you wish to make. Note: Getting the proportions of wax and glycerine right is important: too little paraffin and too much glycerine will result in an incomplete seal, too much wax and too little glycerine will result in nematodes being covered or trapped by paraffin. 2. Transferring nematodes: Pick out the nematode specimens you want with a needle and transfer them to the glycerine drop in the centre of a wax-ringed glass slide. You can usually mount up to ten of them per slide; more will too often result in specimens overlapping or ending up in paraffin. After transferring the required number to a slide, put it under the stereo microscope and using a handling needle arrange all nematodes in the centre so that they touch the side of the slide and are not floating, making sure none overlap with one another. Place three glass supports around the nematodes. 3. Sealing and shuffling: Drop a coverslip over the wax ring and glycerine drop, and put the slide on a moderately hot plate (or a mesh or metal plate above a small flame). Allow the paraffin to melt around the glycerine drop, and allow all air to escape from under the coverslip. Then put the slide back under the stereo microscope, and check that no nematodes are overlapping. If nematodes are overlapping, gently push the coverslip in the required direction to dislodge one of the overlapping nematodes. If the paraffin has set by now, return the slide to the hot plate. You can also re-heat and gently push the coverslip sideways to turn specimens over. Once set, the paraffin will both act as a seal and a separating layer between the coverslip and the glass slide, and ideally your slide will contain just a small circular central area with glycerine and nematodes. 17

18 If some specimens are covered by smudges of paraffin under the coverslip, and/or the paraffin is too thick to observe specimens with high power objectives, put the slide back on the hot plate and allow the wax to heat and spread out further so that it forms a thinner layer. If you want to pick out specimens for transfer to another slide or for use in SEM or cross-sections, gently open the coverslip with a scalpel or thin needle while keeping track of your specimen(s) under the stereo microscope. This may go better if you first heat the slide gently (e.g. leave it on the lamp housing of the microscope for a few seconds). Make sure you do not allow the glycerine to boil during any of these operations, and never push down on the coverslip while the wax is molten. When satisfied with the arrangement of your nematodes on the slide, you can permanently seal off the coverslip with colourless nail varnish. The Noble Art of Picking Worms While you can cheat and use pipettes or micropipettes for most culture procedures, there are no easy ways out when you need to make slides: you have to learn to pick nematodes with an eyelash, a hair, a fine needle or other picking device. Picking worms off fairly dry agar shouldn't be too difficult (unless you have a very agile species), although you may need to use an inconveniently thick or flattened pick if you do not want to damage the agar surface. Picking worms out of a suspension is a different matter, however. Half the trick is getting a good picking device, e.g. a fine and rigid insect needle tapped against the table to bend its tip to a minute hook, or a hand-picked hair from the most recalcitrant and bushy moustache available in your lab. Mount one of these on a handle and prepare yourself for a few memorable hours at the stereo microscope. The other half of the trick is getting the hang of suspended-nematode-dynamics while working at a stereo microscope. Select the specimen of your choice in a Petri dish or cavity block with nematodes in liquid (water, glycerine or whatever is appropriate for the procedure at hand). Try to pull this nematode free from the bottom with a short twitch of the pick, and tease it up to the surface of the liquid until it is more or less horizontal, using one hand to move the pick and the other to change focus. Then position the tip of the pick for the final twitch. For curled worms, try to catch them by the crook of the curved part(s) of their body. Straight worms are more fun (or more trouble), because these will often only come out if you position the pick just below them and at a slight angle to their body axis. With luck, the viscosity and surface tension of the liquid will make the worm stick to the pick instead of pulling it back down. Small straight worms are the best (or worst) because they only have a small surface for viscosity to act upon, and will obstinately refuse to cling to your pick. Teasing a straight, 0.3 mm long nematode out of pure glycerine is a particularly nerdy way of spending long winter evenings, or challenging friends and enemies! The easy way (using Pipettes) 18

19 A generally faster and safer method of picking single specimens from a suspension consists of using a pipette. This will especially be easier if you are not at all used to nematodefishing. Take a fine Pasteur pipette, heat it near the tip above a flame until the glass softens, and pull off the tip smoothly. Break off under stereo microscope the drawn-out end of this glass tip until you have an opening of appropriate size, i.e. about as wide as one to one-half the body length of the nematodes you are going to handle. Fit the pipette with a mouth tube. Gently warm the pipette over a slow flame and while the pipette is still warm, suck up single nematodes. This technique is especially efficient for handling very small nematodes. Further Reading Bloemers, G.F. & Hodda, M. (1995) A method for extracting nematodes from tropical forest soil. Pedobiologia 39: Hooper, D.J., Hallmann J. and Subbotin, S. (2005). Methods of Extraction, processing and detection of plant and soil nematodes. in: Luc, M.; Sikora, R.A. & Bridge, J. (eds.) Plant parasitic nematodes in subtropical and tropical agriculture. Second edition. CAB International, Wallingford: Southey, J.F. (1986 (ed.). Laboratory methods for work with plant and soil nematodes. MAFF, London. 19

20 CHAPTER 2 BIOLOGY OF PLANT PARASITIC NEMATODES Waceke Wanjohi Kenyatta University, Department of Plant and Microbial Sciences (PMS) P. O. Box , Nairobi, Kenya. Objective: Develop an understanding of the general biology of plant parasitic nematodes and its implication to nematode control and management 1. Introduction Plant parasitic nematodes (PPN) are essentially aquatic and spend a greater part of their life cycle in the soil. They infect all plant organs where they feed ectoparasitically or endoparasitically using their stylet. A few are semi- endoparasitic where only the anterior part of the nematode penetrates the root and the posterior part remains outside the root. Endoparasitic nematodes are relatively more damaging than the ectoparasites. Infected plants in general exhibit stunting, chlorosis, wilting and reduced yield, in addition to several below-ground symptoms. Such symptoms are often confused with other soil problems, including compaction and nutrient deficiencies, and nematode damage is often overlooked. Besides nematodes causing diseases on their own, they interact with other nematodes and pathogens to cause disease complexes frequently resulting in more severe diseases. Most PPN exhibit parthenogenetic (males absent, very rare or non functional) and amphimictic (males and females are separate) type of reproduction. Eggs are deposited singly or in masses either in the soil or within plant tissues. Most PPN have four larval stages between the egg and adult, with intervening moults. A life cycle from egg to egg can be completed within 3-4 weeks under optimum environmental conditions; temperature is a key factor in determining the duration of the life cycle. Eggs hatch under favourable environmental factors or in response to root exudates. Passive dispersal aided by several agents plays a significant role in the dissemination of nematodes. In a number of genera, eggs are the survival stages, being protected either in a gelatinous matrix (root knot nematodes) or within the cyst (cyst nematodes). Some PPN enter a reversible anhydrobiotic state to survive desiccation. 20

21 2. Nematode pathology Plant parasitic nematodes are biotrophic parasites which obtain nutrients from the cytoplasm of living root, stem and leaf cells for development, growth and survival. Nematodes have evolved diverse parasitic strategies and feeding relationships with their host plants. They possess a hollow and a protrusible feeding structure, the stylet and a pharynx, which has undergone morphological and physiological adaptations to suit the feeding relationships. Depending on the species, they feed from the cytoplasm of unmodified living plant cells or have evolved to modify root cells into elaborate feeding cells as in root knot nematodes (RKN). The nematodes use their stylet to pierce and penetrate the cell wall of a plant cell, inject gland secretions through the stylet orifice into the cell and withdraw and ingest nutrients from the cytoplasm. Nematodes that enter root tissue also use their stylet to cut openings and/or inject secretions to dissolve (intracellular migration) or weaken (intercellular migration) the cell wall or middle lamella. In general, all PPN damage plants by direct mechanical injury using the stylet during penetration and/or by secretion of enzymes into the plant cells while the nematode is feeding. The physical presence of endoparasitic nematodes inside the host also affects the functioning of the host. As a result of nematode feeding, the architecture and extent of the root system is altered, so that it is less efficient at taking up nutrients and water from soil. The extent of nematode damage depends to a large extent on the inoculum density (level of infestation). Low or moderate numbers of nematodes may not cause much injury but large numbers severely damage or kill their hosts. Feeding relationships The feeding relationships between nematodes and susceptible plants are diverse and the amount of tissue destruction and the degree of plant response are often related to the type of feeding relationship. While some PPN are ectoparasites, others are endoparasites or semiendoparasites. Ectoparasites Ectoparasites feed from root tissue by inserting their stylet from outside the root (Plate 1a b). The group consists of several morphologically divergent families that have evolved different feeding strategies. As a rule, species that have a short stylet feed on epidermal cells e.g. Tylenchorhychus dubius and those with long stylets feed on deeper tissues e.g. Belonolaimus, and Dolichodorus spp. Ectoparasites are either migratory or sedentary. Migratory ectoparasites These nematodes have the most primitive mode of parasitism; they remain outside of the root and use their protrusible stylet to feed either on epidermal cells or cells deeper within the root. With the exception of species of a few genera that feed on root tips, e.g. Belonolaimus, nematodes with this type of feeding strategy generally cause little obvious 21

22 tissue damage. The migratory ectoparasites remove cytoplasm from the parasitized cell, frequently causing their death and then move to another cell to repeat the feeding process. Dorylaimid migratory ectoparasites include Trichodorus spp, Xiphinema index and Longidorus elongatus while Tylenchid migratory ectoparasite includes Tylenchorhychus dubius. Psilenchus, Tylenchus and Atylenchus feed only on root hairs. Sedentary ectoparasites These nematodes feed from a single site or plant cell for a prolonged period of time while remaining outside the root. Sedentary ectoparasites such as Criconemella xenoplax use a single feeding cell as a nutrient source for several days before the nematode moves on to establish another feeding cell. Feeding by C. xenoplax causes little tissue damage compared to Hemicycliophora arenaria and some Helicotylenchus species (migratory ectoendoparasites) which induce terminal galls when feeding on the root tips. Hoplolaimus and Telotylenchus are semi-endoparasites feeding internally and externally on plant root tissues. Endoparasites Endoparasitic nematodes invade the root tissue with part or all of their body. Some feed soon after entering the root while others feed only after migrating to a preferred feeding site (e.g. the cortex and the xylem parenchyma cells) (Plate 1c ) Migratory endoparasites Migratory endoparasites such as Pratylenchus, Hirschmanniella and Radopholus spp, which have a small but robust stylet enter the root and periodically feed as they migrate intracellularly through the root tissue. They primarily inhabit the cortical tissue of the root and retain their mobility and feed on the tissues as they move. This causes extensive destruction of root tissue along the path of the migrating nematode. Sedentary endoparasites Some endoparasitic nematodes become sedentary (Meloidogyne, Globodera, Sphaeronema and Heterodera) and feed from a single cell or a group of cells for a prolonged period of time. For this sustained feeding, the sedentary parasites have evolved very specialized and complex feeding relationships with their host plants. The nematodes invade roots as vermiform second stage juveniles (J2) and development depends upon the modification of the phenotype and the function of the specific root cells to form specialised feeding cells that become permanent source of nutrients for the nematodes (Plate 1d). They modify the root cells of susceptible hosts into elaborate feeding cells including modulating complex changes in cell morphology, function and gene expression. When feeding commences, the juvenile s body grows and it becomes saccate and immobile. In this feeding strategy, destruction of root tissue is usually limited to cells around the feeding site and the nematode. Root-knot nematodes induce giant cells which are enlarged multinucleate cells that are full of cytoplasm and metabolically very active, whereas cyst nematodes form similar cells but are as a result of cell wall breakdown and the formation of a syncytium (Plate 1d). Both types of feeding cell create a transfer cell that enables the rapid transfer of nutrients across the cell to support the development of the female nematode and the production of large numbers of eggs. While the migratory endoparasites primarily inhabit the cortical tissue of the root, the sedentary endoparasites pass through the cortex and invade the vascular cylinder where they induce feeding sites and become sedentary. Unlike 22

23 the sedentary endoparasites which have a fixed feeding site (nurse cells or syncytia) and lose their mobility and become obese, the migratory endoparasites feed on the tissues as they move, retain their mobility and their vermiform shape. Other sedentary endoparasites include Trophotylenchulus obscurus, Tylenchulus semipenetrans, Verutus volvingentis, Cryphodera utahensis, Rotylenchulus reniformis etc. While the J2 is the infective stage in root-knot, cyst and potato cyst nematodes, all stages of ectoparasites and most migratory endoparasites are infective. In Rotylenchulus spp the immature female is the infective stage. Adaptations to mode of feeding Evolutionary adaptations of nematodes for plant parasitism led to the development of the protrusible stylet as well as marked morphological and physiological modifications of the oesophagus to form elaborate secretory glands which are the principal source of the secretions involved in plant parasitism (Plate 2 a-c). These glands enlarged considerably as nematodes evolved from free-living nematodes to obtain nutrients from plants. Tylenchids are well adapted for plant parasitism. In addition to the stylet, they have a well developed oesophagus with a muscular metacarpus containing a triradiate pump chamber and three large and complex specialised secretory gland cells in the pharynx. During feeding, rapid maximum dilation of the pump chamber creates the suction necessary for the nematode to ingest nutrients from the feeding cell through the lumen of the stylet and the oesophagus and force their passage into the intestines. The one dorsal and two sub- ventral glands produce secretions involved in plant parasitism. The dorsal gland cell has a long cytoplasmic extension that extends anteriorly through the metacarpus to terminate into an ampulla, a collecting reservoir for secretory granules in the oesophagus near the stylet knobs. In contrast, the sub- ventral gland cells have short cytoplasmic extensions that terminate in ampullae at the base of the pump chamber in the metacarpus. A sclerotized duct and an elaborate valve with a membrane-delineated end sac connects the ampulla to the oesophageal lumen. The valve controls the release of secretions into the lumen of the oesophagus. The secretions are synthesized in the nuclear region of the glands cells and transported along the microtubules in the cytoplasmic extension to accumulate near the valves in the ampulla prior to their contents being secreted. The association of the neural processes and the neurosecretory cells with the gland cytoplasmic extension and ampulla indicates that the regulated secretion is controlled by the nervous systems. During parasitic development of sedentary endoparasitic species (Meloidogyne spp; Heterdera spp and Globodera spp), the oesophageal gland cells undergo distinct morphological changes. In Meloidogyne spp. the sub-ventral gland cells are the most active glands in infective J2s but as they establish a feeding relationship with the host tissue and increase in body width, the dorsal gland increases and the sub- ventral glands decrease in size. Therefore in adult females, the dorsal gland cell predominates and becomes the functional secreting gland while the sub-ventral are greatly reduced indicating a changing role for the oesophageal gland cells and their secretions at different stages of the nematode life cycle. 23

24 The secretions play a key role in nematode penetration, migration through the root, modification and maintenance of root cells as feeding sites, formation of feeding tubes and /or digestion of host cell cytoplasm to facilitate nutrient acquisition by the nematode. During parasitism of the plant cell, the nematode s stylet penetrates the cell wall but does not pierce the plasma membrane, which becomes invaginated around the stylet tip. The secretions may be deposited outside the plasma membrane or injected directly into the cytoplasm of the host cell through perforation in the plasma membrane at the stylet orifice. The secretions by sedentary parasites transform root cells into metabolically active feeding sites by formation of feeding tubes within the cytoplasm of the parasitized cell. They modify directly or indirectly gene expression to induce profound morphological, physiological and molecular changes in the host cell to enable them to function as a continuous source of nutrients for the nematodes parasitic stages. Removal of nutrients from feeding cells by sedentary nematodes may involve direct withdrawal of the cytoplasmic components (Criconemella xenoplax) or withdrawal facilitated by a feeding tube produced by the parasite (Meloidogyne, Heterodera, Globodera and Rotylenchulus species). 3. Symptomatology Symptoms may vary according to nematode parasitic habits and host parasite relationships, and other factors such as host age and physiological conditions. The symptoms may be seen both above and below- ground. Above ground symptoms Symptoms associated with root nematodes are a direct result of the impaired ability of root systems to take up water and nutrients and thus are essentially similar to symptoms of any root damage that interfers with the physical support and water and nutrient absorption systems. They are thus often similar to mineral deficiencies, inadequate or excessive water supply and generally poor soils. Symptoms are more pronounced if the plants are already affected by other adverse conditions or are attacked by other pathogens. Plants growing under highly favourable conditions may be heavily attacked by nematodes but show few above ground symptoms. The most universal above ground symptoms of a nematode disease are i. Stunting the reduction of growth rate, reduction in amount of foliage and progressive death (die-back) of plants. The stunted and chlorotic plants are distributed in circular to oval areas of variable size in the field but patches of damaged plants may be elongated if infested soil is moved in the direction of cultivation (Plate 3a-b) ii. Chlorosis (yellowing of leaves), poor yield, early senescence, premature dropping of fruits and flowers, fruit malformation. Wilting due to the effect on the functions of roots is also a common symptom of nematode infected plants. iii. Other above ground symptoms are associated with specific nematode species. For example, leaves with dark green spots, angular or cuneiform in shape, with interveinal discoloration and necrosis are associated with Aphelenchoides ritzemabosi on chrysanthemum leaves while twisting and white tips of leaves of rice 24

25 are associated with Aphelenchoides besseyi (Plate 3c). Yellowing and collapse of palm trees followed by a rapid death and a red necrosis in the vascular bundles on the stem forming a red ring in coconut and oil palm is due to infection by Bursaphelenchus cocophilus (Plate 3d). Galls in stems, leaves and seeds of cereals and grasses are caused by Anguina spp. (Plate 3e). Toppling of banana plants especially during fruit bearing is due to Radopholus similis (Plate 3f) Twisting of leaves and raised yellow lesions on stems and leaves on onions and narcissi are by Ditylenchus dipsaci), twisted panicles and empty grains by Ditylenchus angustus on rice, yellowing and rapid death of pine trees (Bursaphelenchus xylophilus) and distorted apical growth and crimpling of leaves and inflorescence (Aphelenchoides besseyi and Aphelenchoides fragariae in strawberry) and so forth. Below-ground symptoms i. Reduced absorption of water and mineral nutrients by the secondary roots. ii. Quantitative and qualitative changes in root exudates such as reduced concentration of amino acids, increased concentration of sucrose, and a disappearance of glucose, threonine, serine, histidine and citric acid in plants infected by some root-knot species. iii. Water movement is affected thus influencing the water potential of leaves, stomatal conductivity, transpiration and root conductivity. Root conductivity can be reduced in beans infected by M. hapla, and in potato plants infected by Pratylenchus penetrans. Globodera rostochiensis causes mineral deficiency (N, P, K and Mg) by affecting ion absorption and reduced water absorption due to poor root development. Calcium concentration may increase due to reduced potassium uptake and dehydration or through the disruption of the endodermis by the nematodes. iv. Reduction of root system especially the secondary feeder roots v. Abnormal development of roots (Plate 4a-h) Overall root galling (Meloidogyne spp. and Nacobbus aberrans) Ulcerations (ulcers/ lesions). Sharply demarcated necroses in different layers of the plant tissues. This results from reactions of phenolic substances in the plants to the secretions discharged by nematodes. Roots with longitudinal necrotic areas are typical of Pratylenchus spp, Radopholus spp; Hirschmaniella spp. infection (Plate 4g) Dry rots usually develop from infestation of fleshy parts of the plants (tubers, root vegetables, stolons) eg by Ditylenchus dipsaci on onions (Plate 4h.), D. destructor on potatoes, R. similis on banana rhizomes. The nematodes cause dry rots in association with secondary invading microorganisms. Excessive branching of secondary roots (M. hapla, Pratylenchus spp. N. aberrans. Localized proliferation of lateral roots (Some Meloidogyne spp and Heterodera spp). Parasitism of young roots stimulates formation of lateral roots. The lateral roots are also infected and the entire root system becomes dendroid and reticulate in appearance. In Heterodera spp. this causes the root-beard disease Swollen, hooked root tip galls (Subanguina spp, Xiphinema spp, Meloidogyne graminicola). Retardation of growth on the root tip. The root 25

26 system is dwarfed and thickened in appearance (Trichodorus, Longidorus, Xiphinema spp,). Roots ending in rounded galls (Longidorus spp. and Hemicycliophora spp). Stubby roots, suppression of root growth (Trichodorus spp and Paratrichodorus) 4. Nematode disease complexes Plant parasitic nematodes can be the sole pathogens or may interact with other plant pathogens or nematodes to cause a disease complex. Nematodes frequently form disease complexes with wilt-inducing and root-rot fungi. Infection by Meloidogyne spp., Pratylenchus and Rotylenchulus reniformis nematodes, for example, increases the incidence and severity of Fusarium, Verticillium spp, Pythium spp. wilt in a number of plants. Meloidogyne spp., Pratylenchus, Anguina and Ditylenchus interact with disease causing bacteria, Clavibacter Pseudomonas and Agrobacterium to increase the severity of the disease. In nematode infested soils, tomatoes were attacked by Ralstonia solanacearum but in nematode free soils they remained healthy. In general, nematodes serve as vectors of other plant pathogens, and/or wounding agents that increase the susceptibility of the host. In general plant parasitic nematodes enhance host susceptibility leading to increased rate of development and severity of wilt and fungal rot diseases. The root exudates from root knot nematode (RKN) infected plants stimulate the fungal pathogens and suppress actinomycetes, which are antagonists of the wilt fungus (Fusarium spp.). The physiological change of RKN- infected plants also enhances penetration by the fungus and wilt development. Root knot nematode infection increases the pathogenicity of the wilt fungus and consequently the severity of the disease. The nematodes establish their feeding sites in the xylem parenchyma cells, bringing about significant changes in the morphology, anatomy and biochemistry of the plant. Giant cells induced by RKN remain in a state of high metabolic activity through continuous stimulation by the nematode. The high concentrations of sugars, hemi - cellulose, organic acids, free amino acids, proteins and lipids benefits the fungal pathogens. The giant cells remain in a perpetual juvenile state which delays maturation and suberisation of other vascular tissues and thus fusarium successfully penetrates and establishes in the xylem elements. Inhibition of phytoalexins by the nematodes implies loss of resistance to the wilt fungus. Inhibition of tyloses formation by the RKN on tomato is a possible mechanism for increased wilt. Tyloses formed in the xylem vessels by the expansion of xylem parenchyma through the pits do not develop from xylem parenchyma cells which are transformed into giant cells or physiologically altered adjacent cells. Nematodes are important vectors of viruses. Xiphinema and Longidorus transmit nepoviruses. Trichodorus and Paratrichodorus vector the tobraviruses. Xiphinema index and X. americanum, for example transmits grapevine fan leaf and tomato ring spot viruses, respectively. Longidorus elongatus transmits the raspberry ringspot virus, Trichodorus cylindricus, the tobacco rattle virus and Paratrichodorus minor, the pepper ringspot virus. The nematodes transmit viruses in a non-circulative manner being the ingestion-egestion type of transmission. The nematode acquires/ingests the virus particles while feeding on the virus infected plant, retains the particles at specific sites within the nematode and egest them by dissociation from the sites of retention. The virus retention sites differ among the 26

27 nematode genera. In Xiphinema, for example, the site is the cuticular lining of the odontophore, the oesophagus and the oesophageal pump while in Longidorus, the site is the inner surface of the cuticular ondontostyle and its guiding sheath. In Trichodorus and Paratrichodorus the site is in the lining of the oesophagus from the most anterior region to the cardia (oesophageal-intestinal valve) but not associated with the onchiostyle. All these surfaces are shed during moulting and therefore the virus particles do not pass from one stage of the nematode to another during development. Bacteria can be transmitted by nematodes externally on their body surfaces or internally within their alimentary canal. Anguina tritici is closely associated with Clavibacter tritici causing the yellow ear rot or tundu in India. The bacterium is associated with the body surface of the juveniles inside the seed galls. Ditylenchus dipsaci transmits Clavibacter michiganense pv. insidiosum which causes wilt in alfalfa. The bacterium is carried on the body of the nematodes into the crown buds and is placed in conducive infection courts. Nematodes have also been reported to transmit fungal pathogens. Anguina tritici is a vector of spores of Dilophospora alopecuri which attacks aerial parts of cereals. The nematode while moving between the leaf sheaths to reach the growing point takes the fungal conidia and deposits them on a growing point. Further, the nematode by feeding on the tender leaves helps in the penetration and establishment of the fungus. All PPN wound plants either by a simple micro-puncture or by rupturing or separating cells. They may thereby either introduce a pathogen on or within their bodies or aid the entry of a pathogen already present on the plant cell surface. Wounding is particularly important for bacteria as they enter plants mainly through wounds. Besides creating wounds the nematodes also modify the host tissue to enrich the substrate nutritionally to the advantage of the bacteria. For example Meloidogyne modifies tomato tissues and makes them more susceptible to bacterial canker (Clavibacter michiganense) and bacterial wilt (Ralstonia solanacearum) especially when the nematodes are inoculated before the bacterium. 5. Mode of Reproduction There are mainly three methods of reproduction in nematodes. i. Amphimixis is also referred to as bisexual reproduction, cross fertilization or gonochorism. The sexes are separate and the females produce oocytes which are fertilized by the sperms from the males. Examples; Anguina tritici, some Meloidogyne species and the mycophagous species, Aphelenchus avenae. In this type of reproduction, the males and females are present in equal numbers, copulation is required and females do not produce sperms. Reproduction using amphimixis is also retained as a facultative ability among some parthenogenetic species and hermaphrodites. In amphimictic species of Hoplolaimina, the spermatheca is usually round and axial and contains elongated sperm, each consisting of a granulated nucleus and a hyaline 27

28 tail portion; the eggs do not mature if no sperm are present in the spermatheca to fertilize them. In parthenogenetic species, the spermatheca is a small and empty sac. ii. iii. Parthenogenesis also referred as autotoky is common where males are very rare, absent or non-functional and are not involved in reproduction. Copulation is optional and females reproduce without sperms. There are two main kinds, meiotic and mitotic parthenogenesis. The 2 types differ in whether or not a first meiotic division takes place in the oocytes. Meiotic parthenogenesis is in Rhabditis, Meloidogyne, Heterodera, Pratylechus and some Longidoridae as well as Aphelenchus avenae. Mitotic parthenogenesis occurs in Rhabditis, Meloidogyne, Helicotylenchus, Pratylenchus. Hermaphroditism (automixis or self-fertilization) is as in Caenorhabditis elegans, in which a single gonad produces both the oocytes and sperms. This together with amphimixis is common in rhabditids and is reported in other free-living nematodes and plant parasites, including criconematids and in several predatory species. It is indicated by the occurrence of hermaphrodite females whose spermathecae contain sperms in the absence of males in the population. The hermaphrodite is morphologically female but has a syngonic (an ovotestis) usually acting protandrically (sperm produced first). After the initial sperm production and their storage in the spermatheca, the gonad switches to oocyte production. A sperm will fertilize any oocyte produced subsequently, thereby achieving automixis. This will continue until the sperm supply is exhausted after which egg -laying may stop in some species. 6. Life cycle and Hatching The life histories of most plant parasitic nematodes are in general quite similar in that all have four larval stages. Eggs may be laid singly or stuck together in masses in a gelatinous matrix secreted by the females. Some females (Heterodera spp.) die and the cuticle tan to form cysts. Many Heterodera spp also produce a proportion of their eggs in a gelatinous matrix (egg mass) attached to the cyst. In RKN all the eggs are laid in an egg sac which may be buried partially within the host-derived root gall which Meloidogyne spp. induce during feeding. Egg masses are also produced by the semi-endoparasitic nematodes such as Rotylenchulus reniformis. Egg sacs and cysts serve to protect the eggs from desiccation and natural enemies. The eggs are typically oval without spines, plugs or excrescences. The uterine egg is semi-fluid and passes through the vagina and vulva with ease. Most Tylenchida are oviparous but a number of entomoparasitic genera have ovoviviparous species in which the eggs hatch in the uterus. Occasionally, in oviparous females, the eggs hatch in the uterus and kill the mother a phenomenon called endotokia matricida. In most species, the egg shell has 3 layers, the outer lipoprotein layer derived from the vitelline layer of the fertilised oocyte and retains the membrane structure. The middle chitinous layer is usually the thickest and provides the eggshell with its structural strength. It has a chitin microfibril core (providing tensile strength) with a collagen-like protein coat (providing the rigidity). The innermost lipid layer represents the main permeability barrier 28

29 of the eggshell. It consists of two or three lipoprotein membranes although in Heterodera schachtii the layer is tetralaminate. The juvenile within the egg develops to adult through four moults, the first moult normally occurring within the egg. The egg develops into a first stage juvenile (J1). The juvenile coils several times within the egg shell and lies still. The J1 grows in size and undergoes the first moult within the egg and then hatches as a J2. The J2 is fully developed except that it lacks reproductive organs and is small in size (Plate 5a-b). Summary of life cycle J2 J1 Egg moults in egg; hatches from egg in Anguina tritici hatches from egg; infective and dauer stage in A. tritici J3 J4 dauer and infective stage in D. dipsaci Adult The J2 undergoes a second moult and becomes a J3 and the J3 undergoes a third moult to become a J4. The J4 undergoes a fourth moult and differentiates into adult females and males and then matures. A life cycle from egg to egg can be completed within 3-4 weeks under optimum environmental conditions. In Longidorus spp. the life cycle takes 2 yrs while in Ditylenchus dipsaci it takes days. Hatching In general given favourable environmental conditions (suitable temperature, oxygen availability and soil moisture levels and in the absence of physiological barriers such as diapause, hatching in most species occurs without requiring specific ques. In a few species, the juvenile hatches when the conditions of the external medium meet particular requirements. For example, in R. reniformis some Meloidogyne, Globodera and Heterodera species, hatching is stimulated by the root secretions from host plants. In certain plant parasitic nematode species, the parasitic life cycle is synchronized closely with that of the host with the aid of environmental and host derived stimuli, to maximize the reproductive success of the nematode. In PPN, the J1 moults within the egg and the resulting J2 hatches. Each egg contains a single juvenile, which hatches by cutting the egg-shell with its stylet by striking it with intermittent rhythmic blows or by rupturing the egg-shell with its tail tip as in Heterodera iri, or through normal rupture of the egg-shell due to juvenile enzymatic secretions and movement. 29

30 The eggs of the cyst nematodes survive in the soil in round (Globodera) or lemon-shaped (Heterodera) cysts each containing several hundred eggs. There are small openings at the neck and the vulval ends of the cyst through which the hatched juveniles escape. Once hatched the J2s of Globodera rostochiensis and G. pallida can survive for <2 weeks without feeding, Meloidogyne javanica and Tylenchulus semipenetrans can persist in the field for months. 7. Nematode dispersal Nematodes can be dispersed actively or passively. Active dispersal Nematodes do not move very far or very quickly by their own locomotory power in the soil. Active nematode migration mostly occurs in the rhizosphere as they are attracted to root exudates. Meloidogyne spp., for example, are attracted to an area just behind the root tip while others such as Pratylenchus are attracted to the root-tips and sometimes further back. The root tip is the region of high metabolic activity from which numerous substances diffuse, some of which act as attractants (gibberellic acid, glutamic acids, tyrosine, amino acids and carbon dioxide), some as repellants and some neither. The best known example of active migration above ground is by Aphelenchoides spp. (e.g. A. ritzemabosi) which moves up the wet external plant parts and then invades the leaves of the host plant. Passive dispersal Dispersal by water Water is a frequent means of passive dispersal. This may include surface run-off such as overland flow, streams, rivers, irrigation canals, percolation and interflow. Infiltration and percolation of water accounts for some downward nematode dispersal but the distance varies with soil properties and precipitation. Dispersal of Radopholus similis, for example, is aided by percolation. Interflow is lateral underground movement of water where percolation water is forced laterally when it comes in contact with an impervious soil layer. Above ground nematodes can be splashed to plants by falling rain or overhead irrigation. Dispersal by wind Wind blowing on bare soils or on low-growing plants or young plants can disperse nematodes e.g. Heterodera schachtii, G. rostochiensis, Criconemoides, Helicotylenchus, Meloidogyne, Pratylenchus, Tylenchorhynchus spp. etc. Phoretic dispersal This includes the involvement of another animal to aid dispersal. Insects are important in dispersing nematodes that attack the aerial parts of plants. Nematodes can pass through the digestive tract of animals and remain infectious in some instances, being dependent on the mobility and the speed of the vector and the survival capacity of the nematode. For 30

31 example, the palm weevil, Rhynchophorus palmarum disperses Bursaphelenchus cocophilus (red-ring of coconut palm). The J3 is deposited onto the palm tree when the weevil is ovipositing her eggs. Bursaphelenchus xylophilus (pine wilt) is transmitted by Monochamus alternatus; a cerambycid beetle. In this instance, the nematode s 4 th stage dauer juvenile is carried on the outside of the beetle under the elytra and in the trachea and deposited into the pine as the beetle feeds. Planting materials Nematodes can spread through planting materials such as seeds, vegetative propagating materials (tubers, corms, bulbs), seedlings and rootstocks. Nematodes spread this way can lead to serious losses in the mature crop or in subsequent crops if nematode build -up is not checked. Dispersal through seeds: Anguina tritici in wheat (Triticum aestivum) Ditylenchus dipsaci in beans (Vicia faba) and onions (Allium cepa) Aphelenchoides arachidis and Ditylenchus africanus in ground nuts (Arachis hypogaea). Tubers: Scutellonema bradys, Pratylenchus coffeae, Radopholus similis, Meloidogyne spp in yams (Dioscorea spp). Bulbs: Ditylenchus dipsaci in onions, garlic and narcissi Rhizomes: Radopholus similis in ginger (Zingiber officinale) and tumeric (Curcuma domestica). Corms: Radopholus similis, Pratylenchus coffeae, P. goodeyi and Helicotylenchus multicintus Seedlings/transplants: Nematodes that are associated with seedlings in nurseries are transferred to the field during transplanting. Dispersal by other means Aphelenchoides besseyi can be spread by contacts between aerial parts of adjacent plants. Leaf drop or wind blown leaves can disperse foliar inhabiting nematodes such as Ditylenchus dipsaci and Aphelenchoides spp. Man s activities also aid in the dispersal of nematodes. E.g. transport of infected plant materials between different countries (international dispersal) or parts of the countries (local spread) or by farm implements between fields and cultivations within fields. 8. Nematode Survival Survival strategies allow endurance of harsh environmental extremes and synchrony with seasonal hosts. There are a number of ways in which nematodes can increase their chances of survival in adverse environmental conditions. They include; 31

32 1. Migrating to escape unfavourable environmental stress. For example, when surface soils are dry the nematodes may migrate to deeper layers of soil. An exodus of nematodes from senescing plants to younger plants 2. High reproductive capacity; example Ditylenchus dipsaci reproduces within the host and builds up high levels of infection. When the host senesces, the nematodes accumulate as 4 th stage juveniles which persist in the soil until they infect a new host (J4 is the infective stage). The J4 are resistant to desiccation and are capable of anhydrobiosis (Plate 6a) 3. Producing survival stages within the life cycle of the nematode is also a survival strategy. Examples: Eggs, infective larvae, cysts, dauer larvae etc as in cyst and root-knot nematodes, J2 of Anguina spp., J3 of several species of Aphelenchoides and J4 of D. dipsaci are capable of surviving long drought periods. 4. Life cycle synchronization. The nematodes develop mechanisms for synchronizing their life cycle with the availability of food or hosts e.g diapause as in Globodera rostochiensis, arrested development and dauer larval formation. 5. Continue activities but modify physiology and behaviour in order to continue living under the changed conditions. Dormancy The nematodes respond to adverse conditions by entering into dormancy which includes diapause and quiescence. Dormancy which can occur at most stages of the nematode s life cycle, is the slowing down or stopping of the nematodes metabolic activities and changing their physiology or structure accordingly while the unfavourable conditions prevail. It ensures that the consumption of energy reserves is kept to a minimum until such a time as they are required for the migration to the host and subsequent invasion or penetration. Diapause is a state of arrested development whereby hatching or activity does not occur until specific requirements have been satisfied even if suitable environmental cues are available. Diapause enables the J2s to overcome cyclic long-term conditions which are unfavourable for hatching and infection e.g winter/summer and/ or the absence of the host and is common in temperate species. Diapause unlike quiescence is temporarily irreversible and requires other triggers to break the dormancy even when all the environmental factors are favourable. For example host-derived stimulus/hatching factor serves as triggers in some nematodes. Thus diapause is associated with a decrease in metabolism and it is not a direct response to unfavourable conditions (unlike quiescence or crytobiosis) but rather to stimuli that heralds the seasonal onset of such conditions. For example, photoperiod- induced diapause in the eggs of Globodera rostochiensis. Diapause may also include a dauer = enduring juvenile formation, for example in RKN and cyst nematodes (Heterodera and Globodera spp.). The length of time that a nematode remains in diapause in nature is for the most part, fixed and is a reflection of the length of the hostile season to which the nematode is subjected and with which it has developed synchrony. 32

33 Diapause allows nematodes to arrest development either before or after exiting the host, the length of the dormancy being dictated by (and in synchrony with) seasonal rhythms, enabling eggs or juveniles to withstand harshness of the external environment. Diapause is therefore not only a means of survival but it is also a means whereby activity of the nematode is made to coincide with that of the host thus increasing the chance of infection. Diapause thus enhances fitness of those nematodes which possess it. Its main function is to act as a timing mechanism to coordinate nematode activity with that of its host in seasonal but otherwise unpredictable environments. Diapause in PPN is categorized into 2 distinct types; obligate and facultative diapause. Obligate diapause- initiated by endogenous factors which are programmed into the life cycle of the nematode and which may or may not be modulated by the host and is terminated by predetermined periods of specific environmental conditions. E.g Meloidogyne naasi and Heterodera avenae Facultative diapause- initiated by exogenous environmental factors and is terminated by either endogenous factors whose duration is dependent on prevailing physical environmental conditions or by the presence of the host. Though more common in Heterodera spp where it provides long-term hatching delays while root- diffusate gives more precise short-term hatching signals, as also evidenced in Globodera and Meloidogyne spp. The ability of Meloidogyne infestations to persist in fallow soil or through periods of drought resides in the ability of females to produce dormant eggs (embryonic diapause) where eggs do not hatch immediately after they are laid. Quiescence= slowed ageing is a spontaneous reversible response to unpredictable unfavourable environmental conditions that results in reduced but measurable metabolism. It occurs at any time of the year and release from quiescence occurs when favourable conditions return. Although quiescence is usually a facultative response occurring only when stress is present, it can also be an obligatory part of the nematode s life cycle and hence referred to as crytobiosis/ anabiosis. Obligate quiescence occurs when the environmental cue affects a specific receptive stage of the nematode life cycle, e. g quiescence of unhatched J2s of several cyst nematodes in the absence of root diffusates while facultative quiescience is nematode stage non-specific. Crytobiosis Crytobiosis = suspended animation takes place if the stress persists or increases and therefore metabolism ceases altogether or it is not measurable. In this situation, all reversible life processes are suspended for a considerable time due to unfavourable conditions. Alterations in metabolism can be triggered by adverse environmental conditions such as i. dessication/dehydration= anhydrobiosis ii. Low and high temperature =cryobiosis and thermobiosis iii. Lack of oxygen = anoxybiosis 33

34 iv. Osmotic shock (high solute concentration = osmobiosis and are reversible once the stress is removed. Anhydrobiosis- adaptation to desiccation Both quiescence and crytobiosis induced by dehydration stress constitute anhydrobiotic survival. In anhydrobiosis, the metabolism comes reversibly to a stand still. Water is an essential component that constitutes 76% of the volume of animals, it s the medium for biochemical reactions and is essential in the structure of biological macromolecules and membranes. In general, therefore, most animals die when they lose more than 15-20% of their body water. Dehydration stress is thus a most important challenge to nematodes since they are basically aquatic in nature. For nematodes parasitising the aerial parts of the plants anhydrobiotic survival is a routine part of their life cycle, involving for the most part specific juvenile stages that are renown for their abilities to survive in this state. The ability of nematodes to survive depends on their inherent ability to withstand desiccation and the stage of nematode development; some nematodes survive better under some moisture conditions than others. Tylenchorhynchus martini survives best in soils between 40-60% field capacity and survival is lowest at 11% and at saturation. The optimum soil moisture range is between 40 80% of field capacity. Dry soils do not allow movement and saturated soils lack oxygen and also inhibit free movement. Excessive moisture kills nematodes through starvation, suffocation or toxins produced by some anaerobes such as Clostridium spp. against Tylenchorhynchus martini. For PPN to enter successfully into anhydrobiosis, they must experience slow and controlled rates of evaporative water loss. Few nematodes are capable of withstanding rapid water loss and prolonged periods of dehydration. Anhydrobiotic PPN can however be divided into 2 broad categories; slow dehydration strategists (SDS) and fast dehydration strategists (FDS). Slow dehydration strategists need a slow rate of water loss to successfully enter anhydrobiosis. They include Pratylenchus thornei, Helicotylenchus dihystera, Scutellonema cavenessi and Aphelenchus avenae. Fast dehydration strategists can survive immediate exposure to low relative humidity and include the anguinids and Ditylenchus dipsaci associated with aerial parts of the plants. They are able to tolerate rapid and repeated cycles of dehydration and rehydration. Anguinids induce galls in the host inflorescence which may provide a barrier to water loss so that their first experience of desiccation may involve a slow rate of water loss. They however will survive fast rates of water loss on subsequent rehydration and desiccation. The unhatched infective juveniles of many cyst nematodes are FDS since the egg shell and the cyst wall may ensure the necessary slow rate of water loss. Fast dehydration strategists can survive rapid loss of water from their surroundings but themselves have mechanisms for ensuring the slow loss of water from their body through behavioural and physiological mechanisms. 34

35 Species Anguina tritici Ditylenchus dipsaci Globodera rostochiensis Scutellonema cavenessi Pratylenchus thornei Helicotylenchus dihystera Longevity whilst desiccated 32 years 23 years 8 years 1 month 1 day 1 day Mechanisms of anhydrobiotic survival They include i. Adaptations to reduce the rate of water loss (slow dehydration) ii. iii. Behavioural adaptations Biochemical and physiological adaptations that help maintain structural and functional integrity at the cellular level i. Reducing the rate of water loss Nematodes have structures that aid anhydrobiotic survival by forming a barrier around the organism. They include the lipid membranes in eggs, the matrix in egg sacs or cuticles of juveniles and adults. The restricted permeability of the nematode egg shell and cyst ensure a slow rate of water loss and may ensure enclosed unhatched J2 survive anhydrobiotically. The permeability of the egg shell of G. rostochiensis decreases as it dries. A lipid layer is the main permeability barrier of the egg shell. The presence of a gelatinous matrix (egg sac) besides the egg shell presents a barrier and allows slow drying of egg masses of the RKN. The gelatinous matrix is reduced to a tough granular mat during drying hence providing the first line of defence in providing a barrier to water loss. When the soil pores around the egg sac drain, the moisture content of the gelatinous matrix around the eggs decreases only slightly thus maintaining a high level of moisture inside. Adults of Rotylenchulus reniformis have a sheath which plays a role in controlling water loss during desiccation. Many PPN remain within the senescing plant host tissue and this provides a physical barrier to slow evaporative water loss. Induction of galls in the inflorescences by anguinids (Anguina tritici, A. agrostis) is a true crytobiotic anhydrobiosis. Survival in anguinids is necessary because in order to infect new hosts, these nematodes must spend sometime exposed to the harsh dehydration regimes associated with soil - air, plant air interfaces before being protected by the plant host itself during the endophytic phase of the life cycle. In anguinids, anhydrobiotic survival involves slow dehydration within the host itself as an initial phase ensuring the development of resistant J2. Slow drying enables anhydrobiotes to prepare themselves metabolically for the onset of anhydrobiosis. It also involves a modification of hosts inflorescence induced by the 35

36 adult nematodes themselves resulting in the formation of galls around the developing mass of juveniles. Slow drying is assured by virtue of mutual protection and by the physical barrier presented by the gall itself. The J2 s of the two nematodes develop tightly packed aggregates (not preceded by swarming within the gall). Coiling of individual nematodes outside of the mass also occurs thus enhancing anhydrobiotic survival. Finally an exodus of nematodes from senescing plants to younger plants has survival importance. In Ditylenchus dipsaci, the resistant J4 are able to control water loss to a much greater extent than other stages in the life cycle because of changes in the permeability characteristics of their cuticle. The nematode is also adept at clumping, coiling and using plant tissue to slow rates of drying. Both J4 and to a lesser extent J3 stages have an intrinsic ability to control water loss through a decrease in permeability of the cuticle as it dries. The nematode also exhibits behavioural adaptations to dehydration. Behavioural adaptations to dessication These are mainly exhibited by FDS and include clumping and coiling. Clumping Clumping is often preceded by swarming which is a coordinated/synchronized movement by a mass of nematodes to specific parts of the host or from one location to another. The swarms therefore consist of nematodes showing synchronized movements and are involved in migration and dispersal and may be followed by aggregation where nematodes form dense masses. As dehydration progresses, the nematodes actively coil before becoming inactive. Coiling may involve the whole of the nematode mass or just those on the periphery. Swarming occurs in bacteriophagous and mycophagous nematodes and PPN. The clumps formed may aid desiccation survival but swarming itself is not triggered by desiccation. Swarming may be triggered by accumulation of toxic waste products, the exhaustion of food supplies and / or the senescence of a host plant. In Ditylenchus dipsaci J4 swarm from senescing host tissue and form clumps/aggregates commonly referred to as eelworm wool and are coiled between scales of infected bulbs (tulips & narcissus) or inside bean pods (Plate 6a). The formation of clumps aids the control of water loss since nematodes on the outside will dry first and form a layer that slows down the rate of water loss from nematodes deeper in the aggregation. This is called the egg-shell effect. Aggregation also occurs in Aphelenchus avenae and anguinids which accumulate in infected host inflorescences and induce them to form galls. Coiling is associated with a wide range of nematodes as a desiccation survival mechanism, which reduces the rate of water loss by reducing the exposed surface area of the nematode. Rotylenchulus reniformis coiled nematodes survive desiccation at 80% and 40% relative humidity. Coiling may follow or occur in conjunction with swarming and aggregation or the nematodes coil individually. The primary stimulus for coiling behaviour appears to be the restriction of lateral movement that occurs in an evaporating water film. Nematodes will also coil in response to increases in temperature and osmotic stress. Biochemical adaptations to dessication 36

37 Biochemical changes also occur during entry into anhydrobiosis, for example, elevated levels of the disaccharide trehalose. Trehalose stabilises membranes during desiccation and rehydration by attaching to the polar head groups of the phospholipids and preventing phase changes that may cause the membrane to become leaky; ( water replacement hypothesis ). Trehalose also stabilises tissues via vitrification by acting as a high viscosity, low molecular mobility medium. Trehalose also prevents protein denaturation oxidative damage and browning reactions as a free radical scavenging agent and as an inert energy source as in A. avenae. There are also desiccation induced heat- tolerant proteins as in A. avenae. Physiological adaptations include change in cuticular permeability and a decrease in surface area by the shrinkage of the body of the nematode as in D. dipsaci J4 which narrows or deepens the grooves of the cuticular annulations. Cryobiosis and thermobiosis adaptation to temperature changes All organisms have an optimum temperature at which their metabolism and hence growth and activity are greatest. Temperature is a major factor limiting distribution of PPN world wide and in different soil depths. Meloidogyne incognita and M. javanica are found in warmer parts of the world unlike M. hapla and can therefore survive better in high and low temperatures, respectively. Nematodes are poikilotherms; their body temperature follows that of the environment. The optimum soil temperature range for activity is C, above and below this range, nematodes become inactive and temperatures below 4 0 C and above 40 0 C may be lethal. Nematodes exhibit a range of responses to temperature changes. The metabolic rate decreases as the temperature decreases due to the lower kinetic energy imparted to reactions but proteins are not denatured by the low temperature and so the effects are potentially reversible. Cryobiosis- Nematodes are more likely to survive very low temperature (< 0 0 C) than high temperature (>50 0 C) since the adverse effects are potentially avoidable or reversible. Nematodes can extend the survivable limits of both high and low temperature by triggering protective responses. Some nematodes enter cryobiosis surviving temperatures below that at which metabolism ceases with some surviving temperatures as low as minus 80 0 C. As the temperature falls, nematodes display cold stupor, cold coma and freezing. If the temperature of an animal is below the temperature at which its body fluids freeze (melting point of their body fluids), it is at risk of freezing. Nematodes survive these conditions by being either freeze tolerant (surviving ice formation within their bodies) or freeze averse (preventing ice nucleation and maintaining their body fluids as a liquid at temperatures well below their melting point, by super cooling). In freeze tolerant nematodes, the temperature at which it freezes (the super cooling point) is well above the temperature at which the animal dies (the lower lethal temperature-llt) and usually close to its melting point, for example Ditylenchus dipsaci and Aphelenchoides ritzemabosi. In freeze averse nematodes, the super cooling point is considerably below its melting point and fairly close to the LLT and it dies once it freezes. In these nematodes such as G. rostochiensis, they avoid freezing by preventing ice nucleation and allowing super cooling. Other adaptations involved in freezing tolerance and cold tolerance include; the production of sugars (trehalose and glycerol) and polyols as cryoprotectants or anti-freezes, avoidance 37

38 of membrane transitions which may be involved in non-freezing injury and production of stress proteins. Thermobiosis-A nematode exposed to increasing temperature is first likely to display heat stupor, where movement becomes disoriented or normal processes/ activities are disrupted, then heat coma, where movement and activity ceases, followed by death at the thermal death point. Nematodes may tolerate heat through the production of heat-shock proteins (stress proteins and molecular chaperones) that both stabilize the target proteins (protecting them against denaturation) and reactivate damaged proteins. For example, embryogenesis and hatching of the eggs of M. javanica and M. naasi can be slowed or stopped at elevated temperature and readily resumed once the temperature stress is removed. Anoxybiosis survival at low oxygen levels Conditions associated with low oxygen tensions are generally a feature of the soil environment caused by an increased level of soil saturation (up to and including flooding) which restricts the rate at which oxygen can diffuse from air into the interstitial spaces of the soil and by the microbial activity placing demands on the available oxygen. Poor aeration induces cryptobiosis in juveniles enhancing anoxybiotic survival. Eggs of PPN and juveniles of many soil dwelling nematodes are able to tolerate low oxygen tension by reducing normal oxidative metabolism levels and show some capacity for facultative anaerobiosis by switching to fermentative as opposed to oxidative respiratory pathways during anoxia. Examples include Caenorhabditis spp, eggs of many Meloidogyne spp and a majority of free-living mycophagus nematodes. This constitutes a physiological adaptation. Certain stages of PPN e.g. J2 of Anguina sp. and all stages of Anguina amsinckiae must endure protracted periods of low oxygen tension and eventually anoxia as the plant senesces and the galls dry. Response to anaerobiotic conditions depends on developmental stages and species involved. Trichodorus christiei, Xiphinema americanum are more sensitive to anaerobiosis than Tylenchulus semipenetrans and M. incognita. Osmobiosis- survival at high salt levels A majority of PPN and free living nematodes are osmoconformers, showing limited ability for osmotic or ionic regulation. High osmotic stress may be produced by drying of the soil, addition of fertilizers etc. Osmobiosis (which protects a nematode from the effects of fluctuations in the external physical environment) is evidenced in cyst nematodes (Globodera rostochiensis and Heterodera goettingiana). The high concentration of trehalose in the perivitelline fluid of the eggs of Globodera rostochiensis imposes an osmotic stress which maintains the enclosed J2 in a state of quiescence. Hatching stimulus results in an increase in permeability of the egg shell, which allows the trehalose to diffuse out of the egg and for water to enter to remove the osmotic stress. Trehalose is a common component of the perivitelline fluid (in the perivitelline space of the egg) of a variety of nematode eggs that do not require specific biotic factors (hatching factors) to break dormancy. Trehalose acts as a cryoprotectant or antifreeze. 38

39 In hyposmotic conditions water enters the body and will have to be removed while in hyperosmotic conditions water will be lost and the nematode stressed. The water lost may induce a state of osmobiosis which enables the nematode to survive the stress period. References Gaugler R. and Anwar B. L. (eds.) Nematode behaviour. CABI Publishing, Oxfordshire, UK. 419pp Lee D. L. (ed.) The biology of nematodes. Taylor and Francis, London UK. 635 pp Luc, M., Sikora R. A. and Bridge J Plant Parasitic Nematodes in Subtropical and Tropical Agriculture CABI Publishing, Oxfordshire, UK. 871 pp Perry R. N. and Wright D. J. (eds.) The Physiology and Biochemistry of Free living and Plant parasitic nematodes. CABI Publishing, Oxfordshire, UK. 438pp Wajid K. M. (ed.) Nematode Interactions. Chapman and Hall, London UK. 377pp 39

40 CHAPTER 3 DIAGNOSIS AND MORPHOLOGY OF PLANT PARASITIC NEMATODES 1. INTRODUCTION Zibusiso Sibanda Formerly Nematology Department Kutsaga Research Station PO Box 1909 Harare. Zimbabwe This chapter provides a basic introduction to the diagnosis of plant parasitic nematodes, particular reference being paid to those groups found in eastern and southern Africa. The information present in this chapter is a summary of a more comprehensive training manual that was prepared for the First Gatsby Regional Nematology Training Course. The detailed manual can be made available on request on CD. Illustrations have been adapted from the published work of other nematologists and their invaluable contribution is acknowledged. COMMON MORPHOMETRIC ABBREVIATIONS There are a number of abbreviations for the various characters measured and ratios calculated when compiling morphometric profiles of nematodes. Some of these values are of more use than others and so an understanding of their potential variability and reliability is of paramount importance. The most commonly used abbreviations are as follows: L = Total body length (head to tail tip). L = Body length from head to anus/cloaca (used when tail is very long and therefore subject to greater variation and the likelihood of breakage). a = Total body length divided by maximum body width. b = Total body length divided by oesophageal length (the oesophagus is defined as head end to oesophago-intestinal junction, i.e. not to the posterior tip of the overlapping gland lobes). b' = Total body length divided by distance from anterior end of body to posterior end of oesophageal gland lobes. c = Total body length divided by tail length. c' = Tail length divided by anal/cloacal body width. V = Position of vulva from anterior end expressed as percentage of body length. Superior figures refer to the extent of anterior and/or posterior gonad or uterine sac, also as percentage of body length. 40

41 V' = Position of vulva from anterior end expressed as percentage of distance from head to anus (i.e., use L, not L). T = Distance between cloaca and anteriormost part of testis expressed as percentage of body length. m = Length of conical part of stylet as percentage of whole stylet length. o = Distance of dorsal oesophageal gland opening behind stylet knobs as percentage of stylet length MB = Distance of median bulb from anterior end expressed as a percentage of total oesophageal length. Caudal ratio A = Caudal ratio B = Length of hyaline tail divided by its proximal width. Length of hyaline tail divided by its width at a point 5µm from its terminus. µm = One thousandth of a millimetre (micron). The following additional parameters are routinely used in the taxonomy of criconematids: R = Total number of body annules. Rex = Number of annules from anterior end to excretory pore. RV = Number of annules from posterior end to vulva. RVan = Number of annules from vulva to anus. Ran = Number of annules from posterior end to anus. 2. GENERAL MORPHOLOGY It is not the object of this module to provide any depth of detail concerning nematode morphology or classification. Nonetheless, it is important to understand some of the fundamentals of nematode morphology in order to more fully understand the biology of nematode-plant interactions. Nematodes are lower invertebrate animals. They are highly diversified and perhaps the most numerous multicellular animals on the earth. Like insects, they are found in almost all types of biotypes and occur in unimaginable numbers and in a wide variety of shapes and sizes. Nematodes are generally free-living in marine, freshwater or soil environments, but a large number of species are parasitic on different kinds of plants and animals. The parasitic species are of considerable agricultural, clinical and veterinary importance as pests of plants and parasites of Man and livestock respectively. Nematodes are found at the bottom of lakes, rivers and at enormous depths in the oceans. Some species can withstand temperatures constantly below freezing point while others live in the waters of hot springs. By 1930 some 4,500 species of nematode had been described. This rose to 9,000 by 1950, and the present-day number of known species of nematodes is well over 15,000. The estimated number of existing species ranges from 500,000 to several million, but the truth is that nobody has much of an idea of their total number. This means that the majority of 41

42 nematode species are not yet known and the taxonomy and phylogeny of this group are thus still very much in the early stages. Nematode morphology is only visible to us after special procedures for extraction and fixation, and by means of complex instruments of observation. These procedures and instruments completely determine our knowledge of nematode morphology, because they impose a strict limit on the level of detail with which we can study nematodes. They may even lead us to substantial errors through the incorrect interpretation of the artefacts which they can generate. All these factors boil down to the following essentials: describing the morphology of nematodes requires a great degree of jargon, because of their diversity and uniqueness, as well as a great degree of caution and training in microscopical observation. The following paragraphs will introduce you to the major descriptive terms in nematode morphology. Although real experience can only be gained by examining fixed and live nematodes for yourself, knowledge of the terminology should at least allow you to identify the main organs and structures in the nematode body, and will thus set you on your way to chart the endless diversity of this phylum. Fig. 1. Anatomy of a plant-parasitic nematode : Rotylenchoides variocaudatus (Dropkin, 1980). 42

43 3. PRINCIPLES OF SYSTEMATICS 2 Taxonomy (Greek taxis = arrangement; nomos = law) deals with the naming and recognizing of taxa, and systematics (Greek systema = whole consisting of parts) is a method of arranging taxa into a hierarchical system of classification. Unfortunately, these two terms are often confused, even by people who should know better. The fashionable term 'biosystematics' is usually used to embrace both concepts. The classification of animals is a basic part of zoology. It serves the purpose of recognizing, and hence distinguishing, animals with respect to each other. Classification at present is based on taxonomic characters, mainly from morphology and comparative anatomy. These taxonomic characters have different values and significance at different grades and categories. For example, there are characters of specific, generic and familial values. Determination of their value, or weighting, is done through using various methodologies as will be discussed here. The monothetic concept in classification is based on the use of a single character or feature as against the polythetic concept which involves the use of several characters. The monothetic concept has often led to ambiguities in classification. Rules of Zoological Nomenclature The taxonomy and classification of animals are governed by rules listed in The International Code of Zoological Nomenclature (ICZN). The Code covers categories from subspecies to superfamily only. It does not cover ordinal, class, or Phylum ranks, nor does it cover the infra-subspecies categories such as form, variety, strain, biotype, etc. Species are based on populations of individuals which can be studied, but genera and higher categories are more abstract, often with extremely vague boundaries and limits. The name of a species is binominal, i.e. it is a binomen consisting of two words, generic and specific. The genus- and species-group names should appear in italics or in a type face different from that of the text. All the names in suprageneric categories are uninomina, i.e. made up of one word and not italicized. All the names of taxonomic categories except species and subspecies names begin with a capital letter. In Tylenchida, taxa are recognized and classified mainly on morphological characters. It is only recently that ecological, biochemical, cytological and embryological characters have been introduced, but still in a very limited manner. The characterization of taxonomic groups involves morphological, biochemical and cytogenetic taxonomical methods while determining their rank is done by three broadly categorized methods: (1) evolutionary systematics (2) cladism (3) phenetics 2 After M R Siddiqi 43

44 A. Morphological Taxonomy: Morphological characters provide more than 90% of the data used in taxonomy and classification of plant and soil nematodes. These characters show considerable variation and care must be taken in using them. Morphometric and allometric measurements often vary greatly under the influence of geographical and ecological conditions. Techniques, types of equipment, methods of observation and personal skills also result in variations. Some characters used to differentiate species of Pratylenchus, e.g. stylet knob shape, length of outer margins of cephalic framework, the shape of the spermatheca and tail tip, tend to vary through host influence. B. Biochemical Taxonomy: Gel electrophoresis has been widely used as a technique in the identification of plant-parasitic species. Evans (1971) was able to distinguish between Ditylenchus destructor and D. myceliophagus populations through the study of their esterase patterns as revealed by gel electrophoresis. Different protein band profiles were obtained by using disc electrophoretic separation of soluble proteins and enzymes of some species of Ditylenchus, Heterodera and Meloidogyne (Dickson et al., 1971). However, these techniques have been used only in a limited manner, although the scope is considerable. Immunological techniques involving DNA hybridization and isoenzyme analysis have been applied to certain aspects of plant nematode taxonomy, although specialized biochemical knowledge, skills and instrumentation are required to use such techniques. The current way forward seems to be via molecular techniques, particularly those employing polymerase chain reaction (PCR) amplification of DNA. C. Cytogenetic Taxonomy: Cytological analysis can be utilised in the differentiation of species and pathotypes. Chromosome number has been used in the differentiation between some species of Meloidogyne and between Radopholus similis similis and R. s. citrophilus, although in the latter case chromosome number has been shown to be more variable than previously thought and therefore useless as a race marker. Chromosome number also differs between nematode groups, for example, Criconematoidea and Hoplolaimidae have a basic chromosome number of n = 5; Dolichodoridae including Belonolaiminae have n = Evolutionary systematics The traditional evolutionary approach involves the reconstruction of phylogeny by using past histories of animals as revealed by the study of their fossils and comparative morphology/anatomy. 2. Cladism The cladistic analysis is a kind of interpretation of similarities in and between taxa. The similarities are established by in-group and out-group comparison of two types of characters or character states. 44

45 3. Phenetics This approach tries to classify organisms on over-all similarities in their structure without bothering to know how they have come about. The classification of Tylenchida has been based almost entirely on structural similarities which have seldom been supplemented with ecological, ethological and physiological data. For this order, the phenetic approach will continue to have a major role to play since identification and classification will have to be based largely on morphological data. The use of a large number of characters, as in numerical taxonomy, has yielded consistent results in developing hierarchical classification. Sokal & Sneath (1963, p. 264) state "One of the attractive properties of taxonomies based on large numbers of characters is their robustness under different statistical treatments. They consider all characters of equal weighting. Key characters, however, are recognisable after a careful over-all comparison has been made. REFERENCES AND ADDITIONAL READING Blackith, R.M. and Blackith, R.E A multivariate study of Tylenchus Bastian, 1865 (Nematoda, Tylenchidae) and some related genera. Nematologica 22, Cobb, N.A One hundred new nemas. Contrib. Sci. Nematol. 9, Coomans, A General principles for the phylogenetic systematics of nematodes. In: Concepts in Nematode Systematics. (Ed. Stone, A.R., Platt, H.M. & Khalil, L.F.). London & New York: Academic Press, pp Dickson, D.W., Huisingh, D. and Sasser, J.N Dehydrogenases, acid and alkaline phosphatases, and esterases for chemotaxonomy of selected Meloidogyne, Ditylenchus, Heterodera and Aphelenchus spp. Journal of Nematology 3, 1-16 Evans, A.A.F Taxonomic value of gel electrophoresis of proteins from mycetophagous and plant parasitic nematodes. Int. J. Biochem. 2, Fortuner, R., Maggenti, A.R. and Ehittaker, L.M Morphometrical variability in Helicotylenchus Steiner, 1945: 4. Study of field populations of H. pseudorobustus and related species. Rev. Nematol. 7, Hennig, W Phylogenetic Systematics. Urbana, USA: Univ. Illinois Press, 263pp Hooper, D.J Identification of plant and soil nematodes. In: Nematodes of Tropical Crops (Ed. Peachey, J.E.). St Albans, UK: Comm. Bur. Helminth., Tech. Commun. No. 40, pp

46 International Code of Zoological Nomenclature, III Edit. International Trust for Zoological Nomenclature, c/o Commonwealth Institute of Entomology, 56 Queen's Gate, London SW7 5JR Jones, F.G.W Parasitism in plant nematodes. In: Plant Nematology (Ed. Southey, J.F) Tech. Bull. (MAFF) No. 7, London, UK: HMSO, pp Mai, W.F. and Lyon, H.H Pictorial key to genera of plant-parasitic nematodes. Fourth edition, revised. Ithaca & London: Comstock Publishing Associates, Cornell University Press, 219 pp Mayr, E Principles of Systematic Zoology. New York, USA: McGraw-Hill, 428pp Moss, W.W. and Webster, W.A Phenetics and numerical taxonomy applied to systematic Nematology. Journal of Nematology 2, Platnick, N.I Cladograms, phylogenetic trees, and hypothesis testing. Syst. Zool. 26, Simpson, G.G Principles of Animal Taxonomy. New York, USA: Columbia University Press, xii + 247pp Sokal, R.R and Sneath, P.H.A Principles of Numerical Taxonomy. San Francisco, USA: Freeman & Co., 359pp Triataphyllou, A.C Cytogenetic aspects of evolution of the family Heteroderidae. Journal of Nematology 2, Triantaphyllou, A.C Cytogenetic aspects of nematode evolution. In: Concepts in Nematode Systematics. (Eds Stone, Platt, H.M. & Khalil, L.F.) Academic Press, pp Williams. T.D Plant-parasitic nematodes. In: Pathologist's Pocketbook. Kew, UK: Commonwealth Mycological Institute, pp SYSTEMATIC SCHEME The following simplified scheme will be followed throughout this manual. It should not be interpreted as the only valid scheme and can be modified according to personal requirements/preferences/whim. Only a small proportion of the genera listed will be dealt with in detail and many will not be mentioned further as they are not of importance in eastern and southern Africa. The scheme is based on the published works of Siddiqi (1986; 2000) and Hunt (1993) and updated where necessary. Synonyms and subgenera are not listed in order to 46

47 enhance clarity. The relevant section of the scheme is repeated for convenience as each major group is considered in the manual. Major nematode genera of economic importance, together with confusable genera, in eastern and southern Africa are printed in bold type. 5. ORDER TYLENCHIDA This order contains the majority of plant parasitic nematodes and is usually divided into three suborders. These are the Tylenchina, Criconematina and Hexatylina, the majority of plant parasites being in the two former groupings. In some schemes there is a fourth suborder, the Aphelenchina, although in this manual the aphelenchs are regarded as belonging to a separate order. In practical respects it matters little which scheme is embraced, the higher classification of nematodes being somewhat arbitrary, particularly at present with the advent of molecular-based phylogenies. SUBORDER TYLENCHINA Diagnosis: Tylenchida. Males with normal oesophagus and stylet (stylet occasionally reduced in some Hoplolaimoidea). Phasmids present, sometimes enlarged, scutellum like and migratory (Hoplolaiminae), in Tylenchoidea papilla-like phasmids or phasmid-like structures present in submedian position on body, in female near vulva, just dorsal to the lateral fields. Cuticle often distinctly annulated, sometimes marked with longitudinal striae or grooves, but annules never retrorse or with scales, spines, appendages or a double cuticle. Cephalic region generally distinctly annulated. Stylet variable in length; conus normally as long as shaft, if elongate then shaft more than 10µm long; knobs variable in shape and size but never large and anchor-shaped as in Criconematoidea of Criconematina, occasionally absent (e.g. Psilenchinae). Precorpus (= procorpus) cylindroid or fusiform; postcorpus or median bulb muscular with valve plates but not amalgamated with precorpus into a large muscular cylinder, or a non-muscular fusiform swelling. Isthmus slender. Oesophageal glands forming a basal bulb or extending over intestine. Oesophagointestinal valve (= cardia) 3-celled, reduced in forms with overlapping glands. Nerve ring circum-oesophageal. Excretory pore generally in oesophageal region. Female: Slender, or obese (spherical, kidney- or lemon-shaped) and may turn into a cyst in some sedentary Hoplolaimoidea. Usually didelphic, amphidelphic (becoming didelphicprodelphic in Heteroderidae and Meloidogynidae). Posterior reproductive branch secondarily reduced or represented by a postvulval uterine sac (e.g. Pratylenchinae, Trophurinae), but in Tylenchoidea as a rule lacking, except for a uterine sac. Vulva a large transverse slit, rarely oval (e.g. some Merliniinae), equatorial or submedian, shifted to lie subterminally or terminally in swollen females. Ovaries generally paired, normally outstretched and with serially arranged oocytes, rachis absent (except in obese females). Males: Oesophagus and stylet normally developed (except some Hoplolaimoidea). Testis single (except some Meloidogyne spp. in which testes are paired, probably due to sex 47

48 reversal; the female primarily being didelphic) outstretched. Spicules paired, rarely dissimilar, cephalated, arcuate, distally round to pointed, never setaceous or U-shaped. Gubernaculum simple or modified; fixed or protrusible. Juveniles: Similar to female in most details occasionally with reduced oesophagus and stylet (e.g. Rotylenchulus) due to rapid development from egg to adult without feeding. Bionomics: Algal and moss feeders, and parasites of lower and higher plants; attacking subterranean parts, not parasitic in above-ground parts of plants or in animals. Key to superfamilies of Tylenchina 1. Phasmids not detectable on tail; phasmid-like structures present much anterior to tail region, dorsal to lateral fields, in female near vulva; tails generally filiform...tylenchoidea Phasmids present in or near tail region (except for migratory scutella of Hoplolaiminae), in lateral fields, not near vulva; tails generally not filiform, if filiform then with distinct phasmids Subventral oesophageal glands enlarged, usually extending past the dorsal gland; sexual dimorphism in anterior region manifest...hoplolaimoidea Subventral oesophageal glands not enlarged, not extending past the dorsal gland; sexual dimorphism in anterior region not manifest...dolichodoroidea 5.1. SUPERFAMILY TYLENCHOIDEA The classification of this superfamily is fairly complex and open to differing points of view. Although there are many nominal genera, it is not usually necessary to identify the group much beyond a general catch-all grouping such as Tylenchus, sensu lato (i.e. in the 'broad sense' and taken to include all the related genera). Most of the genera of this superfamily are associates of algae, moss, lichen and plant roots. They are not root parasites of any agricultural significance and so will be dealt with only superficially. They are, however, commonly encountered in large numbers in washings from soils rich in organic matter and so need to be recognised and separated from the potentially more damaging plant parasitic nematodes. 48

49 These nematodes are considered to be fairly primitive in form, being a conservative group with many ancestral characteristics (e.g. weak feeding apparatus, poorly developed corpus, filiform tail, etc.). FAMILY TYLENCHIDAE Örley, 1880 Diagnosis: Tylenchoidea. Small to moderately large ( mm), not abnormally slender nematodes. Lateral field with 2, 3 or 4 incisures. Labial disc inconspicuous or absent; a perioral disc present in Discotylenchus. Stylet small (6-21µm), conus usually less than half total stylet length, rarely with distinct lumen; basal knobs or swellings small, rounded, or absent (e.g. Neopsilenchus, only subventral knobs absent in Irantylenchus);. Orifice of dorsal gland close to, or at some distance (Boleodorinae, Thadinae) from, stylet base. Precorpus cylindrical. Post corpus or median bulb fusiform or oval, not filling body width, muscular and valvate, or non-muscular, non-valvate; may be absent. Isthmus slender. Basal bulb with glands abutting intestine, with cardia at base. Vulva a transverse slit; lateral membranes rarely present (Aglenchus). Postvulval uterine sac one body width or less offset (except Thadinae), often elongated and directed anteriorly. Ovary single, anteriorly outstretched, oocytes usually in a row. Testis outstretched, tip rarely reflexed. Spicules small, slender, arcuate. Gubernaculum linear or trough-shaped, fixed. Tails generally filiform, similar in both sexes. Bursa adanal, simple, not lobed. Note: The important characteristics of this group include: 1) weakly sclerotised head skeleton 2) stylet and basal knobs weak 3) basal bulb not overlapping intestine 4) genital tract monodelphic, prodelphic 5) phasmid present near vulva, not usually within lateral field 6) tail elongate-filiform in both sexes 7) bursa adanal The body is generally vermiform (worm-like), with the posterior portion slender and tapering sharply to pointed or rounded terminus. The vulva is positioned in the posterior section of the body, although due to the attenuated tail of some genera, the vulval ratio can be greatly affected by variations in development of, or damage to, the filamentous portion. This is resolved by using V' (dividing the length from head to vulva by the length from head to anus 100). On death the body can be slightly curved ventrally (Discotylenchus) to open C-shaped (Tylenchus). The oesophagus is very variable in shape. The procorpus can be with or without median bulb. There is usually a long isthmus and basal bulb. The basal bulb can overlap the intestine, but the gland nuclei remain within the bulb. The head shape is variable and can be used to distinguish genera and species. The stylet is usually delicate and short (average for the family is about 13µm). The genital tract is monodelphic, prodelphic with a post-vulval uterine sac of variable length usually present. The length of the uterine sac is important in identifying 49

50 genera and species. The area around the vulva is important (the presence or absence of cuticular flaps, angle at which the vagina exits via the vulva). The female tail shape is variable between genera, but is usually attenuated and tapering to a pointed terminus. The male tail is similar in shape to that of the female, except for the presence of spicules, gubernaculum and bursa. The spicules are tylenchid in shape, with a simple gubernaculum and the bursa is adanal. The presence of males in populations is dependant on species - some are parthenogenetic and others amphimictic. Bionomics: Associates of algae, mosses, lichens and plant roots, but generally not root parasites of any significance. Key to major subfamilies of Tylenchidae 1. Amphidial apertures prominent, posterior to level of cephalic papillae, partially covered by a cuticular flap...boleodorinae Amphidial apertures rarely prominent, anterior to cephalic papillae, not covered by a cuticular flap Lateral field with 2 incisures (single ridge)...duosulciinae Lateral field with 3 or 4 incisures (2 or 3 ridges), rarely obscure Crustaformeria generally tricolumellate; spermatheca offset; tails elongate filiform...tylenchinae Crustaformeria quadricolumellate; spermatheca axial; tails short, conoid to subcylindrical...thadinae Subfamily TYLENCHINAE Örley, 1880 (Marcinowsky, 1909) Diagnosis: Tylenchidae. Body about mm long. Cuticle finely striated, or coarsely annulated (but smooth in Polenchus). Lateral field with 3 or 4 incisures. Cephalic region continuous or slightly offset. Stylet 6-21µm long, conus shorter than or rarely as long as shaft, with tip appearing solid; knobs distinct (except Irantylenchus). Orifice of dorsal oesophageal gland at about a quarter or less of stylet length behind stylet base. Muscular median oesophageal bulb present or rarely absent (Sakia). Basal bulb offset from intestine. Vulval lips simple, or modified (Aglenchus). Post vulval uterine sac present (except Aglenchus). Ovary single, outstretched, occasionally reflexed at the tip. Testis outstretched; sperm small, rounded. Spicules and gubernaculum typical of the family. Bursa adanal, simple. Tail generally filiform in shape. Type genus: Tylenchus Bastian, 1865 Other genera: 50

51 Tylenchus Bastian, 1865 Coslenchus Siddiqi, 1978 Filenchus Andrássy, 1954 (Meyl, 1961) Key to major genera of Tylenchinae 1. Median oesophageal bulb absent or represented by a non-muscular, non-valvate swelling... Sakia Median oesophageal bulb present, muscular, valvate Subventral knobs of stylet absent......irantylenchus Subventral knobs of stylet present Cephalic region with a perioral disc...discotylenchus Cephalic region without a perioral disc Longitudinal ridges outside lateral fields present......coslenchus Longitudinal ridges outside lateral fields absent Cuticle non-annulated.....polenchus Cuticle annulated Postvulval uterine sac absent; male cloacal lips tubular....aglenchus Postvulval uterine sac present; male cloacal lips not tubular Tail ventrally arcuate or hook-like...tylenchus Tail not ventrally arcuate or hook-like...filenchus Genus TYLENCHUS Bastian, 1865 syn. Tylelenchus Bastian, 1865 (= officially rejected name) Aerotylenchus Fotedar & Handoo, 1979 Areotylenchus Fortuner, 1984 (junior objective synonym of Aerotylenchus) Diagnosis: Tylenchinae. Small to medium sized ( mm), ventrally curved upon relaxation. Cuticle moderately thick (1-2µm), distinctly annulated. Lateral fields each with four incisures. Cephalic region continuous, annulated; framework with light or no sclerotization. Stylet 8-21µm long, with conus comprising between one third and half of stylet length and round, posteriorly sloping basal knobs. Median oesophageal bulb oval, muscular, anterior to middle of oesophagus and with refractive valve plates; basal bulb pyriform. Cardia distinct. Excretory pore usually opposite basal bulb. Vulva a transverse slit, usually at 60-70% of body length, lips not modified; epiptygma or lateral membranes absent. Vagina generally at a right angle to body axis. Postvulval uterine sac about a body width or less long. Spermatheca round to oval, offset. Ovary outstretched. Tail ventrally arcuate, often hooked, regularly tapering to a pointed or minutely rounded 51

52 terminus. Bursa adanal, margins crenate. Spicules cephalated, arcuate, 13-25µm long. Gubernaculum simple, fixed. Cloacal lips slightly raised, anterior one pointed, posterior usually rounded; not tubular. Hypoptygma absent. Type species: Tylenchus davainei Bastian, 1865 Other species: Many other species have been named, but only a fraction of these are regarded as valid, the remainder being species inquirendae et incertae sedis. REFERENCES AND ADDITIONAL READING Andrássy I. (1979). The genera and species of the family Tylenchidae Örley, 1880 (Nematoda). The genus Tylenchus Bastian, Acta zool. hung. 25: Andrássy I. (1980). The genera and species of the family Tylenchidae Örley, 1880 (Nematoda). The genus Aglenchus (Andrássy, 1965) Meyl, 1961, Miculenchus Andrássy, 1959, and Polenchus gen. n. Acta zool. hung., 26: Andrássy I. (1981). The genera and species of the family Tylenchidae Örley, 1880 (Nematoda). The genera Malenchus Andrássy, Acta zool. hung., 17: Andrássy I. (1984). The genera and species of the family Tylenchidae Örley, 1880 (Nematoda). The genera Cephalenchus (Goodey, 1962) Golden, 1971 and Allotylenchus gen. n. Acta zool. hung., 30: Geraert E. and Raski D.J. (1987). A reappraisal of the Tylenchina (Nemata) 3. The family Tylenchidae Örley, Revue de Nématologie 19: Golden A.M. (1971). Classification of the genera and higher categories of the order Tylenchida (Nematoda) In: Zuckerman B.M., Mai W.F. & Rohde R.A. (eds.) Plant parasitic nematodes Vol. 1, London & New York, Academic Press : Maggenti A.R., Luc M., Raski D.J., Fortuner R. and Geraert E. (1987). A reappraisal of the Tylenchina (Nemata) 2. The sub-order Tylenchina. Revue de Nématologie 10: Raski D.J., Koshy P.K. and Sosamma U.K. (1982). A revision of the sub-family Ecphyadophorinae Skarbilovich, 1959 (Tylenchidae: Nematoda). Revue de Nématologie 5: Raski D.J. and Maggenti A.R. (1983). Tylenchidae: Morphological Diversity in a Natural Evolutionary Group. In: Stone A.R., Platt H.M. & Khalil L.F. (eds) Concepts in Nematode Systematics, London & New York, Academic Press :

53 Siddiqi M.R. (1971). Structure of the oesophagus in the classification of the superfamily Tylenchoidea (Nematoda). Indian Journal of Nematology 1: Siddiqi M.R (1986). Tylenchida: Parasites of plants and insects. Wallingford, UK, 645pp. CAB International, Siddiqi M.R (2000). Tylenchida: Parasites of plants and insects. 2 nd edition, CAB International, Wallingford, UK. (in press) SUPERFAMILY DOLICHODOROIDEA Diagnosis: Tylenchina. No marked sexual dimorphism in anterior region. Cuticle prominently annulated, not showing distinct outer and inner layers. Lateral fields each with 2-6 incisures reducing towards extremities, except Belonolaimus which has one incisure. Phasmids small. Cephalic framework mostly with high arches and conspicuous extensions projecting posteriorly, with light to heavy sclerotization; annules generally distinct, basal annule not indented; labial disc prominent in some members of Tylenchorhynchinae and Belonolaiminae which have very long stylets. Stylet long (over 100µm) to short (about 10-12µm), with distinct basal knobs (knobs absent in Psilenchidae). Orifice of dorsal oesophageal gland near stylet base. Corpus with cylindrical precorpus (= procorpus) and a muscular round to oval postcorpus (= metacorpus) having refractive valve plates. Isthmus slender. Basal (terminal) oesophageal bulb present, usually containing the oesophageal glands. Occasionally, the dorsal gland enlarges and overlaps anterior end of intestine, while subventral glands remain small and anterior to the dorsal gland and may or may not overlap intestine. Oesophago-intestinal valve or cardia 3-celled, well-developed, but reduced in forms with overlapping glands. Female reproductive system didelphic, amphidelphic, secondarily becoming pseudo-monoprodelphic by the reduction of the posterior branch in Trophurinae. Vulva a transverse slit, rarely round or oval, median or postmedian, with or without epiptygma; lateral vulval membranes absent (except in subgenus Dolichorhynchus). Vagina at right angle to body axis, cuticularized in Dolichodorinae. Ovaries outstretched, oocytes in one row except in region of multiplication. Female tail rarely less than two anal body widths long, variously modified being conoid, cylindroid, subclavate or elongate-filiform, similar to that of the male in Psilenchidae. Male monorchic, with outstretched testis; sperm small to moderately large, rounded, with little cytoplasm. Bursa enveloping entire tail or, in Psilenchidae, adanal. Spicules symmetrical, cephalated, ventrally arcuate, with or without distal flanges (vela), tip broadly rounded or pointed, with terminal or subterminal pore. Gubernaculum simple or modified, fixed or protrusible. Juveniles essentially similar to female. Migratory ectoparasites of roots. 53

54 FAMILY DOLICHODORIDAE The Dolichodoridae comprises a large number of diverse species found throughout the world although perhaps most common in cooler, more temperate soils. All the species are ectoparasitic on the roots of plants and some species have been shown to be of economic importance on certain crops. In this scheme the lateral line character is maintained as a reliable and easily ascertained means of identifying groups (= genera) of similar nematodes. Personal preference, however, may dictate an alternative scheme involving several large genera, each embracing a large number of species, the number of lateral lines still being utilized, but as a means of division into smaller, more manageable groups of species. Characters used in the identification of Dolichodoridae (i) Body size and death posture: large sized (1.5mm or longer) forms include Dolichodorus, Belonolaimus, Macrotrophurus while medium sized (0.7 - about 1mm) and small sized (under 0.7mm) constitute almost all the rest of the dolichodorids. The body shape is always elongate-cylindrical tapering towards both ends, it is never obese. The death posture is usually straight to arcuate, but is characteristically spiral in Neodolichodorus and some Amplimerlinius. (ii) Cuticle: The cuticle is at least 2 layered, marked by transverse striae making coarse or fine annules (striae absent in Macrotrophurus). The lateral regions are thickened and marked by longitudinal incisures except in Belonolaimus which has a single lateral groove on each side running from head to tail tip. Lateral fields may or may not be areolated, i.e. traversed by the transverse striae, and the areolation may be complete or incomplete. Longitudinal striae may be present all along the body (Tessellus and Scutylenchus) or may be restricted to the anterior end. The character of the presence of either 6, 5 or 3 incisures in the lateral field has been used to distinguish Merliniinae, Quinisulcius, Uliginotylenchus and Divittus, respectively. Triversus and Dolichodorus were differentiated from Tylenchorhynchus and Neodolichodorus, respectively also by 3 versus 4 incisures. The presence of longitudinal cuticular ridges outside the lateral fields has been used to diagnose the genera Trilineellus, Mulkorhynchus, Neodolichorhynchus and Prodolichorhynchus. Localized thickening of the cuticle at the tail terminus has been used to characterize the genera Trophurus, Macrotrophurus, Paratrophurus, Amplimerlinius and Bitylenchus. (iii) Cephalic region: Shape (low, high, offset, continuous, smooth or striated) and degree of sclerotization of the cephalic frame-work are important characters. Several genera do not have a distinct labial disc, but Dolichodorus, Belonolaimus, Sauertylenchus have a rounded labial disc. 54

55 (iv) Stylet: A short stylet (under 35µm) with conus about as long as the shaft is a common feature, although some genera have a very long stylet. Basal knobs are always present, their shape having diagnostic value. A long stylet with the conus much longer than the shaft is found in Dolichodorus, Neodolichodorus, Macrotrophurus and Hexadorus. As a rule, a long stylet has the conus tubular until its tip and a short stylet has a conus that appears to be solid in its anterior third because the aperture is located a little behind the tip. (v) Oesophagus: Most dolichodorids have a tylenchoid oesophagus with a muscular median bulb, a slender isthmus and a glandular, non-overlapping basal bulb separated from the intestine by a cardia. Belonolaiminae and Telotylenchinae, however, have the glands lobe-like and extending over the anterior end of the intestine; the dorsal gland forming most of the lobe. (vi) Intestine: A simple tube with the lumen indistinct. It extends into the tail cavity in some groups (e.g. in Bitylenchus, Trophurus, Dolichodorinae), but never does so in Merliniinae. Serpentine canals or fasciculi may or may not be present. (vii) Tail: One of the most important characters is the size and shape of the female tail. The male tail is always conical and enveloped by a bursa. The tail is relatively long when compared to the anal body width and this is a simple and relatively reliable method for distinguishing members of this group from the hoplolaimids. (viii) Female reproductive system: The shape and position of vulva, presence/absence of epiptygma, sclerotization of vagina and whether the spermatheca is axial or offset and spherical or lobed when distended with sperm are all important characters. As a rule, dolichodorids are didelphic with two equally developed reproductive branches (i.e. amphididelphic), but in Trophurus the posterior branch is rudimentary (i.e. pseudomonodelphic). (ix) Male reproductive system: The testis is single and outstretched and produces small rounded sperm. Shape and size of spicules, gubernaculum and bursa are all important characters. The spicules have a broadly rounded tip with a large central depression in Merliniinae, whereas most other dolichodorids have spicules with a narrow tip and large distal flanges. The gubernaculum is trough-like and fixed in Merliniinae and rod-like and protrusible in Telotylenchinae and many other groups. Family DOLICHODORIDAE Chitwood in Chitwood & Chitwood, 1950 Diagnosis: Dolichodoroidea. Small to large sized (generally about mm), straight, usually arcuate, or more curved. Cuticle strongly annulated (except in Macrotrophurus). Lateral fields each with 1-6 incisures. Phasmids pore-like, caudal. Cephalic region annulated, round. Stylet well developed, knobbed; conus not much shorter than the shaft. Oesophageal glands generally either in a basal bulb abutting intestine, or the dorsal gland is enlarged, forming most of the lobe extending over intestine; subventral glands not enlarged. Didelphic, amphidelphic, rarely (in Trophurinae) pseudo-monoprodelphic. 55

56 Vulva median or submedian, transverse slit-like, rarely transversely oval (e.g. Nagelus); lips simple or modified. Vagina sometimes (Dolichodorinae, Belonolaiminae) cuticularized. Tails dissimilar between sexes. Key to subfamilies of Dolichodoridae 1. Female tail elongate-conoid with narrow, mucronate, spicate or finely drawn out tip except Neodolichodorus in which tail is obtusely rounded, under 2 anal body widths long and with terminal cuticle not abnormally thickened; bursa trilobed...2 Female tail not elongate-conoid or with narrow, mucronate, spicate or finely drawn out tip (except rarely in some Merlinius and Triversus spp.), if under 2 anal body widths long, then with terminal cuticle abnormally thickened; bursa not trilobed (except Dolichorhynchus in which only the terminus is trilobed) Stylet very long (over 50µm), conus much longer than the shaft; labial disc conspicuous...dolichodorinae Stylet not very long (under 40µm), conus about as long as the shaft; labial disc inconspicuous...meiodorinae 3. Cephalic region usually 4-lobed; lateral lip areas reduced; amphidial apertures longitudinal slit-like; oesophageal gland lobe-like, extending over intestine, oesophagointestinal junction less than one body width behind median bulb...belonolaiminae Cephalic region not usually 4-lobed; lateral lip areas not reduced, amphidial apertures pore-like or transversely oval; oesophageal glands in a bulb abutting intestine, if forming a lobe extending over intestine (e.g. Telotylenchinae) then oesophago-intestinal junction more than one body width behind median bulb Posterior ovary degenerate or absent...trophurinae Posterior ovary normal Lateral field with 6 incisures; male with hypoptygma (paired papillae on posterior anal lip)...merliniinae Lateral field with 3-5 incisures; male without hypoptygma Amphidial apertures conspicuous, postlabial; stylet over 80µm long, conus much longer than the shaft......macrotrophurinae Amphidial aperture inconspicuous, labial; stylet under 50µm, conus not much longer than the shaft

57 7. Dorsal oesophageal gland forming a long lobe over intestine, its nucleus located behind oesophago-intestinal junction... Telotylenchinae Dorsal oesophageal gland not forming a long lobe over intestine, its nucleus located in front of oesophago-intestinal junction... Tylenchorhynchinae Subfamily DOLICHODORINAE Chitwood in Chitwood & Chitwood, 1950 Diagnosis: Dolichodoridae. Large sized (1.5-3mm). Lateral field with 3 or 4 incisures, areolated. Cephalic region offset, distinctly annulated, framework strongly cuticularized; labial disc distinct; stylet very long (over 50µm) with conus markedly longer than shaft, and prominent knobs. Intestinal fasciculi and postrectal sac present. Vagina vera cuticularized. Ovaries paired. Female and juvenile tails long, filiform to spicate or, if obtuse to mammillate then less than 2 anal body widths long and with annulated terminus. Male tail short, conical, bursa trilobed with 2 large lateral flap-like lobes and a small terminal lobe; spicules with or without distal flanges; gubernaculum large, protrusible or fixed. Type genus: Dolichodorus Cobb, 1914 Other genus: Neodolichodorus Andrássy, 1976 Key to genera of Dolichodorinae 1. Lateral field with 3 incisures; female tail spicate or filiform... Dolichodorus Lateral field with 4 incisures; female tail obtuse or mammillate.....neodolichodorus Genus DOLICHODORUS Cobb, 1914 Diagnosis:. Dolichodorinae. Body straight to arcuate when relaxed. Lateral field with 3 incisures, completely areolated. Phasmids just postanal. Amphidial apertures in the form of longitudinal slits. Cephalic region prominently 4-lobed with a large offset labial disc. Cuticularization of basal plate of framework massive. Stylet exceptionally elongate (54-170µm), stylet guide (stoma) elongate-tubular. Precorpus swollen with convoluted lumen when stylet is retracted. Median and basal bulbs well developed, joined by a short slender isthmus. Excretory pore well anterior to hemizonid. Vulval lips not modified. Vaginal sclerotization in lateral view symmetrical. Female tail convex-conoid anteriorly then conoid-spicate to filiform. Postrectal intestinal sac only filling convex-conoid region of tail. Bursa large, trilobed. Spicules robust, with large flanges; gubernaculum also robust, protrusible. 57

58 Type species: Dolichodorus heterocephalus Cobb, 1914 REFERENCES AND ADDITIONAL READING Fortuner, R. and Luc, M. (1987). A reappraisal of Tylenchina (Nemata). 6. The family Belonolaimidae Whitehead, Revue de Nématologie, 10, Luc, M. and Fortuner, R. (1987). A reappraisal of Tylenchina (Nemata). 5. The family Dolichodoridae Chitwood, Revue de Nématologie, 10, Siddiqi, M. R. (1986). Tylenchida: Parasites of Plants and Insects. CAB International, Wallingford, UK, 645pp SUPERFAMILY HOPLOLAIMOIDEA Diagnosis: Tylenchina. Small to large nematodes (about 0.5-2mm). Sexual dimorphism in cephalic region present, and may also be present in stylet, oesophagus and body shape, although indistinct in some Pratylenchidae. Cuticle with distinct outer and inner layers, strongly annulated; longitudinal striae may be present but longitudinal ridges outside lateral fields absent. Lateral fields, each with 4 incisures reducing towards extremities, occasionally 3, 2, 1 or none. Cephalic framework well developed, strongly cuticularized and refractive, generally less developed in males. Labial disc present. Cephalic sensilla not on surface. Amphidial apertures pore- or slit-like, just below labial disc. Deirids absent (except Pratylenchoides). Phasmids small, with pore-like apertures, or large, scutellum-like, always lateral in position, in or near anal region, near tail terminus or much anterior to anus at different levels; absent in Aphasmatylenchinae. Stylet well developed, 2-5 times maximum width of lip region; protractors tubular around stylet; conus about as long as shaft, knobs prominent. Orifice of dorsal oesophageal gland close to or at some distance from stylet base. Oesophageal glands not enclosed in a basal bulb, but lobed, overlapping intestine (except Pararotylenchus and some Pratylenchoides spp. in which they form a pseudobulb). Subventral glands enlarged, equal to or usually larger than dorsal gland; nuclei of one or both subventral glands lying posterior to that of the dorsal gland. Postcorporal or median bulb always well developed (except in some males with degenerate oesophagus), muscular, with refractive valve plates. A cellular cardia absent, but oesophago-intestinal junction provided with a small cuticular valvula. Intestinal cell walls and lumen usually indistinct; rectum and anus distinct. Female reproductive system basically didelphic, amphidelphic; posterior branch may be reduced or represented by a uterine sac only. Vulva a transverse slit, lips usually not modified, in swollen females may be located subterminally or terminally; epiptygma present or absent; lateral membranes, if present, not conspicuous. Ovaries outstretched, reflexed or coiled in obese forms. Tails dissimilar between sexes (except some Pratylenchidae). Female tail generally short (less than two anal body widths) but may vary 58

59 to become elongate-conoid, absent in some swollen females. Bursa enveloping tail, subterminal, or absent; usually without phasmidial pseudoribs. Male tail usually short and with a distinct hyaline terminal portion. Spicules paired, similar or dissimilar, cephalated, straight to arcuate, with or without distal flanges, independently protrusible. Gubernaculum fixed or protrusible, with or without terminal titillae; telamon (= capitulum) present in several genera. Type family: Hoplolaimidae Filipjev, 1934 Other families: Heteroderidae Filipjev & Schuurmans Stekhoven, 1941 Meloidogynidae Skarbilovich, 1959 Pratylenchidae Thorne, 1949 Key to families of Hoplolaimoidea 1. Mature female round, pear- or lemon-shaped behind neck, with anus terminal or nearly so; male with stylet larger than that of female and tail non-bursate, very short or absent, develops by metamorphosis (except Meloidodera); third- and fourth-stage juveniles often swollen...2 Mature female not round, pear- or lemon-shaped, with anus not terminal; male with stylet equal to or smaller than that of female and tail bursate, not very short (except Verutus), does not develop by metamorphosis; third-;and fourth-stage juveniles normally not swollen Excretory pore in mature female opposite or anterior to median bulb; labial sensilla of female and juveniles on surface; male with large lip cap and large transverse slit-like amphidial apertures; gall-inciting...meloidogynidae Excretory pore in mature female behind median bulb; labial sensilla of females and juveniles on surface; male with small lip cap and with small oval to round amphidial apertures; notgall-inciting...heteroderidae 3. Juveniles and females with low arched cephalic framework, ecto-or endoparasites of roots...pratylenchidae Juveniles and females with high arched cephalic framework, Ecto- or endo- parasites of roots...hoplolaimidae 59

60 FAMILY HOPLOLAIMIDAE A family containing a wide range of genera usually distinguished initially by their short tails, strong stylets, distinct head sclerotization and high, conoid head region. It includes the group of nematode genera commonly referred to as "spiral nematodes" because of their coiled postures when relaxed. Some genera in the family are amongst the most commonly found and widely distributed nematodes in both temperature and tropical climates. Species of these genera include a number of economically important plant parasites. The main diagnostic characters of the family Hoplolaimidae are: i) female and male tails short, usually less than 2 anal body-widths long ii) head high, conoid; sclerotization distinct iii) strong stylet, usually greater than 20µm long, with well developed stylet knobs iv) oesophageal glands lobed, normally overlapping intestine (exception Pararotylenchus) v) male tail usually with enveloping bursa (exception Rotylenchulus) vi) phasmids usually distinct, pore-like or large (scutella) in differing positions (exception Aphasmatylenchus) vii) slight or marked morphological differences between sexes, some genera with swollen or saccate females viii) vulva usually median or post-median with paired, opposed reproductive tracts (exceptions Rotylenchoides, Acontylus) Family HOPLOLAIMIDAE Filipjev, 1934 (Weiser, 1953) Diagnosis: Hoplolaimoidea. Small to moderately large (usually mm); generally vermiform. Sexual dimorphism manifest in cephalic region. Lateral fields each with 4 incisures, rarely reduced or absent (e.g. Basirolaimus). Deirids absent. Phasmids either small, with pore-like apertures near, or a little anterior to, anus or large, scutellum-like near anus or much anterior to it anywhere on body behind oesophageal region; absent in Aphasmatylenchus. Cephalic region elevated, high arched, framework strongly cuticularized. Oesophageal glands overlapping intestine (except Antarctylus). Ovaries paired or rarely single. Female tail short, rounded (about one body width or less long), or conical (over 2 anal body widths, e.g. Antarctylus) with protoplasmic portion less than 2 body widths long and a distinct hyaline terminal portion. Male tail short (except Aphasmatylenchus). Bursa large, enveloping tail; flaps crenate, sometimes indented at tip, usually lacking phasmidial pseudoribs. Spicules robust or slender, straight to arcuate, with distal flanges which may be reduced. Gubernaculum large, protrusible or fixed; telamon (= capitulum) present or absent. Nominotypical subfamily: Hoplolaiminae Filipjev,

61 Other subfamilies: Aphasmatylenchinae Sher, 1965 Rotylenchinae Golilev, 1971 Rotylenchoidinae Whitehead, 1958 Key to subfamilies of Hoplolaimidae 1. Phasmids present...2 Phasmids absent...aphasmatylenchinae 2. Phasmids scutellum-like...hoplolaiminae Phasmids pore-like Cephalic region large, generally offset and with indented basal annule; orifice of dorsal oesophageal gland less than one-fourth stylet length behind stylet base...rotylenchinae Cephalic region small, generally continuous and with smooth basal annule; orifice of dorsal oesophageal gland more than one fourth stylet length behind stylet base...rotylenchoidinae Subfamily HOPLOLAIMINAE Filipjev, 1934 Diagnosis: Hoplolaimidae. Cephalic region usually offset, first and basal annules marked with longitudinal indentations. Lateral fields sometimes reduced or absent. Phasmids enlarged, scutellum-like near anus or aberrantly placed on body. Stylet knobs robust, compact tulip-shaped or offset, round to anteriorly flattened or concave. Dorsal oesophageal gland orifice about one-fourth or less of stylet length behind stylet base. Didelphic, amphidelphic; ovaries outstretched. Female tail short, rounded. Spicules robust, usually flanged distally. Gubernaculum also robust, usually protrusible. Type genus: Hoplolaimus Daday, 1905 Other genera: Aorolaimus Sher, 1963 Peltamigratus Sher, 1964 (= Aorolaimus for some authors) Scutellonema Andrássy, 1958 Basirolaimus Shamsi, 1979 has been regarded as a separate genus, but Siddiqi (2000) now regards it as a subgenus of Hoplolaimus. In the key below I key the group out separately, but it can be regarded as being Hoplolaimus for generic purposes if so desired. In either event, the crucial character of six oesophageal gland nuclei needs to be observed to enable species identification.. 61

62 Key to genera of Hoplolaiminae 1. Stylet knobs tulip-shaped, each with 1-3 anteriorly directed tooth like projections..2 Stylet knobs not tulip shaped, without tooth like projections Dorsal oesophageal gland uninucleate; lateral fields well developed, each with 4 incisures on most of body except in H. pararobustus; basal head annule divided into small squares...hoplolaimus Dorsal oesophageal gland quadrinucleate; lateral fields poorly developed or absent, each with 1-3 or no incisures at places; basal head annule not divided into small squares...basirolaimus 3. Scutella in or near anal region, close to or opposite each other...scutellonema Scutella much anterior to anus, separated from each other One scutellum prevulval, another postvulvar......aorolaimus Both scutella postvulvar...peltamigratus Subfamily HOPLOLAIMINAE Filipjev, 1934 Genus HOPLOLAIMUS Daday, 1905 syn. Basirolaimus Shamsi, 1979 Nemonchus Cobb, 1913 Hoplolaimoides Shakil, 1973 Diagnosis: Hoplolaiminae. Majority of species large, 1-2mm in length, body lying in a slightly curved position when relaxed, head terminus rounded. Lateral field with 1 to 4 incisures, may be areolated. Head sclerotization very distinct and with a massive cuticular framework. Lip region offset from body, with marked annulations and longitudinal striations; basal annule may be divided into small squares. Stylet knobs massive, tulip-shaped with 1-3 anterior tooth-like projections. Oesophageal glands overlapping intestine, mainly dorsally, with 3 or 6 nuclei (3 in Hoplolaimus; 6 in Basirolaimus). Vulva median with paired, opposed reproductive tracts. Epiptygma indistinct. Female and male tails short, rounded; less than 2 anal body-widths long. Large phasmids (scutella) present, not opposite one another: one anterior to vulva (between vulva and oesophagus) and the other posterior to vulva (between vulva and anus). Spicules massive, may be dimorphic with distal flange of variable size. Gubernaculum large, protrusible. Type species: Hoplolaimus tylenchiformis Daday,

63 Bionomics: Hoplolaimus feed as migratory ectoparasitic, semi-endoparasitic, or endoparasitic nematodes causing root destruction. Some are known to cause serious plant damage and yield loss, including H. colombus on soybean and cotton, H. indicus on rice, H. galeatus on tree seedlings, and H. seinhorsti on cowpea and cotton. Note: Some authorities accept the genus Basirolaimus which differs from Hoplolaimus mainly, and most consistently, in the presence of six nuclei in the oesophageal gland lobe. The six nuclei are not formed by a doubling of the normal three nuclei, but comprise four nuclei in the dorsal gland and one each in the subventral gland lobes. Siddiqi (2000) now regards the genus as a subgenus of Hoplolaimus. Genus SCUTELLONEMA Andrássy, 1958 Diagnosis: Hoplolaiminae. Similar to Aorolaimus and Peltamigratus. Most species less than 1mm in length, body lying in curved, spiral, or C shape position when relaxed, tail terminus mainly rounded. Lateral field with 4 incisures. Head sclerotization well developed; lip region usually offset from body, with annulations and with or without longitudinal striations. Stylet knobs well developed, rounded or cup-shaped. Oesophageal glands overlapping intestine mainly dorsally. Vulva median with paired, opposed reproductive tracts. Female and male tails short, less than 2 anal body-widths long. Differs from other genera in the group by having the enlarged phasmids or scutella located opposite or nearly opposite one another on the tail or near anus. Bursa enveloping tail; usually not indented or lobed. Type species: Scutellonema bradys (Steiner & LeHew, 1933) Andrássy, 1958 Other species: The genus consists of about 50 species, most found in tropical or subtropical countries. Bionomics: Scutellonema spp, are migratory ectoparasites or endoparasites, feeding on roots and tubers. S. bradys is a serious pest of yams (Dioscorea spp.) causing "dry rot disease" of tubers; S. cavenessi is reported to damage groundnuts, S. clathricaudatum can reduce yields of vegetables, and S. brachyurus is recorded as a pest of guar bean and is pathogenic to tobacco. Subfamily ROTYLENCHOIDINAE Whitehead, 1958 Key to genera of Rotylenchoidinae 1. Oesophageal glands extending over intestine 63

64 mostly ventrally and ventrolaterally...2 Oesophageal glands extending over intestine mostly dorsally and dorsolaterally...rotylenchus 2. Posterior ovary non-functional or absent...rotylenchoides Posterior ovary functional...helicotylenchus Genus ROTYLENCHOIDES Whitehead, 1958 Diagnosis: Rotylenchoidinae. Small nematodes around 0.5mm in length, body lying in open C shape position when relaxed. Lateral field with 4 incisures. Well developed head sclerotization. Lip region continuous with body, annulated. Stylet strong with well developed stylet knobs, rounded or cup-shaped. Orifice of dorsal gland less than half stylet length behind base of stylet. Oesophageal glands overlapping intestine mainly ventrally. Vulva posterior at 74-92%, with single functional anterior reproductive tract and short postvulval sac or undifferentiated genital branch. Female and male tails short, less than 2 anal body-widths long. Small, pore-like phasmids at level of anus. Tail terminus rounded to sharply pointed. Type species: Rotylenchoides brevis Whitehead, 1958 Other species: The genus consists of 7 species. Note: According to some authorities this genus should be regarded as a synonym of Helicotylenchus as there is some variation in the genital tract character. Genus ROTYLENCHUS Filipjev, 1936 syn. Calvatylus Jairajpuri & Siddiqi, 1977 Diagnosis: Rotylenchinae. Body length of most species between mm, body in spiral or C shape when relaxed. Lateral field with 4 incisures. Well developed head sclerotization; lip region offset or continuous with body, annulated. Strong stylet and well developed rounded to cup-shaped stylet knobs. Orifice of dorsal oesophageal gland opening located less than 25% of the stylet length behind the knobs. Oesophageal glands overlapping intestine mainly dorsally. Vulva median to post median with paired, opposed reproductive tracts. Female and male tails short, less than 2 anal body-widths long. Phasmids small and pore-like, opposite one another, on tail or near anus. Female tail terminus variable, rounded, curved dorsally or sometimes with small ventral projection. Spicules robust, distally flanged. Titillae and telamon may be present on gubernaculum. Bursa not lobed or indented terminally. Type species: Rotylenchus robustus (de Man, 1876) Filipjev,

65 Bionomics: Feed mainly as ectoparasites or migratory semi-endoparasites. Relatively little known of economic importance. R. robustus can cause damage to peas, carrots, lettuce and spruce seedlings; R. buxophilus can reduce growth of tree seedlings. Genus HELICOTYLENCHUS Steiner, 1945 Diagnosis: Rotylenchoidinae. Similar in general respects to Rotylenchus, although usually less robust. Species vary in size from mm, the body lying in a loose or tight spiral when relaxed. Lateral field with 4 incisures. Lip region not offset from body, annulated; no longitudinal indentations on annules. Stylet strong with well developed stylet knobs, rounded or cup-shapes. Dorsal oesophageal gland opening 25% or more of the stylet length behind stylet knobs. Oesophageal gland overlapping intestine mainly ventrally. Vulva median or postmedian with paired, opposed reproductive tracts. Female and male tails short, less than 2 anal body-widths long. Small pore-like phasmids on tail or near anus. Tail terminus variable, normally more curved dorsally, sometimes with ventral projection. Male tail short, conical with a distinct terminal hyaline portion. Bursa enveloping tail tip, rarely subterminal. Gubernaculum fixed, trough or rod shaped. Type species: Helicotylenchus dihystera (Cobb, 1893) Sher, 1961 Other species: The genus is very speciose and contains over 180 nominal species. These nematodes are amongst the most commonly found in both temperate and tropical soils. Bionomics: Helicotylenchus spp. feed as migratory endoparasites or semi-endoparasites, H. multicinctus is an economically important root parasite of bananas in many parts of the world; H. dihystera has a very wide host range and is reported to cause root necrosis of soybean roots and reduced growth of sugarcane, H. mucronatus has been found causing extensive root and tuber necrosis of bananas and sweet potato. Different species often occur in very large populations in the soil, sometimes associated with reduced growth and yields of crops. Subfamily ROTYLENCHULINAE Husain & Khan, 1967 Diagnosis: Hoplolaimidae. Small sized (usually 0.5mm or less long). No marked sexual dimorphism in body shape in young females and males; only mature female saccate or kidney-shaped. Cephalic region high, continuous, with or without distinct annules. Cephalic sclerotization, stylet and median oesophageal bulb well developed in juveniles and females, regressed in males. Male stylet weaker than that of female. Oesophageal glands in juveniles elongated, overlapping intestine mostly ventrally or laterally. Didelphic, ovaries reflexed or coiled in a mature female. Young female and male tails similar in being elongate-conoid and having a long hyaline terminal portion; tail persists in mature swollen female. Bursa present, low. Juvenile tails tapering to a round tip, 2-3 anal body widths long, hyaline terminal portion smaller than that in young female and in male. 65

66 Type genus: Rotylenchulus Linford & Oliveira, 1940 Other genus: Senegalonema Germani. Luc & Baldwin, 1984 Key to genera of Rotylenchulinae 1. Dorsal oesophageal gland orifice at less than half stylet length behind stylet base; young female with outstretched ovaries; bursa enclosing tail tip...senegalonema Dorsal oesophageal gland orifice at more than half stylet length behind stylet base; young female with ovaries having double flexures; bursa not enclosing tail tip...rotylenchulus Genus ROTYLENCHULUS Linford & Oliveira, 1940 Diagnosis: Rotylenchulinae. Marked sexual dimorphism. Juveniles, males and young females vermiform, arcuate to spiral upon relaxation. Mature female kidney-shaped, with an irregular, less swollen neck, a postmedian vulva and a short pointed tail. Cuticle annulated. Lateral fields each with 4 incisures, non-areolated, obliterated in mature female. Cephalic region high, continuous. Stylet in juveniles and female 2-3 times cephalic region width long. Orifice of dorsal oesophageal gland usually about one stylet length behind stylet base. Subventral glands in the normal position, dorsal gland shifted laterally to subventrally, former much longer than the latter. Immature female: Vermiform, migratory, (has been mistaken for adult stage and formed the basis for the proposal of new genera, Spyrotylenchus, Leiperotylenchus). Ovaries paired, with double flexures. Tail elongate-conoid, with prominent hyaline terminal portion. Male: Stylet and oesophagus regressed. Tail similar to that of young female; bursa subterminal, low, not quite projecting beyond tail contour in lateral view (hence mistakenly reported absent in R. stakmani (= R. reniformis)). Spicules slender, lacking distal flanges. Gubernaculum fixed. Cloacal lips pointed, not forming a tube; hypoptygma absent. Juveniles tail more rounded terminally and with shorter terminal portion than that of female. Type species: Rotylenchulus reniformis Linford & Oliveira,

67 The genus consists of about 10 spp. with R. reniformis being the most widespread and of most economic importance. Most species are confined to tropical or subtropical areas. Bionomics: The migratory juveniles, males and immature females are found in soil. The immature female is the parasitic, infective stage invading roots, penetrating the epidermis and cortex intracellularly and becoming embedded to approximately 1/3 of its length with its head at the endodermis of the stele. The nematode becomes a sedentary semiendoparasite and the body swells, normally to a kidney shape. Feeding on the endodermis produces cellular changes, but galls are not formed. Eggs are laid in a gelatinous matrix surrounding female body on surface of root. Rotylenchulus reniformis has a very wide host range with over 200 plant species being recorded as hosts. It is an economically important plant parasite causing damage and reduced yields to many crops including cotton, sweet potato, tomato, pigeon pea, coffee, mung beans. Alternative key to genera of whole group 1. Mature female swollen or saccate...14 Mature female vermiform Vulva median or postmedian with two reproductive tracts...3 Vulva posterior with single reproductive ract Phasmids enlarged and prominent on tail or along body...4 Phasmids small, on or near tail Both phasmids posterior to vulva...5 One phasmid anterior to vulva, one posterior to vulva Phasmids opposite one another, on or near tail...scutellonema Phasmids widely separated between vulva and anus...peltamigratus 6. Stylet knobs with prominent anterior projections; stylet 33-52µm, body normally lying in slight curve when relaxed...hoplolaimus Stylet knobs normally rounded without prominent projections; stylet 23-36µm, body normally in loose spiral or C shape when relaxed...aorolaimus 7. Oesophageal glands overlapping intestine...8 Oesophageal glands not overlapping intestine...pararotylenchus 8. Oesophageal glands overlapping intestine dorsally or laterally

68 Oesophageal glands overlapping intestine mainly on ventral side...helicotylenchus 9. Opening of dorsal oesophageal gland (d.o.g.) less than ½ stylet length behind stylet knobs...10 Opening of d.o.g. ½ or more stylet length behind stylet knobs Tail tapering to almost pointed terminus, greater than 2 anal body widths in length...antarctylus Tail less than 2 anal body widths in length Oesophageal glands extending over intestine as a short overlap on dorsal side...rotylenchus Oesophageal glands in long lateral overlap of intestine filling body cavity (immature female)...senegalonema 12. Tail tapering to rounded terminus, normally greater than 2 anal body widths in length; slender stylet usually less than 20µm (immature female)...rotylenchulus Tail not tapering, less than 2 anal body widths in length; stylet greater than 20µm...Orientylus 13. Short and variable oesophageal overlap of intestine usually ventral, mature female vermiform...rotylenchoides Long dorsal oesophageal overlap of intestine (immature female)...acontylus 14. Posterior vulva, single reproductive tract, female swollen but not saccate...acontylus Postmedian vulva, two opposed reproductive tracts, Females saccate Dorsal oesophageal gland opening normally greater than one stylet length behind stylet knobs (vermiform immature vulvate females present)...rotylenchulus Dorsal oesophageal gland opening less than one stylet length behind stylet knobs...senegalonema REFERENCES AND ADDITIONAL READING Fortuner, R. (1987). A reappraisal of Tylenchina (Nemata). 8. The family Hoplolaimidae Filip ev, Revue de Nématologie, 10,

69 Siddiqi, M. R. (1986). Tylenchida: Parasites of Plants and Insects. CAB International, Wallingford, UK, 645pp. Siddiqi, M. R. (2000). Tylenchida: Parasites of Plants and Insects. 2 nd edition, CAB International, Wallingford, UK. FAMILY PRATYLENCHIDAE The Pratylenchidae includes some of the most important and damaging nematodes of crops in the tropical and subtropical regions. The family contains a number of genera, some of which are morphologically very similar, thereby making the taxonomy of the group rather complex. Most genera are migratory endoparasites of roots and tubers and thus remain vermiform. However, there are two genera, Achlysiella and Nacobbus, where the female becomes obese and, as a consequence, is restricted to one site within the root tissue. These specialized genera are dealt with separately in the schedule on obese nematodes. The Pratylenchidae is a family under the superfamily Hoplolaimoidea and is characterized by: i) the low, flattened or rounded head ii) the strong sclerotization of the head skeleton iii) the relatively short, stout stylet which is almost usually about 2.5 head widths long iv) the subventral oesophageal glands usually extending in tandem beyond the dorsal gland v) the relatively long, conoid or subconoid tail (c' > 2). Of these genera, three will be considered in more detail, viz. Pratylenchus, Hirschmanniella, and Radopholus. Nacobbus, having swollen females, is, for pragmatic purposes, treated separately in the section 'Obese Nematodes'. Family PRATYLENCHIDAE Thorne, 1949 (Siddiqi, 1963) Diagnosis: Hoplolaimoidea. Vermiform nematodes. Cuticle prominently annulated. Lateral fields each with 4-6 incisures, very rarely areolated behind oesophagus. Deirids absent (except Pratylenchoides). Phasmids pore-like, on tail well behind anus, extending into bursa as pseudoribs except in Hirschmanniella. Female: Amphids pore-like, indistinct, near oral opening which is surrounded by 6 labial pits. Cephalic region low, anteriorly flattened to broadly rounded, annulated; framework strongly cuticularized; labial disc inconspicuous, dumb-bell shaped (with SEM) in type-genus. Stylet strong, length 69

70 not exceeding 3 cephalic region widths (except rarely in Hirschmanniella); conus about as long as posterior part; knobs large, rounded. Orifice of dorsal gland close (usually 2-3µm) to stylet base. Precorpus slender. Postcorpus strongly muscular, with prominent valve plates. Isthmus short. Oesophago-intestinal junction indistinct, with refringent valvula. Oesophageal glands extending over intestine; subventral glands asymmetrical, extending past the dorsal gland, three gland nuclei usually lying in tandem. Vulva a transverse slit, submedian to posterior but not subterminal; lips not modified; lateral membranes absent. Vagina directed inward. Didelphic, amphidelphic or pseudomonoprodelphic. Ovaries outstretched; posterior ovary degenerate in pseudo-monodelphic forms. Spermatheca large, usually rounded, with small round sperm when impregnated. Tail conoid, subcylindrical to elongate-conoid, about twice or more anal body width long, with round to pointed tip which may bear a mucro (Hirschmanniella), generally with inconspicuous hyaline terminal portion. Male: Stylet and oesophagus similar to those in female or reduced as in Radopholinae. Tail elongate-conoid, bursa terminal or subterminal. Testis single, outstretched, spermatocytes in one or two rows. Spicules similar, cephalated, arcuate, pointed with subterminal opening on dorsal or ventral side. Gubernaculum simple, fixed, or complex with telamon or titillae; protrusible. Hypoptygma present or absent. Juveniles resemble females in having similar anterior region and tail. Obligate migratory endoparasites of roots. Nominotypical subfamily: Pratylenchinae Thorne, 1949 Other subfamilies: Hirschmanniellinae Fotedar & Handoo, 1978 Radopholinae Allen & Sher, 1967 Key to subfamilies of Pratylenchidae 1. Oesophageal glands overlapping intestine mostly ventrally; without sexual dimorphism in the anterior region...2 Oesophageal glands overlapping intestine mostly dorsally; with sexual dimorphism in the anterior region...radopholinae 2. Tails similar between sexes; phasmids near terminus...hirschmanniellinae Tails dissimilar between sexes; phasmids not near terminus...pratylenchinae 70

71 Simplified key to major genera of Pratylenchidae 1. Oesophageal glands overlapping the intestine dorsally; male with knob-like head and degenerate oesophagus...radopholus Oesophageal glands overlapping the intestine ventrally, or mostly so; male with low rounded or flattened head and normal oesophagus Female with median vulva and two genital tracts (amphididelphic)...hirschmanniella Female with posterior vulva (V = 70%) and a single genital tract (monoprodelphic)...pratylenchus Subfamily PRATYLENCHINAE Thorne, 1949 Diagnosis: Pratylenchidae. No marked sexual dimorphism in anterior region. Small sized (under 1mm long). Lateral field with 4-6 incisures. Cephalic region low, usually flattened, with 2-3 annules. Stylet generally less than 20µm long. Oesophageal glands lobe-like, about 3 body widths or less long, mostly on ventral side of intestine. Ovaries paired or single. Female tail 3 anal body widths or less long, subcylindrical to conoid, lacking a mucro. Male tail conoid, arcuate, completely enveloped by a bursa. Spicules with subterminal pore on dorsal or ventral side. Gubernaculum fixed. Hypoptygma generally present. Endoparasites of roots causing typical lesions, rarely attacking aquatic or marsh plants. Type-genus: Pratylenchus Filipjev, 1936 Other genus: Zygotylenchus Siddiqi, 1963 Genus PRATYLENCHUS Filipjev, 1936 Diagnosis: Pratylenchidae. Small nematodes (less than 1mm long) dying slightly curved ventrally on application of gentle heat. No marked sexual dimorphism in form of anterior region. Head region low, flattened, usually appearing as a flat, black cap under the stereomicroscope. Lip region divided into 2, 3 or 4 annules and continuous with the body contour; strongly cuticularized. Stylet 20µm or less in length (i.e. about twice, or slightly more, of the head width), moderately cuticularized and with rounded or anteriorly concave knobs. Oesophagus equally developed in both sexes, median bulb well developed; oesophageal gland lobes overlapping the intestine ventrally. Female: vulva well posterior, usually at 70-80% of body length; genital system with a single anteriorly directed tract and a variable post-vulval section which may show some differentiation, but is never functional (mono-prodelphic); spermatheca oval or round and usually filled with sperm in bisexual species; tail sub-cylindrical or more or less conoid with a broad to 71

72 narrowly rounded or truncate terminus which may be smooth or annulated. Male: tail short, dorsally convex-conoid; bursa extending to tail tip; spicules slender, arcuate. Type species: Pratylenchus pratensis (de Man, 1880) Filipjev, 1936 Other species: Almost 100 species have been described. They are often distinguished by rather subtle characters, thus making this a difficult genus to approach for the non-specialist. Bionomics: Migratory endoparasites with all stages found in the root cortex. Low soil populations can be associated with high root populations. The nematodes feed mainly on cortex cells and form cavities containing 'nests' or colonies of nematodes of all stages. Discolouration of affected tissues is usually pronounced. Above ground symptoms of attack include chlorosis and stunting. Some species reproduce sexually while others are parthenogenetic. The life-cycle can be completed in three to four weeks and the nematodes can survive in the absence of host plants for several months. Most of the important species are polyphagous, although P. goodeyi may be restricted to banana. The major species are P. brachyurus, P. coffeae, P. goodeyi, P. penetrans and P. zeae Distribution: P. brachyurus, P. coffeae and P. zeae are widely distributed in tropical areas; P. penetrans mainly in cooler regions of the tropics; P. goodeyi on banana in Crete and the Canary Islands and in the cooler, montane areas of Cameroon, Ethiopia, Kenya, Tanzania, Uganda and Burundi. Subfamily RADOPHOLINAE Allen & Sher, 1967 Key to genera 1. Posterior ovary normal...2 Posterior ovary degenerate or absent Deirids absent; all gland nuclei behind oesophago-intestinal junction...radopholus Deirids present; at least one gland nucleus opposite or anterior oesophagointestinal junction...pratylenchoides 3. Female cephalic region conoid-rounded; stylet robust, 20µm or longer, generally with tulip-shaped knobs; male with asymmetrical cephalic region and a subterminal bursa...hoplotylus Female cephalic region broadly rounded; stylet not robust, less than 20µm long with knobs not tulip-shaped; male with symmetrical 72

73 cephalic region and a terminal bursa Subventral oesophageal glands partially extending on ventral side of intestine; male oesophagus not degenerate...apratylenchoides Subventral oesophageal glands not extending on ventral side of intestine; male oesophagus degenerate...radopholoides Genus RADOPHOLUS Thorne, 1949 Diagnosis: Pratylenchidae. Small nematodes (less than 1mm long) dying more or less straight or slightly curved ventrally on application of gentle heat. Marked sexual dimorphism in form of anterior region: female head region low, rounded, continuous or slightly offset. Male cephalic sclerotization, stylet and oesophagus reduced; female cephalic sclerotization strong, stylet and oesophagus well developed. Median bulb in female oesophagus well developed and oesophageal glands overlapping the intestine mostly dorsally. Female: vulva median, with two functional and equally developed genital tracts (amphididelphic); spermathecae rounded and with sperm in bisexual species; tail elongate, conoid (about 60µm long in R. similis). Male: tail elongate, conoid, ventrally arcuate; bursa not reaching to tail tip in R. similis and most other species; spicules slender, arcuate. Type species: Radopholus similis (Cobb, 1893) Thorne, 1949 Other species: Over 20 species have been placed in this genus, but some belong to other genera such as Achlysiella or Zygradus. Achlysiella is discussed in more detail in the section Obese Nematodes. Bionomics: Migratory endoparasites of root and corm/tuber tissues. In roots, the feeding activities are restricted to the cortex causing cavitation, discolouration and severe damage, thus allowing secondary invasion by other micro-organisms. The adult male is nonfeeding. The major species is R. similis which currently has two recognised host races or biotypes. R. similis attacks banana and many other plants. R. similis citrophilus (recognised as a separate species by some authorities on differing chromosome count and minor morphological details) was proposed for a citrus race, but is no longer recognised at specific or subspecific level. The importance of another citrus attacking species, R. citri, has yet to be demonstrated under field conditions. 73

74 Distribution: The majority of species have been described from the Australasian region. However, R. similis is now found world-wide in tropical regions and occurs virtually everywhere that banana is grown. The citrus race, formerly known as R. similis citrophilus, is recorded from Florida. Radopholus citri was described attacking citrus in Java and may represent a threat to other citrus growing areas if introduced. REFERENCES Café Filho, A. C. and Huang, C. S. (1989). Description of Pratylenchus pseudofallax n. sp. with a key to species of the genus Pratylenchus Filipjev, 1936 (Nematoda: Pratylenchidae). Revue de Nématologie, 12, Luc, M. (1987). A reappraisal of Tylenchina (Nemata). 7. The family Pratylenchidae Thorne, Revue de Nématologie, 10, Siddiqi, M. R. (1986). Tylenchida: Parasites of Plants and Insects. CAB International, Wallingford, UK, 645pp. Siddiqi, M. R. (2000). Tylenchida: Parasites of Plants and Insects. 2 nd edition, CAB International, Wallingford, UK. (in press). FAMILY MELOIDOGYNIDAE The Meloidogynidae (colloquially known as 'root knot' nematodes) comprises a diverse and widespread group of nematodes amongst which are a number of economic importance. Typically they are sedentary, semi-endoparasites, the females swelling enormously and laying large numbers of eggs into a protective gelatinous matrix, The J2 is the infective stage and after invading a root becomes sessile and initiates the development of specialized trophic cells to nurture the subsequent stages. The nematode also releases chemicals which initiate gall formation. Vermiform males are produced in many species, often when food supply is a restrictive factor. Many species are extremely polyphagous whereas others are more host specific. The greatest number of species occur in the tropics and sub-tropics, although a few species have a temperate distribution and a single generation a year. Diagnosis: Meloidogynidae. Sexually dimorphic. Mature female sedentary, swollen, globular or pear-shaped, cuticle thin, white, annulated. Non cyst forming. Tail absent, anus and vulva terminal surrounded by characteristic pattern of striae on the cuticle (perineal pattern). Excretory pore anterior to median bulb and near to stylet knobs, head skeleton hexaradiate. Stylet <25 m long, with well developed basal knobs; procorpus cylindrical followed by spherical metacorpus with well developed musculature and 74

75 cuticular valve plates; procorpus and metacorpus not amalgamated. Oesophageal glands overlapping intestine ventrally. Genital tracts paired, elongated, anteriorly directed and much convoluted. Eggs laid in an external gelatinous matrix. The eggs are usually unembryonated and not retained in the female body. The first moult occurs within the egg, the second-stage juveniles hatching and being the infective stage. Female: Sedentary, swollen, globular or pear-shaped, cuticle thin, white, annulated, tail absent, anus and vulva terminal surrounded by characteristic pattern of striae on the cuticle (perineal pattern), excretory pore anterior to median bulb near to stylet knobs, head skeleton hexaradiate. Stylet <25µm long, well developed basal knobs: procorpus cylindrical followed by spherical metacorpus with well developed musculature and cuticular valve plates, procorpus and metacorpus not amalgamated. Oesophageal glands overlap intestine ventrally. Genital tracts paired, elongated and convoluted. Eggs laid in gelatinous matrix, usually unembryonated and not retained in the female body. First moult within the egg, second-stage juveniles hatch and are infective. Male vermiform, migratory, stylet well developed, <33 m; cephalic framework well developed, hexaradiate; short bluntly rounded tail; no bursa, usually one, but sometimes two testes; paired slender spicules, simple gubernaculum. Intersexes or sex reversal may occur, particularly in response to nutrient stress. Second-stage juveniles vermiform, infective, migratory; head skeleton hexaradiate, stylet slender, knobbed, stylet <23 m long. Hyaline region of tail less than half the tail length. Heat relaxed form straight to arcuate. The 3rd and 4th stage juveniles occur within the root and are swollen, sedentary, with a blunt terminus and no stylet. They develop within the cuticle of 2nd stage juveniles, the tail spike of which is retained. Type species: Meloidogyne exigua Goeldi, 1892 syn. Heterodera exigua (Goeldi, 1892) Marcinowski, 1909 Other species: The genus contains a large and ever increasing number (>70) of species with several new species being proposed each year. Many of the species are becoming difficult to differentiate using classical taxonomic techniques and biochemical or molecular techniques are being increasingly employed to try to circumvent the problem. Many of the taxonomic problems are undoubtedly due to the fact that most species reproduce by mitotic parthenogenesis. Note: The genus Hypsoperine is sometimes recognised as being distinct from Meloidogyne (e.g. Siddiqi, 1986). It differs by having the anus and vulva located on a cone-like elevation or protuberance on the mature female and in having the excretory pore of the J2 anterior to the hemizonid as opposed to posterior. In addition, the females often lie parallel to the root axis in contrast to Meloidogyne. Bionomics: Obligatory, sedentary endoparasites of plant roots inciting gall formation and a complex trophic system of giant cells in the root cortex. Many species exhibit extreme polyphagy, thus making control by crop rotation difficult. 75

76 MORPHOLOGICAL CHARACTERS RECOMMENDED FOR USE IN SPECIES IDENTIFICATION Most important characters J2: tail length hyaline tail length tail shape Mature female: stylet length stylet shape excretory pore position as ratio of stylet length perineal pattern Male: stylet length stylet cone length stylet shape head shape (light microscope) distance of dorsal oesophageal gland orifice (DGO) from stylet base Supplementary characters J2: head shape stylet shape presence of inflated rectum or not Female: Male: body shape head shape tail shape spicule shape REFERENCES AND ADDITIONAL READING CIH Descriptions of Plant Parasitic Nematodes Sets 1-8. CAB International, Wallingford, UK. Jepson, S. B. (1987). Identification of root-knot nematodes (Meloidogyne species). CABI Wallingford, UK. 265 pp. 76

77 Luc M., Maggenti A.R. and Fortuner R. (1988). A reappraisal of the Tylenchina (Nemata) : 9. The family Heteroderidae Filipjev & Schuurmans Stekhoven, 1941 Revue de Nématologie 11, Sasser, J. N. and Carter, C. C. (1986). An advanced treatise on Meloidogyne. Vol. I, 422pp.; Vol. II, 223pp. Siddiqi, M. R. (1986). Tylenchida: Parasites of Plants and Insects. CAB International, Wallingford, UK, 645pp. Taylor, A. L. (1987). Identification and estimation of root-knot nematode species in mixed populations. Florida Department of Agriculture and Consumer Services SUPERFAMILY CRICONEMATOIDEA Many of the criconematids are commonly referred to as ring nematodes because of the pronounced annulation that is so typical of many species. They form one of the most distinct groups of plant parasitic nematodes, but are not always well represented in extractions because of their sluggish nature - sieving techniques usually recover far more than tray methods. Most species are ectoparasitic on plant roots, those with longer stylets being able to feed deep within the root cortex. In some genera the mature female has a tendency to become obese. Those nematodes with truly obese females (e.g. Tylenchulus) are dealt with under the section Obese nematodes to facilitate comparison with other nematodes sharing a similar trophic habit. Although not systematically accurate, this approach does have the advantage of being pragmatic. The following systematic scheme contains the major generic names commonly found in the literature. However, there is a long history of flux in the systematics of the Criconematinae and Macroposthoniinae, a flux that continues in the present day, some workers splitting the subfamilies into numerous small genera, others preferring a broader approach and retaining only a few, somewhat loosely defined genera. In this introductory account the latter course is adopted for simplicity, although some of the other generic names are mentioned in passing. The superfamily may be conveniently divided into three families that are easily differentiated from on another on morphology: the Criconematidae, or ring nematodes; the Hemicycliophoridae, or sheath nematodes and the Paratylenchidae, or pin nematodes. The taxonomy of this group may involve features of the juvenile stages (e.g. whether the cuticle is spined or not), and this can cause problems when material is limited or where there is a mixed population. Males are degenerate and usually short-lived. They are usually rather uncommon in extractions, although they can be abundant under certain circumstances. As a consequence, it is the female morphology that is most utilized for diagnostic purposes, the presence of males usually being indicated by the presence of sperm in the female spermatheca. 77

78 Key to subfamilies of Criconematidae and Hemicycliophoridae 1. Stylet knobs anchor-shaped (anteriorly concave)...criconematidae (2) Stylet knobs sloping backwards...hemicycliophoridae (4) 2. Double cuticle present, annules normal......hemicriconemoidinae Double cuticle absent, very heavily annulated nematodes, annules retrorse Juveniles with cuticular scales or spines, adults usually with scales or spines......criconematinae Adults lacking cuticular scales or spines and juveniles usually lacking such structures......macroposthoniinae 4. Females with a double cuticle, the outer being well-developed and not membranous. Head annules not separated......hemicycliophorinae Females without a double cuticle or, if present, the outer sheath membranous, much thinner than the body cuticle and closely adpressed. Head annules well separated Caloosiinae FAMILY CRICONEMATIDAE Characters of Criconematinae and Macroposthoniinae The nematodes belonging to these subfamilies are characterized by the following: i) Small stout bodies with cuticle marked by deep striae forming conspicuous annules which are either plain or bear scales or spine-like retrorse projections which may or may not be arranged in longitudinal rows. ii) The spear or stylet is usually greatly elongated, µm long, with very long conus and large, anteriorly directed basal knobs. iii) The precorpus or procorpus and the median bulb of the oesophagus are not demarcated but amalgamated. iv) The isthmus is very short and the basal bulb is small and spathulate. v) The female gonad is monoprodelphic without a posterior uterine sac. vi) Males degenerate, stylet absent, lateral fields present, bursa greatly reduced or absent. vii) Phasmids absent. 78

79 Some characters of taxonomic importance The morphology of this group is markedly at variance with other plant parasites and this in turn means that the characters used in the delineation and identification of this group differ somewhat. The major characters are listed below: 1. The length and width of the body. 2. The number of body annules (R). 3. The number of annules between the labial disc and base of stylet knobs (Rst). 4. The number of annules between the labial disc and the excretory pore (Rex). 5. The number of annules from tail terminus to vulva (RV). 6. The number of annules from tail terminus to anus (Ran). 7. The number of annules between vulva and anus (Rvan). 8. The shape, size, number and arrangement of cuticular outgrowths (scales, spines, etc.) on the annules. 9. Anastomoses of annules may or may not be present. 10. Submedian lobes/lips. Submedian lobes when present, are visible in lateral as well as dorso-ventral view, but can be observed best in an en face view. There are 4 submedian lobes, their size being variable in different species. These lobes may be connected dorsally and ventrally. In some species 6 lips (4 submedian lobes and 2 lateral) are present instead of submedian lobes. Often the lateral lips are reduced or absent. 11. Labial disc. Submedian lobes and labial plates surround a labial disc with an I- shaped oral opening. This disc may be elevated. 12. Amphids. Amphidial openings are oval or slit-like, observed better in an en face view. 13. Spear or stylet consists of a very long conical part (conus), a short cylindrical part and the basal knobs. The basal knobs are always projecting forward (anchorshaped). 14. Genital tract. The female genital tract is mono-prodelphic and lacks a posterior uterine sac. It consists of vulva, vagina, uterus, oviduct and the ovary. The single ovary consists of the usual germinal and growth zones. Its length depends on the age of the female and in older females it may extend into the oesophageal region. The oviduct is a short tubular region that enters the uterus between spermatheca (if present) and quadricolumella. The vagina is oblique to the body axis and directed anteriorly. 15. Vulva when viewed ventrally may be open, closed or with an overlapping anterior lip. 16. Tail is greatly variable, may be conical pointed or conical rounded or short rounded. If annules are ornamented their size usually increases towards the tail end. 17. Males are rare. The following features are of importance: shape of body upon killing, body length and width, shape of head, tail, position of excretory pore, length and shape of spicules, gubernaculum, number of lines in the lateral fields and the bursa. 79

80 18. Juveniles have smooth, crenate or spined posterior edges to the annules. Sometimes early stages show crenate or spined annules but the older stages do not. The number of annules on the juveniles may be more than those of the adults. Male juveniles do not possess a stylet. Subfamily CRICONEMATINAE Diagnosis: Criconematidae. Cuticle ornamented in juveniles, and usually the adults, with scales or spine-like outgrowths. These are usually arranged in longitudinal rows. Many genera have been described in this group, but the major ones are: Criconema Hofmänner & Menzel, 1914 Bakernema Wu, 1964 Crossonema Mehta & Raski, 1971 Ogma Southern, 1914 Key to genera - adult females 1. Spines or scales arranged in longitudinal rows...ogma Spines or scales absent or not arranged in longitudinal rows Spines or scales absent...criconema Spines or scales present Cuticle outgrowth membranous, hardly discernible Head not separate and bearing similar appendages to other annules...bakernema Cuticle outgrowths stronger, well-defined Head separate, smooth or fringed...crossonema Subfamily MACROPOSTHONIINAE Diagnosis: Criconematidae. Cuticle smooth in juveniles and females, lacking spines or scale-like outgrowths, but may occasionally be finely crenate. The most commonly cited genera are: Criconemoides Taylor, 1936 Discocriconemella De Grisse & Loof, 1965 There has been considerable doubt about the type species of Macroposthonia and Criconemoides and both genera have been synonymized under Criconemella by some authors whilst others disagreed. It should be noted that there was also an attempt to replace the use of both Macroposthonia and Criconemella by the name Mesocriconema. A recent ruling from the International Commission on Zoological Nomenclature re-established Criconemoides as a valid genus and this name should now be used instead of Macroposthonia, Criconemella or Mesocriconema. 80

81 Key to genera - adult females 1. Head annule disc or saucer-shaped...discocriconemella Head annule normal, not disc or saucer-shaped...criconemoides Genus CRICONEMOIDES Taylor, 1936 (after Ebsary, 1982) Diagnosis: Macroposthoniinae. Female body mm. Annules , smooth to crenate, anastomoses present or absent. Head annules normal, not discoid. Submedian lobes present or absent, if present may be fused to varying degrees or separate. Labial plates present or absent. Vulva open or closed, anterior vulval lip ornamented or not. Vagina straight or sigmoid. Stylet well developed, µm. Oesophagus about 30% of body length. Tail shape variable. Juveniles: annules smooth to crenate. Males: head end rounded to conoid, lateral incisures 2 to 4, usually 4, bursa distinct. Type species: Criconemoides morgensis (Hofmänner in Hofmänner & Menzel, 1914) Taylor, Other species: Many described and mostly difficult to identify. Most species have been placed in Macroposthonia, Criconemella or Mesocriconema at one time or another although a recent ruling by the International Commission on Zoological Nomenclature re-instated Criconemoides as a valid genus, thereby relegating the other generic names to junior synonymy. Subfamily HEMICRICONEMOIDINAE Diagnosis: Criconematidae. Female and juveniles sausage-shaped with rounded, coarse annules. Cuticle double, but outer sheath usually fairly closely adpressed. Stylet strong with anchor-shaped basal knobs. There is only one genus. Genus HEMICRICONEMOIDES Chitwood & Birchfield, 1957 Diagnosis: With the characters of the subfamily. Type species: Hemicriconemoides wessoni Chitwood & Birchfield,

82 FAMILY HEMICYCLIOPHORIDAE Diagnosis: Criconematoidea. Females and juveniles with a double cuticle, annules not retrorse and lacking scales or spine-like structures. Stylet strong with rounded basal knobs. Males lacking a functional stylet and oesophagus. Bursa present. There are four nominal genera: Hemicycliophora de Man, 1921 Aulosphora Siddiqi, 1980 Colbranium Andrássy, 1979 Loofia Siddiqi, 1980 Some authors do not accept Aulosphora or Loofia, both being regarded as junior synonyms of Hemicycliophora. Key to genera - adults 1. Vulva lips round, low, not modified. Body not recessed behind vulva; spicules arcuate...2 Vulva lips modified, pointed, elongate. Body recessed behind vulva; spicules semicircular or hooked-shaped Female head offset by deep groove. Vulva and anus sub-terminal; bursa almost reaching tail tip.colbranium Female head not offset. Vulva and anus not subterminal; bursa short, not almost reaching tail tip......loofia 3. Vulval lips less than 3 body annules long, often divergent. Spicules semi-circular in shape; pre- and post-cloacal regions of bursa in ratio 1:1...Hemicycliophora Vulval lips longer than 3 body annules, almost parallel. Spicules U- or hookshaped; pre- and post-cloacal regions of bursa in ratio 3-4:1...Aulosphora Subfamily HEMICYCLIOPHORINAE Genus HEMICYCLIOPHORA de Man, 1921 (after Siddiqi, 1980) Diagnosis: Hemicycliophoridae. Body just behind vulva deeply recessed. Vulva lips modified, elongate but less than 3 annules long, usually divergent. Female tail elongate, tapering, filiform, cylindrical or, rarely, hemispherical. Spicules semi-circular. Penial tube well developed but less than a body width long, directed outward and forward. Body 82

83 just in front of penial tube recessed. Pre- and post-cloacal regions of bursa almost equal. Type species: Hemicycliophora typica de Man, 1921 FAMILY PARATYLENCHIDAE The Paratylenchidae or pin nematodes comprises a small group of criconematid nematodes characterized by: i) a criconematid-type oesophagus and monoprodelphic genital system ii) a well developed stylet, which may be very long and slender iii) fine annulation iv) male stylet and oesophagus non-functional and degenerate Identification to species level is a difficult process as the nematodes are so small and many of the characters are subjective. Note Gracilacus is sometimes regarded either as a junior synonym or subgenus of Paratylenchus, the only consistent differences between the two genera being stylet length (perhaps an arbitrary character) and a tendency in Gracilacus females to become obese (although this state is not yet confirmed for all species). Key to subfamilies 1. Isthmus short, bursa absent or very weakly developed...paratylenchinae Isthmus elongate, bursa present, well developed...tylenchocriconematinae Subfamily PARATYLENCHINAE Diagnosis: Paratylenchidae. Female body slender or saccate. Stylet well developed and may be extremely long and attenuate. Female genital tract monoprodelphic, outstretched. Male bursa absent or very weakly developed. Key to genera 1. Mature female obese with cuticular ornamentation in the form of minute refractive tubercles. Tail very short and blunt...cacopaurus Mature female usually slender, but if obese then without cuticular ornamentation. Tail not very short and blunt Female stylet shorter than 38µm...Paratylenchus 83

84 Female stylet 40µm long or longer...gracilacus Genus PARATYLENCHUS Micoletzky, 1922 Diagnosis: Paratylenchinae. Small nematodes, less than 0.5mm long and usually dying in a fairly closed 'C' shape. Stylet well developed, but shorter than 38µm long. Vulva well posterior, genital tract monoprodelphic, outstretched. Tail long, conoid, with a pointed or finely rounded tip. Male oesophagus and stylet degenerate, bursa absent. Type species: Paratylenchus bukowinensis Micoletzky, 1922 Other species: Many species have been described, but they are very difficult to identify because of their minute size and the difficulty of assessing some of the character states even the number of lateral lines can be difficult to ascertain with certainty. Bionomics: Often occur in very large numbers and seem to be particularly associated with woody plants. REFERENCES AND ADDITIONAL READING Andrássy, I. (1979). Revision of the subfamily Criconematinae Taylor, 1936 (Nematoda). Opusc. zool. Budapest., 16, Ebsary, B. A. (1982). Bakernema yukonense n. sp. (Nematoda: Criconematidae) with keys to the species of Criconemella and Discocriconemella. Canadian Journal of Zoology, 60, Raski, D. J. and Luc, M. (1987). A reappraisal of Tylenchina (Nemata). 10. The superfamily Criconematoidea Taylor, Revue de Nématologie, 10, Siddiqi, M. R. (1986). Tylenchida: Parasites of Plants and Insects. CAB International, Wallingford, UK, 645pp. Siddiqi, M. R. (2000). Tylenchida: Parasites of Plants and Insects. 2 nd edition, CAB International, Wallingford, UK. (in press) SUPERFAMILY ANGUINOIDEA Diagnosis: Hexatylina. Small to large sized ( mm), in some genera adults may be obese and sedentary. No marked sexual dimorphism in anterior region. Cuticle with fine transverse striations, often appearing smooth. Lateral field plain, or with 4, 6 or more incisures. Deirids usually present. Phasmids absent. Cephalic region low, cap- 84

85 like, smooth with indistinct or no annulations, generally continuous with body contour; framework hexaradiate, faintly cuticularized. Oral opening a small round pore surrounded by 6 labial papillae; amphids indistinct, oval, pore-like, slightly dorsosublateral, labial but at some distance from oral opening. Stylet small (under 15µm), with small rounded knobs. Orifice of dorsal gland close to stylet base. Median oesophageal bulb present or absent, with or without refractive valve plates. Oesophageal glands tend to be enlarged, forming a basal bulb, or rarely, the dorsal gland extends over intestine dorsally and laterally; no stem-like extension at base of basal bulb (except Cynipanguina which has a different type of extension). A tricellular cardia absent; two anteriormost intestinal cells often acting as a valve. Gonads single, anteriorly outstretched, may be reflexed or coiled in swollen adults; germ cells may be arranged about a rachis. Vulva a large transverse slit, posteriorly located; lateral vulval membranes rarely distinct (Pterotylenchus). Spermatheca axial, elongated, sac-like (except Pseudhalenchus). Sphincter valve between oviduct and uterus may be present. A postvulval sac often as long as or longer than body width present (absent in Diptenchus), may serve as storage for sperm, often with a small rudiment of posterior reproductive branch. Sperm round, large, with a prominent translucent cytoplasmic vesicle around the nucleus. Spicules robust, anteriorly expanded, separate or fused medially, tip often truncate or broadly rounded. Gubernaculum simple, trough-like, not protrusible, rarely absent (Nothanguina). Bursa moderately large, usually subterminal, but may extend to terminus (Sychnotylenchidae) or be adanal. Tails similar between sexes (except when bursa is terminal), usually elongateconoid, may be cylindroid or filiform; juvenile tails often elongate-conoid to filiform. Fungal feeders; parasites of lower (mosses, seaweeds) and higher plants, attacking stems, leaves, floral parts and seeds, almost always inciting galls; root parasitism known only for Subanguina radicicola which incites and inhabits galls; also associates of insects (Sychnotylenchidae) but not parasitic in insects or other animals). Nominotypical family: Anguinidae Nicoll, 1935 (1926) Relationship: Members of this superfamily are closely similar to those of the Paurodontidae and free-living forms of the Sphaerularioidea (suborder Hexatylina), but differ in lacking an entomoparasitic phase in their life-history and a stem-like extension from the oesophageal base (except in Cynipanguina). Traditionally Anguina and Ditylenchus have been classified in the Tylenchidae, but they differ from the members of this family in their different origin and evolution from fungal feeding forms and in having the two anteriormost cells of the intestine modified to act as a valve (a tricellular cardia is absent in this group), a prominent crustaformeria, large sized sperm with a prominent cytoplasmic vesicle and an absence of phasmids. Bionomics: The genera and species of the family Anguinidae are mainly fungal feeders or parasites of stems, leaves, flower parts and seeds where they usually incite galls. The only known root parasitic species is Subanguina radicicola, which incites and inhabits root galls on various grasses. 85

86 Important characteristics of the family Anguinidae: equal. 1) the cephalic region is low and smooth; framework hexaradiate, sectors almost 2) the basal oesophageal bulb is either small or large, offset from intestine; the dorsal gland may become enlarged and extend over intestine as a lobe. 3) Vulva is generally less than 85% of body length; post vulval uterine sac present or rarely (Diptenchus) absent. 4) Ovary outstretched or reflexed; oocytes may be arranged around a rachis in obese females. 5) The tail is similar between the sexes; female tail rarely subcylindrical, never cylindrical or hooked. 6) The bursa varies from adanal to subterminal, never encircling tail tip. Key to families of Anguinoidea 1. Terminal excretory duct abnormally widened and cuticularized: parasites of marine plants (algae)...halenchidae Terminal excretory duct not abnormally widened and cuticularized; not parasites of marine plants Female tail cylindroid or subcylindroid, dissimilar to that of male; bursa enveloping tail terminus; associates of insects...sychnotylenchidae Female tail conoid to filiform, rarely subcylindroid, similar to that of male; bursa not enveloping tail terminus; very rarely associates of insects...anguinidae Family ANGUINIDAE Nicoll, 1935 (1926) Diagnosis: Anguinoidea. Adults from about 0.4 to 3mm long, slender or obese. Cephalic region low, smooth; framework hexaradiate, sectors almost equal. Muscular valvate median bulb present or absent. Basal oesophageal bulb small or large, offset from intestine, or the dorsal gland may become enlarged and extend over intestine as a lobe. Excretory duct not abnormally widened or cuticularized. Vulva generally at less than 85% of body length. Postvulval uterine sac present, or rarely (e.g. Diptenchus) absent. Ovary outstretched or reflexed; oocytes may be arranged about a rachis in obese females. Tails similar between sexes, female tail conoid to filiform, rarely subcylindrical, but never cylindrical or hooked. Bursa variable from adanal to subterminal, never enclosing tail tip. Fungus feeders or parasites of stem, leaves, flower parts and seeds where they usually incite galls, not root parasites (except Subanguina radicicola which incites and inhabits roots galls). Nominotypical subfamily: Anguininae Nicoll, 1935 (1926) 86

87 Key to subfamilies of Anguinidae 1. Postcorpus bulboid, muscular, valvate...anguininae Postcorpus not bulboid, muscular, or alvate Female spiral, obese; gubernaculum absent...nothanguininae Female not spiral, obese; gubernaculum absent...nothotylenchinae Subfamily ANGUININAE Nicoll, 1935 (1926) Diagnosis: Anguinidae. Adults slender or obese, straight, arcuate or strongly curved when relaxed. Precorpus cylindroid. Postcorpus muscular, with distinct valve plates. Basal bulb enclosing oesophageal glands present, dorsal gland may form a lobe extending over intestine dorsally or laterally (Pseudhalenchus, Safianema, some Anguina). Vulva lacking lateral membranes. Postvulval uterine sac well developed (absent in Diptenchus). Crustaformeria with 4 or more rows of cells. Ovary outstretched or with tip reflexed once or twice. Gubernaculum present. Type genus: Anguina Scopoli, 1777 Key to genera of Anguininae 1. Crustaformeria with more than 20 cells; female generally obese (partially obese in several Subanguina spp.)...2 Crustaformeria with less than 20 cells (generally 16 cells); female slender, occasionally partially obese Oesophageal base with a long digit-like extension...cynipanguina Oesophageal base without a digit-like extension Gametocytes usually arranged about a rachis; female strongly obese, spirally urved...anguina Gametocytes not arranged about a rachis; female generally not strongly obese or spirally curved...subanguina 4. Dorsal oesophageal gland forming a long lobe extending over intestine, with nucleus lying posterior to oesophago-intestinal junction...5 Dorsal oesophageal gland not forming a long lobe extending over intestine, with nucleus anterior to oesophago-intestinal junction Lateral field with 4 incisures; spermatheca rounded...pseudhalenchus Lateral field with 6 incisures; spermatheca elongate-saccate...safianema 6. Postvulval uterine sac absent...diptenchus Postvulval uterine sac present...ditylenchus 87

88 Genus ANGUINA Scopoli, 1777 Diagnosis: Anguininae. Medium to large sized (1-2.7mm), obese; mature female curved generally in one to one-and-a-half spirals. Median oesophageal bulb muscular. Basal bulb in adults enlarged, continuous or offset from isthmus by a constriction, base usually extending over anterior end of intestine. Ovary with one or two flexures anteriorly due to excessive growth; oocytes in multiple rows, arranged about a rachis. Crustaformeria a long tube formed by a large number of cells in multiple irregular rows. Spermatogonia in multiple rows. Bursa subterminal. Second-stage juvenile generally resistant and is the infective stage. Obligate plant parasites inciting galls in seeds of cereals and grasses, stems, leaves and inflorescence of various monocotyledonous plants; type species causes wheat seed galls (ear cockles); only A. amsinkiae and A. balsamophila parasitize dicotyledonous plants. Type species: Anguina tritici (Steinbuch, 1799) Filipjev, 1936 Bionomics: Within the genus Anguina, there are several species of economic importance. The second stage juvenile (J2) is the resistant and infective stage and can survive desiccation for thirty years or more. The life-cycle of an Anguina species can be exemplified by Anguina tritici. A. tritici (the type species) infects grasses, wheat and barley. The nematodes initially feed ectoparasitically on the growing points and leaf bases until they are able to enter the inflorescences as the embryo seeds develop. The nematodes then develop to adults, which feed and mate inside the grain. The female lays a large number of eggs (between a few hundred to 32,000). The adults die in the galled grain, the entire cavity of which is occupied by the J2 stage which gradually desiccates. As a result of infection by the nematode, the grain is destroyed. The life cycle resumes the following season when rain softens the gall, allowing the J2 nematodes to revive by absorbing water. They then migrate in search of germinating host seedlings which they invade. A. tritici can easily be controlled by efficient seed cleaning techniques which remove the galls from the grain and thus avoids the possibility of sowing them with seed for the next crop. Genus DITYLENCHUS Filipjev, 1936 syn. Anguillulina (Ditylenchus) Filipjev (Schneider, 1939) Diagnosis: Anguinidae. Body usually under 1.5mm long, not curving strongly when relaxed; mature adults slender. Lateral field with 4 or 6 (occasionally more) incisures which may be indistinct. Median bulb muscular, with valve plates. Isthmus not marked off from basal bulb. Basal bulb a thin elastic sac containing oesophageal glands; base of bulb may extend over intestine but the nuclei of glands remain within the bulb anterior to the oesophago-intestinal junction. Intestine with 2 normal sized but often hyaline cells at its anterior end, possibly acting as a valve; lumen of intestine not considerably narrowed in anterior region. Ovary outstretched, with 1 or 2 rows of oocytes. Vagina at right angle to body axis or nearly so, not directed forward. Postvulval uterine sac present. Testis outstretched. Bursa adanal to subterminal. Tails elongate-conoid to subcylindrical or filiform. Fungal feeders and parasites of higher plants, several species including the type species are capable of attacking aerial parts. 88

89 Type species: Ditylenchus dipsaci (Kühn, 1857) Filipjev, 1936 Bionomics: Many species in this large genus are fungal feeders and are not known to attack higher plants. There are, however, several important plant parasitic species which are capable of causing extensive loss to agricultural crops. The most famous species is D. dipsaci, known as the stem nematode or stem and bulb nematode. This species attacks over 450 plant species and occurs as biological races or biotypes. The fourth stage juvenile (J4) is the survival stage and in drying plant tissues, the nematodes tend to aggregate, forming clumps of "eelworm wool". The nematode is a migratory endoparasite of bulbs and the green parts of plants. It can be devastating on onion and broad bean crops. Another species, D. angustus, the Ufra nematode, attacks rice and can be particularly damaging on deep water rice in Bangladesh and Vietnam. The nematodes invade and feed on the developing rice seedlings and on mature plants are found in the tender parts of the stem, leaf sheath and flower spike. Both these species have four lateral lines, but D. destructor, a member of the D. triformis group, has 6 incisures in the lateral field. Members of this group are mainly fungal feeders, although D. destructor attacks potato tubers and bulbous iris. D. myceliophagus attacks and destroys the mycelia of cultivated mushrooms. REFERENCES AND ADDITIONAL READING Brzeski, M. W. (1981). The genera of the Anguinidae (Nematoda: Tylenchida). Revue de Nématologie, 4: Fortuner, R. and Maggenti, A. R. (1987). A reappraisal of the Tylenchina (Nemata). 4. The family Anguinidae Nicoll, 1935 (1926). Revue de Nématologie, 10: Fortuner, R. and Raski, D. J. (1987). A review of Neotylenchoidea Thorne, 1941 (Nemata : Tylenchida). Revue de Nématologie, 10: Siddiqi, M. R. (1986). Tylenchida: Parasites of plants and insects. CABI International, Wallingford, UK, 645pp OBESE NEMATODES Plant parasitic nematodes with obese or swollen females occur in a number of unrelated families within the Tylenchida, although the phenomenon is best represented, and reaches its highest evolutionary development, in the Heteroderidae and Meloidogynidae. Nematodes with obese females usually protect their eggs, either by forming a cyst or cystlike structure in which the eggs are retained, or by secreting a gelatinous matrix in which the eggs are embedded. Where a gelatinous matrix is produced, the number of eggs laid is often fairly small, the eggs themselves being relatively large and the ensuing juveniles rapidly moulting to the adult stages without feeding. However, in other nematodes, such as 89

90 Meloidogyne, large numbers of eggs, perhaps in excess of 500, are laid into the matrix and the juvenile stages are also parasitic. Nematodes belonging to the Heteroderidae and Meloidogynidae (ie. the cyst and root knot nematodes) are dealt with separately in this course, this section concentrating on some of the other genera commonly found parasitizing the roots of crops. For the sake of completeness, a list of the families and the obese genera they contain is appended. Family TYLENCHULIDAE Skarbilovich, 1947 This is a small group of interesting nematodes, the most important genus being Tylenchulus, although both the distribution and significance of Trophotylenchulus in tropical soils are certainly underestimated. All genera are sedentary, semi-endoparasitic root parasites and are often associated with tree crops. Tylenchulus semipenetrans is a serious pest of citrus plantations and causes the disease known as 'slow decline'. This nematode has a world-wide distribution and is virtually ubiquitous in citrus growing areas. It has been disseminated on the roots of infested planting material and is often a serious pest of established orchards. Key to genera 1. Adult female spirally coiled; excretory duct directed posteriorly in relation to the pore...2 Adult female not coiled, excretory duct directed anteriorly from the pore Excretory pore well behind oesophageal region...trophotylenchulus Excretory pore in oesophageal region...trophonema 3. Adult female with circumoral disc, excretory pore at less than 65% of body length...ivotylenchulus Adult female without oral disc, excretory pore at more than 65% of body length...tylenchulus Genus TYLENCHULUS Cobb, 1913 Diagnosis: Criconematoidea. Mature female swelling posteriorly with the bulk of the body protruding from the root surface and the attenuated anterior section embedded in the cortex. They are small and range in length from mm. Female excretory pore far posterior at about 68-85% of the body length and located slightly anterior to the vulva, the duct being directed anteriorly. Female head skeleton weak; stylet slender. Oesophagus of modified criconematid form, the median bulb being well developed and the nonoverlapping basal bulb distinct. Vulva posterior; genital tract mono-prodelphic and coiled. Male vermiform with delicate stylet and degenerate oesophagus; bursa absent. Juveniles vermiform with a posteriorly located excretory pore and a long, pointed tail (they die straight and strongly resemble Tylenchus under the stereomicroscope). Type species: Tylenchulus semipenetrans Cobb,

91 Other species: T. furcus Van den Berg & Spaull, 1982 Bionomics: Sedentary semi-endoparasites, mainly attacking tree crops. The second stage female juveniles are the infective stage and penetrate the root cortex. Male juveniles moult to the adult without feeding. Feeding by the females induces the development of 'nurse' cells in the cortex, a trophic specialization. These cells eventually disintegrate into a mass of necrotic tissue and the epidermal layer is sloughed. The eggs are deposited in a gelatinous matrix which is secreted by the excretory system (hence the posterior excretory pore). Heavily infested roots will be coated with such egg masses and, as soil particles adhere to the gelatinous material, the roots have a dirty appearance after gentle washing when compared to healthy roots. Very large populations of T. semipenetrans can be found on the roots of infected citrus and immense numbers of vermiform stages in the rhizosphere. The gradual decline in vigour and cropping ability caused by the citrus nematode causes the disease known colloquially as 'slow decline'. T. furcus was recorded on sugar cane and grass roots in South Africa. Family HOPLOLAIMIDAE Filipjev, 1934 Genus ROTYLENCHULUS Linford & Oliveira, 1940 Diagnosis: Hoplolaimoidea. Sexually dimorphic. Immature female found free in the soil, vermiform and dying C-shaped when heat-relaxed. Head region rounded to conoid and continuous with body contour. Head skeleton of medium development, stylet moderately strong with rounded basal knobs. Dorsal oesophageal gland orifice well posterior to stylet knobs ( the stylet length). Glands well developed with a long, more or less lateral overlap. Vulva usually posteriorly located (58-72%), genital tracts didelphic, each with a double flexure. Tail conoid with a rounded terminus. Mature female found on roots; swollen to kidney shaped body protruding from root, anterior part irregular. Vulval lips protuberant, genital tracts convoluted. Male vermiform, free living in soil. Head skeleton, stylet and oesophagus reduced, but still conspicuous. Tail pointed, spicules curved, bursa not reaching tail tip. Juveniles free living in soil and resembling immature female in general respects. Type species: Rotylenchulus reniformis Linford & Oliveira, 1940 Bionomics: The eggs are laid in a gelatinous matrix. On hatching, the juveniles moult to the immature female or male without feeding. The immature female is the invasive stage, but only the anterior part penetrates the root tissue, the posterior section remaining in the soil and swelling ie semi-endoparasitism. About 50 eggs are deposited in the gelatinous matrix which is produced by specialized vaginal cells. R. reniformis has an extremely wide 91

92 host range, is almost ubiquitous in tropical and subtropical soils and is reported to cause damage to a number of crops. REFERENCES AND ADDITIONAL READING Siddiqi M.R. (1986). Tylenchida: Parasites of plants and insects. CAB International, Wallingford, UK, 645pp. 6. ORDER APHELENCHIDA The order Aphelenchida is for nematodes with a tylenchid type oesophagus comprised of a mouth stylet, cylindrical procorpus, swollen muscular metacorpus (median bulb) with distinct refractive crescentic thickenings and a terminal glandular region which usually overlaps the intestine. Some controversy exists over whether the aphelenchs deserve ordinal status or are merely a suborder of the Tylenchida. The two groups show substantial differences in fundamental morphological characters, but the weighting of such differences is open to subjective interpretation. Members differ from Tylenchida in the fact that the dorsal oesophageal gland duct opens into the anterior part of the median oesophageal bulb in aphelenchs, not into the lumen of the procorpus as in tylenchs. Females, except of some insect parasitic forms, remain vermiform; the vulva is in the posterior third of the body, there is a single prodelphic (anterior) ovary and there is often a post vulval sac. The mouth stylet is often weak, usually under 20µm long, rarely with well developed basal knobs, but there are some striking exceptions with stylets greatly in excess of 100 m. The Aphelenchida is divided into two superfamilies Aphelenchoidea and Aphelenchoidoidea. The Aphelenchoidea has the two families Aphelenchidae and Paraphelenchidae, each of which is based on a single genus. Species within this group, although often associated with diseased plant tissues, seem to be fungivorous and, like some Aphelenchoides species, occasionally cause damage in mushroom culture. The Aphelenchoidoidea has more families, the exact number depending on the author involved. The Entaphelenchidae are obligate insect parasites and the Seinuridae are mainly predatory. Many of the others, especially Parasitaphelenchidae, Ektaphelenchinae, Cryptaphelenchinae and Schistonchinae are insect parasites or associates in some cases particularly of bark boring beetles. However, within Parasitaphelenchidae, Rhadinaphelenchus cocophilus and Bursaphelenchus xylophilus (Bursaphelenchinae) are involved in serious disease of palm and pine trees respectively. Acugutturus parasiticus Hunt, 1980 (the only species in Acugutturinae), an ectoparasite of the American cockroach, and other ectoparasites of noctuid moths are unique in the Aphelenchida because of the µm long stylet. The rarely occurring Anomyctus xenurus (the only species in Anomyctinae) also has a long stylet (30-35µm) and is possibly a predatory nematode. The plant parasitic "bud and leaf" nematodes are in the genus Aphelenchoides (Aphelenchoidinae), but only three species (A. besseyi, A, fragariae and A. ritzemabosi) are of general importance and a few more are occasionally associated with plant damage. 92

93 Order APHELENCHIDA Siddiqi, 1980 Diagnosis (modified after Siddiqi, 1980): Soil dwelling or insect associates; trophic habit mycetophagous, phytoparasitic, predaceous or entomoparasitic. Body small to long (0.2mm - 2.5mm), vermiform, rarely obese except in some insect parasites. Cuticle thin, usually finely annulated and with lateral fields marked by 0 to more than 12 incisures. Cephalic region low, rounded, continuous or offset and with weak or moderate sclerotization. Stylet always present, length usually 10-20µm, but exceptionally up to 185µm; conus usually shorter than the shaft, but much longer in certain insect ectoparasitic forms. Basal swellings or knobs usually weakly developed or entirely absent. Oesophagus comprising a narrow, cylindrical procorpus, a strongly-developed, offset, ovoid to rounded rectangular median bulb with crescentic valve plates and well developed oesophageal glands forming a dorsally overlapping lobe in all genera apart from Paraphelenchus where the glands are small and enclosed in a non-overlapping basal bulb. All three gland orifi (ie. including the dorsal gland orifice) located within the median bulb. Isthmus usually short or absent. Nerve ring circum-oesophageal or circumintestinal. Intestine cellular with distinct lumen. Rectum usually distinct except in some insect associates. Anus a broad transverse slit with an overhanging anterior lip, but absent or degenerate in some insect parasites or associates. Vulva posterior at 60-98%, usually in the form of a transverse slit or, exceptionally, an oval pore (Aphelenchus). Genital tract monoprodelphic, usually outstretched, but occasionally with a double flexure. Spermatheca axial, if present. Post-uterine sac usually present and may act as a spermatheca. Male genital system monorchic, outstretched. Spicules typically rosethornshaped with prominent apex and rostrum, or derived therefrom, but elongate and cephalated in Aphelenchus and Paraphelenchus. Gubernaculum usually absent, but well developed and elongate in Aphelenchus and Paraphelenchus. Bursa usually absent, but a true peloderan bursa with supporting ribs is present in Aphelenchus only, although some genera may have a terminal flap of cuticle (= bursa). Type genus: Aphelenchus Bastian, 1865 Key to superfamilies 1. Spicules slender, cephalated. Gubernaculum well developed, elongate; V- shaped in cross-section. Lateral fields with six or more incisures. Oesophagus with distinct isthmus and nerve ring circum-oesophageal...aphelenchoidea Spicules robust, rosethorn-shaped or derived therefrom; typically with a dorsal and ventral limb joined by a transverse bar. Gubernaculum absent or, if present, consisting of a small structure near the distal tip of the dorsal limb of the spicule; never elongate or V- shaped in cross-section. Lateral fields usually with four or fewer incisures, exceptionally six. Oesophagus lacking a distinct isthmus which, if visible at all, is a short stump less than the distance from the valve plates to the base of the bulb in length; nerve ring circumintestinal...aphelenchoidoidea 93

94 Superfamily APHELENCHOIDEA Fuchs, 1937 (Thorne, 1949) Diagnosis: Aphelenchina. Cephalic region low, flattened, continuous with body contour. Lateral fields with six or more incisures. Oesophagus with a distinct isthmus; glands either in a dorsally overlapping lobe (Aphelenchidae) or restrained within a nonoverlapping basal bulb (Paraphelenchidae). Nerve ring circum-oesophageal. Vulva in the form of either a transverse oval pore (Aphelenchidae) or a transverse slit (Paraphelenchidae). Female tail short, sub-cylindroid to conoid and with a broadly rounded terminus which may be mucronate. Spicules slender, ventrally arcuate; cephalated. Gubernaculum well developed, elongate; V-shaped in cross-section. Bursa present or absent; if present then well developed, peloderan and supported by four pairs of ribs (only three pairs reported by Thorne (1961) in A. eremitus). Type genus: Aphelenchus Bastian, 1865 Type family: Aphelenchidae Fuchs, 1937 (Steiner, 1949) Other family: Paraphelenchidae T. Goodey, 1951 (J. B. Goodey, 1960) Key to families 1. Male with prominent peloderan bursa supported by four pairs of ribs. Female vulval aperture in the form of an oval pore. Oesophageal glands forming a long dorsally overlapping lobe...aphelenchidae Male lacking a bursa. Vulva in the form of a transverse slit. Oesophageal glands small, retained within a non-overlapping basal bulb..paraphelenchidae Family APHELENCHIDAE Fuchs, 1937 (Steiner, 1949) Diagnosis: Aphelenchoidea. Soil dwelling or found in decaying plant material. More than six lateral lines (usually 10 to 12). Vulval aperture in the form of a transverse oval pore. Oesophageal glands free, forming a dorsally overlapping lobe. Female tail short, cylindroid with a rounded tip. Male bursa well developed, peloderan in form and with four pairs of supporting ribs, one pair adanal and the other three postanal. Genus APHELENCHUS Bastian,

95 Diagnosis: Aphelenchidae. Medium sized to fairly long nematodes ( mm). Heat relaxed form more or less straight to ventrally arcuate. Cephalic region low, rounded and slightly offset. Cuticle finely annulated. Lateral fields broad with numerous incisures, usually in excess of ten, exceptionally fewer (A. sparsus only has four to six, for example). Stylet slender with slight basal swellings. Procorpus cylindrical, narrowing just before joining the large, ovoid, median bulb with centrally placed, crescentic valve plates. Oesophageal glands in a dorsal lobe. Nerve ring just behind the median bulb and more or less opposite the excretory pore. Vulva posterior at about 70-80% of the body length. Vulval lips slightly protuberant and body often narrowing sharply just behind the vulva. Vagina sloping anteriorly. Genital tract monoprodelphic, outstretched. Developing oocytes mostly in a single row. Post-uterine sac extending for up to half the vulva/anus distance. Tail short, cylindroid, sometimes slightly ventrally concave, ending in a broadly rounded terminus. Male, when present, with paired, slender spicules which are ventrally arcuate and slightly cephalated proximally. Gubernaculum linear. Bursa well developed, extending from the proximal region of the spicules to the tail tip and supported by four (exceptionally three) pairs of ribs; one pair preanal and the other three in a subterminal group. Tail short, conoid, tapering to a narrowly rounded point. Type species: Aphelenchus avenae Bastian, 1865 Bionomics: A. avenae is common throughout the world in many soils and rotting plant tissues, it readily reproduces on a wide range of fungi including cultivated mushroom, but does not seem to cause primary damage to higher plants. Family PARAPHELENCHIDAE T. Goodey, 1951 (J. B. Goodey, 1960) Diagnosis: Aphelenchoidea. Soil dwelling. Usually six to eight lateral lines. Oesophageal glands small, enclosed in an abutting basal bulb which is amalgamated with the isthmus. Vulva in the form of a transverse slit. Female tail short, sub-cylindroid and usually with a mucronate terminus. Male bursa absent, four to five pairs of caudal papillae Type genus: Paraphelenchus Micoletzky, 1922 (Micoletzky, 1925) Superfamily APHELENCHOIDOIDEA Skarbilovich, 1947 (Siddiqi, 1980) Diagnosis: Aphelenchina. Cephalic region usually high and offset from body contour. Lateral fields with four or fewer incisures (very exceptionally six). Stylet often with basal knobs or swellings. Oesophagus with isthmus rudimentary or absent. Nerve ring 95

96 circumoesophageal. Oesophageal glands in a dorsally overlapping lobe. Vulva in the form of a transverse slit. Female tail conoid to a pointed or narrowly rounded terminus which may be mucronate or otherwise adorned. Spicules robust, rosethorn-shaped or derived therefrom; usually with a prominent apex and rostrum. Gubernaculum absent or indistinct; if present it is a small structure located at the distal tip of the dorsal limb of the spicules and is never elongate, linear or V-shaped in cross-section. Caudal papillae usually numbering two or three pairs, occasionally as many as five pairs. Bursa absent around the cloaca, but a small terminal flap of cuticle is present in the Parasitaphelenchidae. Type genus: Aphelenchoides Fischer, Type family: Aphelenchoididae Skarbilovich, 1947 (Paramonov, 1953) Key to families 1. Stylet of both sexes very long (50-180µm), attenuated; the conus constituting the majority of the stylet length. Ectoparasites of insects...acugutturidae Stylet usually about 10-20µm long, never over 35µm long and never attenuate with the conus constituting the majority of the stylet length Mature females with swollen body, endoparasitic in the haemocoel of beetles. Three adult forms: male; immature female; mature parasitic female....entaphelenchidae Mature females not swollen or endoparasitic. Only two adult forms in the life cycle Females with functional anus and elongate tails more than four anal body widths long, often becoming attenuate or filiform, but may be more cylindroid with a rounded or spathulate terminus. Male tail elongate conoid with a spicate terminus...seinuridae Females with short or medium conoid tails usually less than four anal body widths long, but if longer and with a filiform or spicate terminus then the female anus is non-functional Males with small bursa-like flap of cuticle at tail tip...parasitaphelenchidae Males lacking such a bursa Females lacking a functional anus and rectum, the intestine ending as a blind diverticulum in the tail region. Stylet typically with a wide lumen...ektaphelenchidae Females with a functional anus and rectum, intestine not ending as a blind diverticulum. Stylet usually robust, with a narrow lumen and basal knobs or swellings...aphelenchoididae 96

97 Family APHELENCHOIDIDAE Skarbilovich, 1947 (Paramonov,1953) Diagnosis: Aphelenchoidoidea. Stylet slender, with narrow lumen and usually with small basal knobs or swellings. Post-uterine sac usually present. Female tail of medium length, conoid, with pointed or rounded, often mucronate, terminus. Spicules paired, separate, rosethorn-shaped or derived therefrom. Gubernaculum absent. Bursa absent. Key to subfamilies 1. Cephalic region low, rounded, lacking an obvious oral disc...aphelenchoidinae Cephalic region high, almost spherical, and with a prominent cuticularized oral disc...anomyctinae Subfamily APHELENCHOIDINAE Skarbilovich, 1947 Type genus: Aphelenchoides Fischer, 1894 Key to genera 1. Tail tip bearing four pedunculate tubercles with fringed margins. A vulval flap, formed by the posterior extension of the anterior lip, may be present...laimaphelenchus Tail tip not as above, but may be variously mucronate. Vulval flap never present Vulva well posterior, at about 78-93% with the mean value in excess of 80%. Male tail initially short conoid and then narrowing rapidly to a digitiform or short filiform extension...3. Vulva more anterior at about 60-75% with the mean value less than 80% and usually between 65-70%. Male tail conoid, evenly tapering Post-uterine sac absent, female tail subconoid, tapering evenly to a pointed terminus. Apex of spicules greatly elongated and continuing the line of the dorsal limb. Associates of nitidulid beetles...sheraphelenchus Post-uterine sac present, well developed. Female tail dome-shaped with a terminal spike. Apex of spicule small, knob-like. Associates of scolytid beetles...ruehmaphelenchus 4. Stylet very robust, about 17µm long and with massive, rounded basal knobs. Cuticular annules coarse (1.7µm)...Megadorus 97

98 Stylet not with the above combination of characters, although it may be robust and as long as 21-24µm. Cuticular annules fine Stylet robust, 21-24µm long with well developed knobs. Found associated with fig wasps or inside figs...schistonchus Stylet and other characters not as above Lips higher than wide, stylet short, robust and with rounded knobs. Conus distinctly shorter than the shaft. Spicules strongly curved and with the apex and rostrum well developed...tylaphelenchus Not with the above combination of characters, spicules rosethorn-shaped, apex and rostrum low and rounded or absent...aphelenchoides Genus APHELENCHOIDES Fischer, 1894 Diagnosis: Aphelenchoidinae. Small to long nematodes, usually between 0.4 to 1.2mm in length. Heat relaxed females die straight to ventrally arcuate whereas the males assume a 'walking-stick' shape with the tail region sharply curled ventrally. Cuticle finely annulated. Lateral fields often with four incisures but may be two or three. Cephalic region usually rounded in form and slightly offset. There are six equally sized lips and the cephalic skeleton is weak. Stylet slender, usually with basal knobs or swellings, often about 10-12µm long and usually less than 20µm long. Procorpus cylindrical, leading to a well developed ovoid or spherical median bulb with central valve plates. Oesophageal gland lobe well developed and lying dorsal to the intestine. Nerve ring and excretory pore posterior to the median bulb, although the excretory pore may be anterior or posterior to the nerve ring. Vulva postmedian, usually at between 60-75% of the body length, only very exceptionally more posterior. Genital tract monoprodelphic, typically outstretched, but may reflex. Post-uterine sac usually present and often containing spermatozoa, but may be absent. Tail conoid with a variable terminus which may be bluntly or finely rounded, digitate or bifurcate or with a ventral projection. One or more mucrons of various shapes may be present. Male tail strongly hooked ventrally to form the characteristic 'walking-stick' form, conoid in shape and tapering to a variable terminus. Spicules thorn-shaped, paired and separate. The rostrum and apex are usually well developed, but may be almost absent. Typically there are three pairs of caudal papillae, one pair adanal, one pair subterminal and the other in between. Bursa absent. Type species: Aphelenchoides kuehnii Fischer, syn. A. (Aphelenchoides) kuehnii Fischer, 1894 (Filipjev, 1934) Bionomics: Although over 100 species have been described in this genus, few have been implicated as important plant parasites. The most important are A. besseyi on rice and strawberry, A. fragariae on strawberry, ferns and other ornamentals and A. ritzemabosi on chrysanthemum, strawberry and many other crops and weeds especially composites. A. arachidis infests the testa of groundnut seed, A. blastophthorus is occasionally reported on 98

99 scabious and A. subtenuis from bulbs. All of these, exceptionally A. ritzemabosi, can also be cultured on fungi as can A. composticola which can cause serious damage to cultivated mushroom. Some other species often found associated with diseased plants are A. bicaudatus, A. saprophilus and A. hamatus and these also feed on fungi. REFERENCES AND ADDITIONAL READING Allen, M. W. (1952). Taxonomic status of the bud and leaf nematodes related to Aphelenchoides fragariae (Ritzema Bos, 1891). Proceedings of the Helminthological Society of Washington 19, Anderson, R. V. and Hooper, D. J. (1980). Diagnostic value of vagina structure in the taxonomy of Aphelenchus Bastian, 1865 (Nematoda: Aphelenchidae) with a description of A. (Anaphelenchus) isomerus n. subgen. n. sp. Canadian Journal of Zoology 58, Baranovskaya, I. A. (1981). [Plant and soil nematodes (Aphelenchoididae and Seinuridae)] In Russian. Moscow, Isdatelstvo Nauka 233pp. [See Helm. Abs. B, 51 No. 1208] Dean, C.G. (1979). Red ring disease of coconut. Tech. Comm. No. 47. Commonwealth Agricultural Bureau, UK, 70pp. Franklin, M.T. (1957). Aphelenchoides composticola n. sp. and A. saprophilus n. sp. from mushroom compost and rotting plant tissues. Nematologica 2, Franklin, M.T. (1978). Aphelenchoides and related genera. In: Southey, J. F. Edit. Plant nematology. MAFF, RB407; London HMSO pp Goodey, Y. (1961). Soil and freshwater nematodes. Rewritten by J.B. Goodey. London, Methuen 544pp. Hooper, D.J. and Clark, S.A. (1980). Scanning electron micrographs of the head region of some species of Aphelenchoidea (Aphelenchina: Nematoda). Nematologica 26, Hunt, D.J. (1993). Aphelenchida, Longidoridae and Trichodoridae: Their systematics and bionomics. CAB International, Wallingford, UK., 372pp. Husain, S.I. and Khan, A.M. (1967). On the status of the genera of the superfamily Aphelenchoidea (Fuchs, 1937) Thorne, 1949 with descriptions of six new species of nematodes from India. Proceedings of the Helminthological Society of Washington 34, Massey, C.L. (1974). Biology and taxonomy of Nematode parasites and associates of bark beetles in the United States. Agriculture Handbook No. 446 Washington, D.C., United States Department of Agriculture. 233pp. 99

100 Nickle, W.R. (1970). A taxonomic review of the Aphelenchoidea (Fuchs, 1937) Thorne, 1949 (Nematoda, Tylenchida). Journal of Nematology 2, Nickle, W.R. and Hooper, D.J. (1991). The Aphelenchinae bud, leaf and insect nematodes. In: W.R. Nickle Ed. Manual of Agricultural Nematology New York: Marcel Dekker. pp Paramonov, A.A. (1964). [Principles of Phytonematology. Vol. 2. Taxonomy of phytonematodes] In Russian. Moscow, Izdatelstvo Akad. Nauk SSR 446pp. (See also printed and published by Indian National Science Documentation Centre for U.S. Dept. Agric. & Nat'nl Sci. Foundation Washington, pp. Siddiqi, M.R. (1980). The origin and phylogeny of the nematode orders Tylenchida Thorne, 1949 and Aphelenchida n. ord. Helminthological Abstracts Series B, Plant Nematology 49, Thorne, G. (1961). Principles of nematology. New York, McGraw-Hill 553pp. Yin, K., Fang, Y. and Tarjan, A.C. (1988). A key to the species in the genus Bursaphelenchus with a description of Bursaphelenchus hunanensis sp. n. (Nematoda: Aphelenchoididae) found in pine wood in Hunan province, China. Proc. Helminthol. Soc. Wash. 55, ORDER DORYLAIMIDA SUPERFAMILY DORYLAIMOIDEA FAMILY LONGIDORIDAE Although Longidorus elongatus (de Man) was first described in 1876, it was not until the last 25 years or so that longidorids received much attention from nematologists. Since about 1960 many new species have been described and extensive studies have been carried out on the biology of selected species, usually those implicated in nepovirus transmission. Despite the fact that longidorids are large nematodes they tend to be under-recorded because of the use of unsuitable extraction techniques, the nematodes remaining in the soil and not actively participating in the extraction process. The best method is probably the immersion-sieving technique and it is always a good idea to supplement the standard tray extractions with this method. Longidorids are root ectoparasites often congregating in large numbers at, or just behind, root tips where their feeding gives rise to a characteristic galling. The Longidoridae is the only family in the Dorylaimida known to contain numerous important plant-parasitic nematodes. 100

101 Morphology Longidorids conform to the basic dorylaimoid type, the most obvious difference being the greatly elongate odontostyle and odontophore and to some extent their long, relatively slender bodies. The oesophagus is in two parts - a narrow anterior section and an expanded, muscular, basal portion containing the glands and gland ducts. The female genital system varies from didelphic to monodelphic or mono-opisthodelphic, with most variations in between. The vulva is usually median or anterior to median. The male tail region often curls sharply ventrad on death due to the contraction of the oblique copulatory muscles. The body cuticle is usually thin but tends to be thicker at the neck region and on the tail where radial striations may be prominent. Body pores consist of a lateral series and, especially in the oesophageal region, a ventral and dorsal series may also be present. The amphidial apertures vary from broad slits to obscure pores but are always lateral and just behind the lip region. Family LONGIDORIDAE (Thorne, 1935) Meyl, 1961 Diagnosis: Dorylaimoidea. Long to very long, slender nematodes ranging from 1.5 to 12mm in length. Cuticle smooth. Cephalic region rounded, continuous with body contour or offset. Lips amalgamated with the usual circlets of papillae. Amphidial apertures ranging from small pores to broad transverse slits. Amphids large, pouch-like or stirrup-shaped. Lateral chords broad with one to three rows of lateral body pores. Dorsal and ventral series of body pores usually present. Odontostyle greatly elongate, attenuate, µm long. Odontophore elongate, sometimes with three strong, basal flanges. Junction of cheilostome and stomodaeum marked by a strongly cuticularized guide ring varying in position from near the lip region to near the odontostyle base. Oesophagus in two parts: an anterior, narrow, tubular section and posterior, short cylindrical, bulb containing longitudinal valve plates. Three oesophageal glands - one dorsal and two ventrosublateral. Female vulva located anteriorly to post-median in position, usually median. Genital tracts typically amphididelphic reflexed, but may be pseudomonodelphic or mono-opisthodelphic. Uterus of some species of Xiphinema and Xiphidorus with various cuticularized structures in the lumen and/or attached to the walls. Spicules large, dorylaimoid with lateral accessory guiding pieces. Tail shape variable, but generally similar in each sex. Type genus: Longidorus Micoletzky, 1922 (Filipjev, 1934) Type subfamily: Longidorinae Thorne, 1935 Other subfamilies: Xiphidorinae Khan, Chawla & Saha, 1978 (Jairajpuri & Ahmad, 1992) Xiphinematinae Dalmasso, 1969 Bionomics of Longidoridae: Ectoparasites found in the rhizosphere of a wide variety of plants. Some species of Longidorus and Xiphinema have been implicated in vectoring 101

102 certain plant viruses. The direct feeding damage includes root tip galling and stunting of the root system. Distribution of Longidoridae: Longidorus is widespread in Europe, Asia, Africa and North America, but is probably introduced to South America. Paralongidorus is widespread in Africa and Asia and Xiphinema is found world-wide, but with particular diversity in southern Africa. Xiphidorus and Paraxiphidorus are restricted to South America and Longidoroides is found in southern and eastern Africa and India. Key to subfamilies 1 Dorsal gland nucleus elongate, smaller than those of the ventrosublateral glands and located at some distance posterior to its orifice...2. Dorsal gland nucleus round, larger than those of the ventrosublateral glands and located adjacent to its orifice...xiphinematinae 2. Amphidial aperture a minute slit (pore-like), odontostyle with furcate base, guide ring located near to odontostyle/odontophore junction, male copulatory supplements few in number (less than 8) and with an hiatus between the adanal pair and the ventromedian series...xiphidorinae Not with the above combination of characters...longidorinae The family can be divided into two major subfamilies, the Longidorinae and Xiphinematinae, using a suite of characters. The major differential characters are: Character Longidorinae Xiphinematinae guide ring usually around anterior usually around posterior part of odontostyle part of odontostyle amphidial aperture pore-like or slit-like slit-like odontostyle base simple forked odontophore base simple heavily flanged dorsal oesophageal well posterior to its just posterior to its gland nucleus orifice, small orifice, large 102

103 The following genera in each subfamily are discussed in more detail: Longidorinae Longidorus Micoletzky, 1922 (Filipjev, 1934) Paralongidorus Siddiqi, Hooper & Khan, 1963 (included under Paralongidorus are Siddiqia Khan, Chawla & Saha, 1976 and Inagreius Khan, Both these genera fit well under Paralongidorus and differ only in having an offset head. Inagreius differs from Siddiqia only in having a cup-like bilobed amphidial pouch. Xiphinematinae Xiphinema Cobb, Key to genera 1. Odontostyle base forked, odontophore base strongly flanged...xiphinema Odontostyle base simple, odontophore base may be thickened but not strongly flanged Amphidial aperture pore-like...longidorus Amphidial aperture slit-like...paralongidorus MAJOR DIFFERENTIAL CHARACTERS Longidorus and Paralongidorus 1) body length or 'L' can be useful to divide species into broad categories 2) the lip region can be expanded from the body contour; low and rounded and continuous with the body contour or continuous and more or less truncate 3) tail shape can be almost hemispheroid and bluntly rounded; short conical with a broadly rounded terminus or short conical with a more sharply tapering terminus 4) odontostyle length 5) position of guide ring in relation to anterior end. Can be expressed as number of labial widths posterior to head end 6) tail length, c and c' are of importance 7) amphidial pouch shape can vary from saccate to bilobed. The ratio 'a' can be of use but 'b' is of little importance and 'V' tends to be about the same for all species. Xiphinema 1) as for Longidorus, 'L' can be useful to divide species into broad categories, e.g. all the X. americanum group have short bodies, but can be rather variable 103

104 2) 'V' is useful as the vulva can be rather anterior in the monodelphic species. 'V' is fairly constant although geographical variation can be considerable 3) odontostyle length - usually fairly constant within a population, but can vary considerably between populations. The modern trend is to combine the odontostyle and odontophore lengths and use the 'total stylet length' as the morphometric character 4) female genital tract - very important as variations in structure are used to sub-divide species into a number of groups: (a) (b) mono-opisthodelphic - a single posteriorly directed tract with no trace of any anterior branch pseudo-mono-opisthodelphic - as above but some tissue of the anterior branch remains. This tissue can be: (i) a short undifferentiated uterine sac (ii) a longer organ showing some differentiation but lacking an ovary (c) didelphic - (i) one branch anteriad and one posteriad, both functional (ii) as above but with a small, non-functional anterior ovary (d) monoprodelphic - posterior branch absent leaving an anterior genital tract. Only one species recorded in this group. This is almost certainly an erroneous observation. In addition there can be uterine differentiation of a cuticular nature. The true 'Zorgan' is a spherical, muscular structure separated from the uterine tissues by sphincters and containing a number of cuticularized pieces. Pseudo-Z organs are cuticular spines or globular structures, often scattered along the uterine wall and are possibly more widespread than presently recorded. Presence or absence of these structures can be important but careful observation is essential for correct determination. 5) tail shape and length are very important. The shape can vary from short and bluntly rounded right through to elongate and filiform. Ratio c' can vary from 0.5 to 20 and is a very useful parameter. Tail shape can be divided into the following broad groups: (i) tail smoothly rounded, hemispherical (ii) tail rounded but spatulate or clavate (iii) tail hemispheroid with a terminal bulge, peg or mucron (iv) tail short, conical, digitate (c' <2.5) (v) tail regularly short conical (c' <2.5) 104

105 (vi) tail long conical (c' ) (vii) tail very long, attenuated (c' >7.5) Other features of the tail, such as the presence or absence of a blind terminal canal are also used. 6) heat-relaxed form (habitus) - fairly constant, species varying from dying in a fairly tight spiral through various degrees of curvature to almost straight 7) lip region - whether offset, continuous, expanded etc. Can be useful but also tends to be rather subjective at times 8) presence or absence of males 9) shape of juvenile tails - may be more useful when full data on juvenile tail shapes are available. Subfamily LONGIDORINAE Thorne, 1935 Genus LONGIDORUS Micoletzky, 1922 Diagnosis: Longidorinae. Body long to very long (3 to >10mm) and slender. Heat relaxed form varying from more or less straight to C-shaped. Lateral chords broad and with one or two rows of lateral body pores. Cephalic region rounded; continuous or offset. Lips fused and with the usual arrangement of papillae. Amphidial apertures in the form of small, inconspicuous pores which lead back to well developed pouch-like amphid fovea. Odontostyle elongate, needle-like; not heavily cuticularized. Dilatores buccae absent. Guiding apparatus with a simple ring usually situated within a couple of head-widths of the anterior end, but exceptionally further posterior, perhaps at up to 40% of the odontostyle length. Junction of odontostyle and odontophore simple. Odontophore about two thirds of the odontostyle in length, moderately cuticularized, thickening slightly in the posterior region, but lacking basal flanges. Odontostylet protractor muscles attached to base of odontophore and running parallel to the cephalic region. Oesophagus comprising a narrow, cylindrical anterior section, which is looped back on itself when the odontostylet is in the retracted position. There are three glands: dorsal and two ventrosublateral. The nucleus of the dorsal gland is situated some distance posteriorly to the orifice and is smaller than the ventrosublateral nuclei. Nerve ring located around the narrow anterior section of the oesophagus; a second nerve ring, located more posteriorly, occurs in some species. Hemizonid prominent. Intestine simple, prerectum well developed and several anal body widths long. Anus in the form of a transverse slit. Vulva a transverse slit, median in position. Vagina well developed, muscular, at right angles to the body axis and leading to a substantial ovejector. Genital tract amphididelphic, reflexed. Tail short, dorsally convex-conoid to a finely rounded terminus, or broadly rounded. Several pairs of caudal pores present. Tail similar in shape to that of the female. 105

106 Type species: Longidorus elongatus (de Man, 1876) Micoletzky, 1922 Bionomics: Ecto-parasites of plant roots, particularly soft rooted forms. Genus PARALONGIDORUS Siddiqi, Hooper & Khan, 1963 Diagnosis: Longidorinae. Long, slender nematodes up to 12mm in length. Heat relaxed form more or less straight to C-shaped. Cephalic region continuous with body contour or expanded and offset by a distinct groove. Amphidial apertures in the form of transverse slits. Amphidial fovea elongate, funnel-shaped. Odontostyle long, attenuate and may be strongly cuticularized. Junction with odontophore ranges from plain to markedly furcate. Odontophore usually lacking well developed basal flanges, but showing distinct development in exceptional species. Guiding ring markedly posterior to the lip region, but not usually at more than a third of the odontostyle length. Guiding sheath not extending anterior to the guide ring. Oesophagus comprising a narrow tubular section anteriorly which expands abruptly into a posterior bulboid section. Nucleus of dorsal oesophageal gland small, rounded; situated some distance behind its orifice. Nuclei of anterior ventrosublateral glands more developed than that of the dorsal gland. Female vulva median to post-median in position. Genital tracts amphididelphic, reflexed. Uterine spines or other cuticularizations not reported. Tail short, rounded; may be conoid or hemispheroid. Spicules paired, massive; dorylaimoid in form with accessory guiding pieces. Tail similar to that of the female. Type species: Paralongidorus sali Siddiqi, Hooper and Khan, 1963 Other species: About 50 other species have been proposed. Subfamily XIPHINEMATINAE Dalmasso, 1969 Genus XIPHINEMA Cobb, 1913 Diagnosis: Xiphinematinae. Body long to very long, 1.5 to 6mm, and fairly stout. Heat relaxed form straight, ventrally arcuate, C-shaped or an open spiral. Cuticle smooth. Lateral chords broad with one or two rows of lateral body pores. Dorsal and ventral series of body pores may be present, particularly in the oesophageal region. Cephalic region rounded, continuous or offset. Lips fused with the usual circlets of papillae. Amphidial apertures broad slits extending for almost the entire lip width. Amphid fovea stirrup- or funnel- shaped. Odontostyle elongate, needle-like; heavily cuticularized. Dilatores buccae present. Guiding apparatus tubular with a strongly cuticularized posterior ring and, apparently, a lightly cuticularized anterior ring (really just a fold in the guiding sheath). The guide ring proper is posteriorly located 106

107 near the odontostyle/odontophore junction. Proximal end of odontostyle appearing forked at its junction with the odontophore which is strongly developed with three massive posterior flanges to which the protractor muscles attach. Oesophagus comprising a narrow, cylindrical anterior section, which is normally looped back on itself, leading to an expanded cylindroid expansion containing the glands. Dorsal gland nucleus located at the same level as the orifice, more developed than ventrosublateral nuclei. Nerve ring around the anterior section of the oesophagus. Hemizonid prominent. Intestine simple, pre-rectum well developed and several anal body widths long. Anus in the form of a transverse slit. Vulva located anteriorly to post-median, in the form of a transverse slit. Vagina well developed, muscular; at right angles to the body axis or posteriorly directed in some forms with an anterior vulva. Ovejector prominent. Genital tract variable; often amphididelphic reflexed, but as the vulva migrates anteriorly the anterior branch progressively regresses, first becoming non-functional, then a remnant and finally completely absent (= mono-opisthodelphic). Some species display cuticularized structures in the uterus. Rarely these cuticularizations are found in a Z organ, a specialized structure with thick walls and circular muscles which is constricted at both ends by a sphincter. More commonly the cuticularizations take the form of spines or variously shaped structures in the uterus. Tail form very variable e.g. short hemispheroid, with or without a digitate process, medium to long conoid, initially conoid and then attenuating to a filiform terminal section. Male genital tract diorchic, opposed. Spicules paired, massive, dorylaimoid in form with distal accessory guiding pieces. Tail of similar form to that of the female. Type species: Xiphinema americanum Cobb, 1913 Other species: Well over 240 species have been described and are currently regarded as valid. The taxonomy of the genus is increasingly complex, the best approach being the polytomous or multiple entry key as used by Loof and Luc (1990, 1993). The X. americanum-group is possibly only accessible via molecular studies. Bionomics: Ecto-parasites of plant roots and capable of attacking woody plants. Some species are virus vectors. REFERENCES AND ADDITIONAL READING Coomans, A. (1975) Morphology of Longidoridae. In: Nematode Vectors of Plant Viruses. Edited by Lamberti, F., Taylor, C.E. & Seinhorst, J.W. Plenum Press, pp Hunt, D.J. (1993) Aphelenchida, Longidoridae and Trichodoridae: Their Systematics and Bionomics. CAB International, 372pp. 107

108 Lamberti, F. (1975) Taxonomy of Longidorus and Paralongidorus. In: Nematode Vectors of Plant Viruses. Edited by Lamberti, F., Taylor, C.E. & Seinhorst, J.W. Plenum Press, pp Loof, P.A.A. and Luc, M. (1990) A revised polytomous key for the identification of species of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) with exclusion of the X. americanum-group. Systematic Parasitology, 16: Loof, P.A.A. and Luc, M. (1993) A revised polytomous key for the identification of species of the genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) with exclusion of the X. americanum-group: Supplement 1. Systematic Parasitology, 24: Luc, M. (1975) Taxonomy of Xiphinema. In: Nematode Vectors of Plant Viruses. Edited by Lamberti, F., Taylor, C.E. & Seinhorst, J.W. Plenum Press, pp Luc, M. & Dalmasso, A. (1975) A 'lattice' for the identification of species of Xiphinema. In: Nematode Vectors of Plant Viruses. Edited by Lamberti, F., Taylor, C.E. & Seinhorst, J.W. Plenum Press, pp ORDER TRIPLONCHIDA FAMILY TRICHODORIDAE The first trichodorid was described by de Man in 1880, when he named Dorylaimus primitivus, a species which subsequently became a member of Trichodorus, a new genus erected by Cobb in Trichodorids are relatively distinctive under the stereomicroscope because of their plump, cigar-shaped bodies which are rounded at both ends, but the lack of an easily recognizable stylet often leads the inexperienced to mistake them for non-parasitic nematodes. Live trichodorids are very sluggish in their movements and often appear to be moribund. When heat - relaxed they either die straight or slightly ventrally arcuate with varying degrees of curvature in the male tail region. The body length varies from mm, but is usually less than 1mm. The tail is extremely short and rounded in both sexes, particularly so in the female where the anus is subterminal. The cuticle is thick and smooth and is lacking in transverse striae when viewed under the light microscope. It is loose and wrinkly and may swell abnormally in some species after fixation. The cephalic region is rounded and slightly offset, the outer ring of papillae sometimes being particularly prominent. The amphidial apertures are located just posterior to the lip region and take the form of short, gaping, ellipsoid structures about one third to half the head width in size with the sensilla sac just behind the amphid pouch or fovea. The excretory pore is small and fairly inconspicuous and is in the oesophageal region or, rarely, a few body widths posterior. In the female, lateral body pores may be present or absent and their presence or absence within a body width of the vulva ('advulval' pores) is regarded as a reliable generic character. The stoma is cuticularized and forms a simple guiding tube around the anterior part of the onchiostyle, the proximal portion appearing as a ring or collar. The onchiostyle 108

109 differs in origin from both the odontostyle of the dorylaims and the stomatostylet of the tylenchs and aphelenchs. The anterior section consists of a dorsal tooth which is predominantly solid, but proximally may appear to be hollow. This is formed within the region of the stoma and is attached posteriorly to an extension which is fused to the dorsal wall of the pharynx and serves for attachment of the protractor muscles. The onchiostyle is used as a pick to penetrate cell walls and no food passes through its structure in contrast to the hollow odontostyle and stomatostylet of the dorylaims and tylenchs/aphelenchs respectively. Onchiostyle length varies from about 25µm to 150µm. The oesophagus comprises two parts: a narrow, tubular anterior section and a posterior, poorly muscular bulboid expansion which accommodates the oesophageal glands; bulb lumen and orifi of glands obscure. The shape of the oesophagus is noticeably different to that of dorylaims, the posterior section gradually expanding to form a spathulate bulb as opposed to the cylindrical form in the latter group The vulva is median to postmedian (V = 50-60%) in Trichodorus and Paratrichodorus, but far posterior in Monotrichodorus (V = 75-86%) and Allotrichodorus (V = 80-90%). The lips are non-protuberant and the vulval aperture takes the form of a small pore or a slit, either transverse or longitudinal, and often located in a shallow depression. The vagina may be relatively short and with weakly developed, inconspicuous musculature, as in Paratrichodorus or extending further into the body with thicker walls and strong musculature as, for example, in Trichodorus. Distally, at the junction of the vulva and vagina, there is a cuticularized ring which may be weak or strong in development and which, in lateral view, appears as two, variously shaped, cuticularized bodies, one anterior and one posterior to the vaginal lumen. The male genital tract is monorchic and outstretched. The spicules are paired, separate and either ventrally arcuate, or modified therefrom, as in Trichodorus, or more or less straight with the distal tips curved ventrad. In Trichodorus, where a bursa is lacking, the copulatory muscles extend anteriorly for several spicule lengths and, on contraction, produce the typical J-shaped heat-relaxed form. Conversely, in Paratrichodorus the bursa is well developed, the muscles extend for less than a spicule length and, as a consequence, the heat-relaxed male dies more or less straight. A bursa may be absent, as in Trichodorus, well developed as in most Paratrichodorus or merely represented by a flattening and thickening of the ventrosublateral cuticle of the cloacal region. Bionomics: Trichodorids are polyphagous migratory ectoparasites of the roots of perennial and woody plants. Interest in the biology of trichodorids increased markedly in the sixties and seventies when they were shown to be capable of transmitting plant viruses. Root pathology includes browning of epidermal tissues and cessation of root elongation ( stubby root disease). Cortical cells are only fed upon if made accessible by root cracking or collapse of epidermal tissues as a result of a mass attack. Cytoplasmic streaming to the feeding site occurs even after onchiostyle withdrawal. Cells can recover if the nematode abandons a site soon after penetration, thus allowing the possibility of virus transmission from infected vectors), their major pest status stems from their known ability to transmit three tobraviruses: tobacco rattle virus (TRV); pea early browning virus (PEBV) and pepper ring spot virus (PRV). TRV is currently known from Europe, Japan and USA, 109

110 PEBV only from Europe and PRV only from South America. Virus particles are selectively adsorbed on the lining of the oesophagus and dissociation occurs as the nematode saliva is injected into the host, although the root cell must not be killed or damaged if transmission is to be successful. All juvenile stages can vector the viruses, but need to be reinfected after each moult as the virus retaining cuticle of the oesophagus is sloughed. Adult vectors can remain infective for long periods. Trichodorids appear to be most widespread and abundant in light sandy soils although this does not preclude their occurrence in heavier soils. They appear to be highly susceptible to mechanical damage and as a result are usually found below the depth of cultivation. SUPERFAMILY TRICHODOROIDEA THORNE, 1935 (SIDDIQI, 1974) Diagnosis: Body mm long, plump, females cigar-shaped, males straight or J- shaped on heat relaxation. Cuticle thick, smooth; may swell abnormally on death. Amphidial apertures wide, gaping ellipses; sensilla sac separated from the fovea only by a constriction. Onchiostyle distally solid, dorsally convex, attached to the dorsal wall of the pharynx. Simple anterior guiding ring present. Oesophagus consisting of a narrow anterior section expanding into a posterior bulboid section of spathulate or pyriform shape containing five glands - one dorsal, two anterior ventrosublateral and two posterior ventrosublateral. Distinct excretory pore present, located either within the oesophageal region or slightly posterior. Prerectum absent. Vulva small, median to slightly postmedian or more posterior in position depending on whether the genital system is amphididelphic, reflexed, or monoprodelphic, reflexed. Uterus a simple tube; oviduct consisting of two cells. Spermatheca present or absent. Female anus almost terminal. Male genital tract monorchic, outstretched. Spicules straight or curved with the spicule protractor muscles forming a capsule around the proximal half of the retracted spicules. Bursa present or absent. Tail very short and rounded. Type genus: Trichodorus Cobb, 1913 Type family: Trichodoridae Thorne, 1935 (Siddiqi, 1961) Other families: Monotypic. Family TRICHODORIDAE Thorne, 1935 (Siddiqi, 1961) Diagnosis: Trichodoroidea. With the characters of the superfamily. Type genus: Trichodorus Cobb,

111 Keys to genera Female: 1. Female genital tract amphididelphic...2. Female genital tract monoprodelphic Vagina extending halfway into body, musculature well developed; lateral body pores within one body width of vulva; cuticle not abnormally swollen on fixation...trichodorus Vagina extending for up to a third of the corresponding body width; no lateral body pores within one body width of vulva; cuticle abnormally swollen on fixation...paratrichodorus 3. Lateral body pores present near vulva...monotrichodorus Lateral body pores absent...allotrichodorus Male: 1. Diagonal copulatory muscles extending far anterior to head of retracted spicules. Tail region sharply curved (habitus J-shaped) on heat relaxation...trichodorus Diagonal copulatory muscles either within the range of the retracted spicules or only extending about one body width anterior to spicule head. Tail region almost straight and not sharply recurved on heat relaxation Three ventromedian copulatory supplements more or less within the range of the retracted spicules, bursa present...allotrichodorus Only one or two ventromedian copulatory supplements within the range of the retracted spicules, bursa present or absent Spicule protractor capsule weakly developed, bursa usually prominent, diagonal copulatory muscles within range of the retracted spicules...paratrichodorus Spicule protractor capsule prominent, bursa absent, diagonal copulatory muscles extending just anterior to retracted spicules...monotrichodorus Genus TRICHODORUS Cobb, 1913 Diagnosis: Trichodoridae. Body plump, cylindrical with rounded ends. Heat relaxed females die ventrally arcuate, the males J-shaped with the tail region more sharply curved ventrad. Cuticle not swelling strongly on fixation. Oesophagus consisting of a narrow anterior section which expands posteriorly to form a spathulate bulb. Bulb usually non-overlapping, but in some species a ventral overlap develops whereas in others the 111

112 intestine extends dorsally along the bulb to form an overlap. Posterior ventrosublateral nuclei located anterior to the oesophago-intestinal junction and with the dorsal nucleus usually at the same level. Vulva a median pore or a transverse or longitudinal slit. Vagina extending into the body for about half the corresponding diameter. Vaginal musculature well developed and prominent and sclerotization usually strong. Genital tract amphididelphic reflexed; spermatheca present, although weakly developed in a few species. Anus subterminal; tail rounded. Spicules more or less ventrally arcuate, never straight; either smooth or with various ornamentations, bristles, etc. Gubernaculum present. Bursa absent (but the lateral cuticle may appear as a bursa T. cylindricus). Tail short, rounded, with one pair of ventrosublateral papillae and a pair of caudal pores. Type species: T. obtusus Cobb, 1913 (species inquirenda) Other species: About 50 species have been described and are currently regarded as valid. Bionomics: Migratory polyphagous ectoparasites of plant roots found mainly in sandy or sandy-loam soils in temperate regions. In addition to causing stubby root symptoms from their direct feeding activities, some species are known to be virus vectors and are of considerable economic importance in Europe and the USA. Distribution: Almost world-wide (Africa, Asia, Australia, Europe, North America, Central America), but predominantly in the more temperate regions of Europe and North America. Some of the present distribution is probably due to nematodes being introduced with nonindigenous plants. Genus PARATRICHODORUS Siddiqi, 1974 Diagnosis: Trichodoridae. Body plump, cigar-shaped, heat relaxed form more or less straight in both sexes. Cuticle swelling strongly after heat relaxation and/or acid-fixation. Lateral body pores present in about half the species, but typically not present within a body-width of the vulva (reportedly present in four or five species). Onchiostyle dorsally convex. Oesophagus consisting of a narrow anterior section expanding posteriorly to form a bulb which usually overlaps the intestine ventrally. Posterior ventrosublateral nuclei located near to the oesophago-intestinal junction, the dorsal nucleus usually being near the oesophageal enlargement. An extension of the intestine along the dorsal side of the bulb may be present or absent. Vulva a minute median pore or small transverse or longitudinal slit. Vagina extending into the body for up to one third the corresponding diameter, musculature weakly developed and inconspicuous; sclerotization poorly developed. Genital tract amphididelphic, reflexed. Spermatheca present or absent. Anus subterminal, tail rounded. Males lacking or rare in about 40% of the nominal species. Spicules normally straight; transversely striated, except at the extremities; gubernaculum present. Bursa present, but may be inconspicuous. Tail short, rounded. 112

113 Type species: Paratrichodorus tunisiensis (Siddiqi, 1963) Siddiqi, 1974 Bionomics: Migratory polyphagous ectoparasites of plant roots. In addition to causing stubby root symptoms from their direct feeding activities, some species are known to be virus vectors, but information on the vast majority of species is lacking. Distribution: World-wide, but mainly in tropical and subtropical regions. The genus may well have been introduced relatively recently to Central and South America with nonindigenous plants. REFERENCES AND ADDITIONAL READING Decraemer, W. (1991). Stubby root and virus vector nematodes: Trichodorus, Paratrichodorus, Allotrichodorus and Monotrichodorus. In: Nickle, W.R. (ed.), Manual of agricultural helminthology, Marcel Dekker, New York, pp Decraemer, W. (1995). The family Trichodoridae: Stubby root and virus vector nematodes. Kluwer Academic Publishers, 360pp. Hunt, D.J. (1993). Aphelenchida, Longidoridae and Trichodoridae: Their systematics and bionomics. CAB International, Wallingford, UK, 372pp. Siddiqi, M.R. (1974). Systematics of the genus Trichodorus Cobb, 1913 (Nematoda: Dorylaimida), with descriptions of three new species. Nematologica, 19: Siddiqi, M.R. (1980). On the generic status of Atlantadorus Siddiqi, 1974 and Nanidorus Siddiqi, 1974 (Nematoda: Trichodoridae). Systematic Parasitology, 1: Siddiqi, M.R. (1983). Phylogenetic relationships of the soil orders Dorylaimida, Mononchida, Triplonchida and Alaimida, with a revised classification of the subclass Enoplia. Pakistan Journal of Nematology, 1: Siddiqi, M.R. (1992). Studies on plant-parasitic nematode genus Monotrichodorus, with descriptions of three new species from South America and Paratrichodorus paramirzai sp. n. from India. Afro-Asian Journal of Nematology, 1:

114 CHAPTER 4 MANAGEMENT OF PLANT PARASITIC NEMATODES J.W. Kimenju, Dept. of Plant Science and Crop Protection, University of Nairobi, P.O.Box 30197, Nairobi, Kenya INTRODUCTION It is estimated that plant parasitic nematodes account for about 12% yield losses on a worldwide scale. The losses can be both quantitative and qualitative in nature. In response to the losses, nematologists and growers have developed several strategies for their control and management. The terms pest control and pest management are often used interchangeably but they have different meanings. Control can be defined as an effort to kill unwanted organisms at a particular time or place. It implies a specific act or a few acts within a limited time frame leading to a marked reduction in either the pest population or the damage caused by the pest Management, on the other hand can be defined as the act of managing, including the whole system of prevention and treatment of a pest or disease. Management implies several pest control tactics in concert over an extended period of time. Control can refer to those specific tactics which are applied to reduce or eliminate nematode populations while nematode management can be reserved for those efforts employed to reduce the numbers of nematodes to non-injurious levels characterized by the use of multiple control procedures. The main principles guiding nematode management programs are: (i) (ii) (iii) (iv) (v) (vi) (vii) Most plant parasitic nematodes have a wide host range. Dispersal of plant-parasitic nematodes is usually passive but may be active or aided by vectors. The principal dispersal agents of plant parasitic nematodes are water, man, wind and arthropods. Soil, water and plant residues are the main reservoirs of plant parasitic nematodes Knowledge of the factors that affect nematodes is invaluable in their management. Control of nematodes induced diseases is usually directed at inhibition of the nematodes themselves. Control strategies should be more preventive rather than curative and aimed at preventing build-up of high population densities. (viii) Sustainable management of plant parasitic nematodes requires that all viable strategies be combined into integrated pest management packages. 114

115 The knowledge that the initial population density will determine the expected yield is, in many cases, the reason to reduce the initial population level. Some plant parasitic nematodes, however, have such a high multiplication rate that even low densities can cause remarkable damage. For such nematodes one has to try to reduce the number of infected plants by eliminating the infection sources and by therapy of infected plants. The reduction of nematode population levels can be obtained by the following methods: (i) Killing the nematodes by starvation. (ii) Directly killing the nematodes by a chemical or any other technique applied before the crop is sown or planted. (iii) Using chemicals that prevent the nematodes from feeding. Sustainable management of plant parasitic nematodes can be achieved through an integration of different tactics that fall into five broad strategies: - (i) (ii) (iii) (iv) (v) Preventing introduction and spread of nematodes Cultural practices, particularly cropping systems, fallowing, resistant cultivars and organic amendments. Physical agents especially heat Chemicals (nematicides) Biological control PREVENTING INTRODUCTION AND SPREAD OF NEMATODES Exclusion is the first control to consider in nematode management. It is easier to deal with nematodes before they become established in agricultural fields than it is to subsequently eradicate or manage them. Many nematode species are carried in seed or propagating stock (endoparasites); others can be present in soil adhering to tools or farm machinery. The speed at which nematodes can establish themselves in an agricultural field depends on their initial population and the frequency at which suitable host plants are grown in the same field. Polyphagous (wide host range) nematodes are usually favoured in most situations where different hosts are alternated. Prevention of nematode spread can be considered at different levels; the farm, the nation and the world. At farm level, a judicious choice of the propagation material must be the basis for each crop. For this reason the multiplication sites (nurseries) should be established on unsuspected or disinfested land. If the production of nematode free plants appears to be impossible, one must apply methods that disinfest the soil such as thermotherapy and chemotherapy. One of the ways nematodes are disseminated from one field to another is the soil adhering to farm machinery. In many instances, new infection sites are found at the points in the field where the farmer starts ploughing. Cleaning the farm machinery would prevent the fields from infection. Irrigation is 115

116 another way of nematode dissemination. Nematodes are spread by water in both the field and in greenhouses. Sedimentation of the nematodes in a basin or a reservoir can reduce their presence in the water and limit their spread. Filtration of irrigation water is also gaining popularity as a nematode management strategy especially in capitalintensive production systems. To protect the individual farmer and to limit the nematode presence to a restricted area, different countries restrict circulation of contaminated plant material. Legal measures aiming at the reduction of the infections of dangerous plant parasitic nematodes are frequent. At the international level, important phytosanitary problems are governed by quarantine regulations. Quarantine refers to regulatory actions aimed at preventing or retarding the introduction, establishment and spread of dangerous pests. Quarantine pests are those of potential national economic importance that are not yet present in the country or endangered region or that are present but not widely distributed. Consignments must be free of quarantine pests (Zero tolerance). The 10 most frequently cited nematodes in the quarantine list of 125 countries are (frequencies between brackets): Globodera rostochiensis (51), Ditylenchus dipsaci (23), Heterodera schachtii (16), Ditylenchus angustus (13), Aphelenchoides fragariae (13), Ditylenchus destructor (12), Radopholus similes (11), Meloidogyne javanica (11), Aphelenchoides ritzemabosi (11), and Aphelenchoides besseyi (9). Exclusion of plant material is confined to plants known to be hosts of pests or of biological races or strains of pests with extremely high risk for the importing country, and originating from countries where the pest is known to occur. Soil accompanying plant material is undoubtedly a serious quarantine risk. It is, therefore, prohibited by most quarantine regulations. Spot Treatment Spot treatment entails marking out spots of high nematode density and treatment of the same to reduce spread of the nematodes. The strategy is based on the fact that nematodes are hardly evenly distributed in a field. Treatment of the spots may be achieved through application of high amounts of organic substrates or chemical nematicides. CULTURAL PRACTICES Crop rotation Crop rotation is a simple nematode management method which also forms part of good agricultural practices. Plant parasitic nematodes are obligate parasites: they need a host plant for both their development and multiplication. Each species of phytonematodes has a range of hosts, which may be wide, but does not include all crop plants. Nematodes numbers increase on favorable and decline on unfavorable hosts. In crop rotation for management of a nematode species, susceptible crops are rotated with immune or resistant crops. The susceptible crop is usually the most profitable and the 116

117 rotation crops less profitable. A rotation should be planned so that the nematode population is at its lowest level when the principal or the most profitable and most susceptible crop is planted. For example, tomato is a profitable crop but susceptible to all the common species of Meloidogyne. After a tomato crop is harvested, the root-knot nematode population in the soil is usually high. A second tomato crop would be severely damaged. If the nematode species present is not M. hapla or race 1 of M. arenaria, peanuts can follow a tomato crop without the risk of damage. While the peanuts are growing, the nematodes cannot reproduce. Instead, many of the juveniles in the soil die or become non-infective because of starvation and the attacks of predators, fungi and other natural enemies. Examples of poor host crops that are commonly used in rotations to suppress root-knot nematodes include maize, onions, wheat, rhodes grass, and asparagus. Crop rotation is associated with major limitations. Non-host plants are not always interesting from the financial point of view and they may be unknown to the farmer. Sometimes rotation crops require special capital investments, which may not fit in the farm budget. For all these reasons, rotations must be well tested before their practice can be extended. Moreover, there is always a risk that rotations can enhance multiplication of nematodes that were not hazards to the main crop. Early planting: Plant parasitic nematodes are mainly harmful in the early stages of the crop, when the root system is not well developed. In the temperate region growers can sow or plant in cool periods, so the crop gets started before the nematodes are active. By the time the soil warms and juveniles of cyst nematodes (G. rostochiensis, G. pallida and H. schachtii) invade growing roots, plants have already accumulated reserves and can withstand attack. Continued early plantings, however, may select strains of plant parasitic nematodes adapted to the system. Although tropical conditions are generally unfavourable for the cyst nematodes, high altitude zones experience temperate-like climate and are therefore prone to cyst nematode colonization. Desiccation: Nematodes in an active state are very sensitive to desiccation. When transferred directly from water to an environment with low relative humidity or high osmotic pressure, they are killed in a few minutes. On the other hand slow drying of nematodes usually induces anhydrobiosis, increasing their resistance to adverse environmental conditions including dryness and toxic substances. In arid and semiarid areas, 80% mortality can be achieved by rapid desiccation of the soil over a short period of time. In such areas, ploughing at intervals of 2 4 weeks during the dry season can reduce populations of root-knot nematodes substantially. This exposes eggs and juveniles in roots and deeper layers of the soil to rapid desiccation and may be sufficient to significantly increase the yield of a subsequent crop. Intermittent irrigation during the period of desiccation improves nematode control, owing to the increased susceptibility of reactivated nematodes to desiccation. Flooding: Flooding of the soil cuts off its oxygen concentration to practically zero within one or two days. Carbon dioxide begins to increase, following flooding, due to reduction 117

118 by anaerobic bacteria. Other chemical changes in flooded soil are: denitrification, accumulation of ammonia, reduction of iron, manganese and sulphates and production of organic acids, methane and hydrogen sulphide. Flooding has been practiced as an economical method of nematode control in bananas grown on peaty clay soil in Surinam. In that country, banana fields become heavily infested by Radopholus similis after 4-5 years. The banana plants are then destroyed and the fields are flooded for 4 5 months and then replanted with hot-water-treated rhizomes. Soil disinfestation by flooding is mainly caused by an indirect effect due to an increase of toxic substances produced by anaerobic microbiological activity in the soil, rather than by the direct effect of lack of oxygen. Flooding is restricted in adoption because it is only viable in the valley bottoms and in regions that are endowed with enormous water resources. It may also be used to explain the general decline in nematode numbers after excessively wet seasons. Soil amendments: Many substances can be added to soil to increase organic matter: manure from domestic animals, sewage sludge from municipal waste disposal facilities, crop residues after harvest, and cover crops. Products obtained after the processing of agricultural products are also suitable for incorporation into the soil: cottonseed hulls, and oil cakes. Organic soil amendments are known to improve the structure and water holding capacity of the soil. Plants developing in a substrate rich in organic matter usually grow more vigorously and thus tolerate damage by harmful organisms including nematodes. Breakdown of organic matter releases compounds that may be toxic to nematodes. In particular, decomposing residues of plant tissues release simple organic acids such as acetic, propionic and butyric acids. These may remain for several weeks in concentrations sufficient to kill some plant parasitic nematodes with little or no effect on free-living species. In addition, organic substrates stimulate build-up of indigenous microorganisms some of which are antagonistic to phytonematodes. For instance, addition of chitin to the soil, derived from crustaceans, stimulates the growth of actinomycetes, some of which are antagonistic to plant parasitic nematodes. It also stimulates increase of fungi with enzymes capable of digesting chitin. Many of these fungi penetrate cyst nematodes to attack chitinous walls of eggs. During degradation of organic matter by microorganisms, toxic metabolites that kill nematodes are released. The lower efficacy of amendments as compared to nematicides is outweighed by their cheapness and relative availability. However, amendments are usually bulky and need to be applied in large quantities. That makes them more appealing to small-scale farmers who have limited financial resources but with access to locally produced materials. The most widely studied materials in East and southern Africa include crop residues, animal manures (chicken, cow, goat, and pig manures), compost, green manures and industrial wastes such as sawdust, sugarcane bagasse, and molasses. 118

119 Antagonistic Plants Several plant species have been characterized as being antagonistic to plant parasitic nematodes. The most intensively studied include marigolds, asparagus, sunnhemp, mustard and neem. The antagonism results mainly from release of root exudates that have nematicidal properties. Nematode-antagonistic plants have shown high potential in nematode management when used in sequential or multiple cropping systems. Available information shows that marigolds and sunnhemp are more effective than fallow in root-knot nematode suppression. Over 50% decline in juvenile numbers has been reported in fields left under marigolds for three months. Wide-scale usage of the plants is, however, restricted because most of the plants have little or no market value. When used as companion crops, most of them exhibit a strong weed effect resulting in reduced yields of the principal crop. PHYSICAL METHODS Like all organisms, nematodes have limited resistance to physical stresses exerted upon them. Physical control methods exploit this quality. However, nematodes are usually well protected in host tissues or soil. Therefore physical methods of control require high energies. Moreover, there is only a small difference in heat tolerance between plants and nematodes. Control by heating Nematodes are very susceptible to heat. Few nematodes resist temperatures higher than 60 0 C for a duration of 30 minutes. Soil disinfestation by means of vapour has been practiced in intensive crop production systems for almost a century. It is still the most appropriate way to transfer heat to the soil, because the intensity is relatively low (100 0 C) and quantity is high. Heat distribution is relatively good. The steam moves as water vapour to the point where it is needed and then condenses on soil particles cooler than itself. Sheet steaming is widely used because of its low capital and labour cost. In this system, steam is blown under a plastic sheet 0.25 mm thick, anchored at the edges by sand bags or chains and left for at least eight hours to penetrate into the soil. The drier the soil is, initially, the more condensed water it can absorb and the deeper the heat can penetrate. Steam penetration is slower in loam than in sandy soils, therefore one is advised to cultivate the soil before steaming. To improve heat penetration to the deeper soil layers, a permanent system can be developed in which the steam is blown in through pipes buried at a depth of 60 cm, and left to move to the soil surface. Comparing different steaming techniques, Dutch researchers obtained the best results with negative pressure steaming. With this method, steam is blown under a plastic sheet and pulled into the soil by negative pressure, created in the soil by an exhaust fan, which sucks the air out of the soil through buried perforated drainpipes at a depth of 60 cm. Most plant parasitic organisms are destroyed at a temperature of 60 0 C for 30 minutes. Higher temperatures induce chemical changes in the soil, which may have adverse effects on subsequent growth. The release of an excessively large amount of manganese may 119

120 cause toxicity in crops, especially in soils with low ph. Plant growth may also be affected by an increase in levels of nitrite and ammonium nitrogen after steam sterilization. Moreover, at high temperatures most of the soil microflora is destroyed, creating a biological vacuum that may support the rapid development of residue and re-contaminating organisms. Soil Solarization The technique of soil solarization was developed in Israel. The field is tilled to fineness and irrigated in mid-summer and while soil moisture is still at or just below field capacity, a clear thin polyethylene sheet is then spread over the soil and its edges buried. The sheet is left undisturbed for periods ranging from two to nine weeks depending upon several factors including the level of solar radiation. The polyethylene sheet is removed at the end of the solarization period and the field is available for normal use. Irrigation can be done simply by flooding or by sprinklers or underground drip irrigation systems. The choice of the mulching material is important. The material should have a high transparency to permit short-wave solar radiation to reach the soil, but it should be impermeable to long-wave radiation (greenhouse effect) and to water vapour and gases leaving the ground. Transparent mulching has been found to be more effective than black, since it transmits most of the incident radiation to the soil, whereas the black polyethylene tends to heat up. The heating of soil under polyethylene is generally less near the edges of the plot. Thus it is preferable to treat larger plots with continuous sealed mulch. The optimum period of solarization depends on factors such as the quantity of solar radiation available locally, the soil characteristics, the type of mulching materials, the thermal sensitivity of the target organism and the available time in the cropping system for solarization. The principles of solarization include:- (i) Accumulation of heat in polyethylene mulched soil by transmission of short wave solar radiation and prevention of loss of long wave radiation from the soil. (ii) High soil moisture improves the thermal conductivity of the soil. (iii) Greater thermal sensitivity of hydrated than of desiccated organisms. (iv) Shift of the biological equilibrium in favour of the natural enemies of plant pathogens. (v) Production of toxic gases and release of toxic mineral ions. (vi) Prolonged exposure to high temperature kills or weakens the pathogen, rendering them more vulnerable to their natural enemies. (vii) Water vapour condensing under the mulch reduces heat loss and may concentrate solar radiation. (viii) Control of weeds that serve as alternative hosts of nematodes and other pathogens. Soil solarization can be combined with other methods of control. It has been observed that control of nematodes with combinations of solarization and either 1, 3- dichloropropene, ethylene dibromide, metham sodium, ethoprophos or formaldehyde is better than with any of these treatments alone. Covering of soil with clear polythene is a 120

121 mandatory step after application of metham sodium to reduce escape of gases from the soil. It should be noted that the polythene is too expensive for use by most small-scale farmers in East and southern Africa. The method has high potential in treatment of small areas such as nurseries where disease free planting materials are produced. Disinfestation by heating with electricity Under intensive horticultural production in greenhouses, soil may be heated using buried electrical resistances. Observations have demonstrated that when the soil temperature is kept at 50 0 C for one hour, root-knot nematodes are effectively controlled. The costs are reduced when the heating takes place in the warm season. Although the method is restricted in adoption due to the cost of energy, it has practical value in nurseries producing high value planting materials especially for export. Such high value produce include chrysanthemum flowers, carnations and roses. Field burning The most primitive way of heating soil is by burning stubble over it. However, experiments have showed that burning of dry leaf litter, 10 cm thick, killed root-knot nematodes to a depth of only 9 cm. In some cases, burning of straw and stubble after harvest may be effective in protecting the next crop from severe attack especially by foliar nematodes. Burning of crop resides is, however, disastrous because it denies the soil muchneeded organic matter. It also poses environmental hazards, coupled with the risk of fire spreading to non-target areas. As earlier stated addition of organic substrates into the soil helps to establish biological control against nematodes and also improves plant growth as a result of enhanced nutrients and water holding capacity. Disinfestation of planting material using heat Hot water treatment has been successfully used in the control plant parasitic nematodes. The treatment is useful as a preventive control measure in propagating material (seeds, rootstocks, corms, tubers, rhizomes) that might harbour endoparasitic nematodes. The chances of success in hot water treatment increases as the difference between thermal sensitivity of the host and nematodes increases, with the latter being more sensitive. The smaller the difference is, the more accurately the temperature and time of the treatment must be controlled. It is good to note that both plants and nematodes are less susceptible to high temperatures when they are at dormancy or dehydrated. To improve the efficiency of hot water treatments it is necessary to displace trapped air by presoaking the propagules in water, since the air acts as an insulator. Presoaking for about two hours before hot water treatment is a common practice to control Aphelenchoides besseyi in rice seed. Hot water treatment of rhizomes of bananas to control Radopholus similis is improved by peeling all necrotic tissue from the corms before treatment. Control by irradiation The negative consequences of UV, gamma and X-rays on nematode reproduction, motility and morphology are well documented. Gamma-rays have multiple effects which may include sterilization, delayed gonadal growth, delayed egg-hatching and morphological 121

122 abnormalities. The movements of the nematodes are also known to become sluggish. The irradiation effects depend on the developmental stage of the nematode and also on both the irradiation doses and the nematodes species. Eggs may develop normally after a UVtreatment but the juveniles hatching from irradiated eggs exhibit reduced growth and reproduction is usually inhibited. Increasing doses of UV cause an increase in mortality of the second stage juveniles The use of UV-, X- and gamma rays for soil and substrate disinfestations appears to be impractical because the soil and substrate acts as a buffer and absorbs most of the liberated energy. The irradiation of nematode infected plants is also not feasible because plants are more susceptible than nematodes. Ionizing rays can, however, be applied in disinfestation of nutrient solutions used in hydroponic-type systems. A small UV-unit can treat about 2500-liter water per hour. The technology is no doubt beyond the reach of small-scale growers especially the subsistence farmers. It can be recommended for adoption in hightechnology flower production systems. Resistance Growing resistant cultivars provides an ideal method for maintaining nematode population densities below damaging levels. Resistant cultivars have several advantages over other methods applied nematode management: (i) It can completely prevent nematode reproduction, unlike some of the alternative methods of control such as crop rotation. (ii) Their use requires little or no additional technology and is cost effective. (iii) It allows rotations to be shortened. (iv) Adoption of resistant cultivars is not associated with any toxic residues. For some low-value crops, breeding resistant cultivars is probably the only practical longterm approach to nematode control. This is also true for high-value crops, which are very expensive to establish and / or maintain with frequent nematicide treatments. Besides their resistance to plant parasitic nematodes, resistant cultivars need to be tolerant; those that are intolerant suffer extreme damage if grown in heavily infested soil. Tolerant cultivars that are not resistant tend to increase the nematode population densities to damaging levels. Resistance Resistance describes the effects of host genes that restrict or prevent nematode multiplication in a host species. Tolerance of damage is independent of resistance and relates to the ability of a host genotype to withstand or recover from the damaging effects of nematode attack and yield well. Resistance induced by the nematode depends on the nematode biology. Ectoparasites generally have a necrotrophic relationship with the plant. The injected saliva liquefies the cytoplasm that accumulates around the stylet tip and is rapidly ingested, usually killing the host cell. The nematode then moves to a new cell and repeats the process. Such behaviour limits the possibilities for effective induced resistance. Migratory endoparasites may have 122

123 a necrotrophic type of feeding; in some species, however, the feeding is partly biotrophic as it involves the induction of favourable changes in cells adjacent to the feeding site. Resistant cultivars are available to species within this group, especially the stem and leaf parasites. Sedentary endoparasites are obligate biotrophs. Nematodes within this group are the most damaging to plants and resistance to them is widespread. Tolerance Cultivars of a crop are regarded as differing in their tolerance if the decrease in their growth and / or yield due to damage by nematodes differs significantly when they are grown in uniformly infested soil. Where cultivars of different yield potentials are being compared, the proportional effect on yield must be compared in adjacent, uniformly uninfested and infested conditions. Resistance and tolerance are independent attributes of plants but resistance may confer tolerance especially if it decreases the incidence of nematode attack or parasitism. Equally, mechanisms of tolerance exist that are independent of resistance. Tolerance is probably widespread and important in wild plants but is likely to be lost during crop breeding. Various mechanisms of tolerance have been suggested. They include: differences in numbers of roots, compensatory root growth, delayed senescence and enhanced water uptake. Tolerance is independent of resistance and it appears to be largely nonspecific. The use of tolerance is a valuable strategy for many crops or situations where alternative control measures are not available. However, tolerance of cyst and root-knot nematodes generally needs to be combined with a degree of resistance, otherwise populations will be increased to densities where even tolerant cultivars are damaged. Susceptible and tolerant genotypes may, however, be useful in rotations following resistant genotypes as a means of reducing the rate of selection of virulence. BIOLOGICAL CONTROL Many natural enemies attack plant parasitic nematodes in the soil and reduce their populations. They include bacteria, rickettsia, fungi, protozoa, tardigrades, tubellarians, nematodes, enchytraeids, mites, and insects. It is important to determine the nature and extent of such attacks on nematode multiplication in order to establish whether these enemies can be exploited to reduce damage and increase crop yield. There are two types of biological control: induced, where the biological control agent has been applied by man, and natural, where the agents have increased to suppress nematode multiplication without being specifically introduced. The term suppression is used to indicate a reduction in the numbers of nematodes not simply a reduction in disease symptoms; it may be general or specific if only one or two organisms are involved. Biological control should not be considered as a replacement for the chemical control. Growers that require rapid kills when their fields are found to be heavily infested have few 123

124 alternatives to chemical treatment. Biological control is slow acting and cannot fulfill this requirement. It, therefore, fits very well in an integrated nematode management system. Introduced organisms that must become established in soil and spread are likely to be affected greatly by environmental conditions. Control is also density-dependent, with a greater proportion of nematodes escaping attack when they are at low densities. Hence, rarely do natural enemies eradicate pests. Equilibrium populations are established that should be below the economic threshold for damage to the crop. Natural enemies as biological control agents Pasteuria penetrans Pasteuria penetrans is an obligate parasite of some plant parasitic nematodes. Nematodes become infected in soils when they get in contact with the endospores, which adhere to their cuticle. For instance, infected second -stage Meloidogyne juveniles enter roots and begin feeding before the spores germinate. A germ tube penetrates the cuticle and gives rise to a vegetative microcolony that fragments and proliferates throughout the nematode body cavity. Eventually females become filled with spores and egg production is prevented. Pasteuria penetrans is very virulent and has reduced Meloidogyne populations, in pots, by 99% in only three weeks. Pasteuria penetrans can survive several years in air-dried soil apparently without loss of viability and is little affected by soil conditions or a range of nematicides. Spores adhere to many plant parasitic nematodes, but only 20 30% germinate, and attachment may not necessarily lead to infection. The concentration of spores and the period of nematode activity in soil, therefore, determines the number of nematodes killed. P. penetrans spores are non-motile and are spread in soil chiefly by water movement and by tillage practices. Although P. penetrans appears to have considerable potential as a biological control agent, its commercial exploitation is prevented by lack of methods for large-scale production. Figure 1. Root knot nematode juvenile encumbered with spores of P.penetrans Nematode trapping fungi Fungi that produce adhesive network traps are good saprophytic competitors and grow rapidly in vitro but are less efficient at trapping nematodes than some that are slower growing and capture their prey on adhesive knobs and branches or in constricting rings. 124

125 Most trapping fungi do not seem capable of rapid colonization and are considered poor competitive saprophytes that do not readily establish when added to soil. A carbohydrate source other than nematodes is necessary to support mycelial growth. Different species of trapping fungi vary in their ability to capture nematodes, but there is little evidence of specificity for particular prey species, and once traps are produced, most types of nematodes are caught (Fig. 2). The limited trapping activity and its non-specific nature have meant that these fungi are difficult to exploit as control agents, and control has been inconsistent. A commercial strain of Arthrobotrys irregularis has been produced on rye grain and marketed as Royal 350. It is recommended that the fungus be applied at 1.4 t/ha at least 1 month before planting and incorporated into the soil. Reduced root galling caused by Meloidogyne spp. and increased tomato yield has been reported. Arthrobotrys robusta, sold under the trademark Royal 300, is used for the control of nematodes damaging mushrooms (Ditylenchus myceliophagus). It is introduced into the compost on rye grain at a rate of 1% with the mushroom spawn. In one trial, Royal 300 increased yield by 20% and reduced the final populations by 40%. A A B B C D Figure 2. Mycelial network of a nematode trapping fungus, Arthrobotyrs oligospora (A), nematodes trapped by the fungus (B & C) and a nematode trapped in adhesive mycelial columnar of Monacrosporium sp (D) 125