Mycorrhizal Technology for Reclamation of Saline Waste Land of Indian Thar Desert

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2 Mycorrhizal Technology for Reclamation of Saline Waste Land of Indian Thar Desert By DR.NISHI MATHUR Head, Department of Biotechnology Mahila P.G.Mahavidyalaya Kamla Nehru Nagar, Jodhpur, Rajasthan,INDIA 2016 InternationalE - Publication

3 International E - Publication 427, Palhar Nagar, RAPTC, VIP-Road, Indore (MP) INDIA Phone: , Mobile: contact@isca.co.in, Website: Copyright Reserved 2016 All rights reserved. No part of this publication may be reproduced, stored, in a retrieval system or transmitted, in any form or by any means, electronic, mechanical, photocopying, reordering or otherwise, without the prior permission of the publisher. ISBN:

4 DEDICATED TO MYBELOVED PARENTS Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert iii

5 CONTENTS S.NO. NAME OF CHAPTER PAGE NO. 1. INTRODUCTION MATERIAL AND METHODS RESULTS DEVELOPMENT OF ARBUSCULAR MYCORRHIZAE DISCUSSION SUMMARY REFERENCES Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert iv

6 Chapter 1 Introduction Land is facing serious threats of deterioration due to unrelenting human pressure and utilisation incompatible with its capacity. The information on land degradation is needed for a variety of purposes like planning reclamation programs, rational land use planning, for bringing additional areas into cultivation and also to improve productivity levels in degraded lands. Land degradation has numerous environmental, economic, social and ecological consequences. There can be rather serious effects in terms of soil erosion, loss of soil fertility and thus reduced plant growth or crop productivity, clogging up of rivers and drainage systems, extensive floods and water shortages. It is estimated that some forms of land degradation constituting 75% of the earth s usable landmass affect 4 billion people in the world. About 15% of the world population is effected by land degradation which is likely to worsen unless adequate and immediate measures are taken to arrest the degradation processes. The largest category is land affected by water and wind erosion, which account for 80 percent of degraded followed by salinization / alkalization and waterlogging. Reliable time series data are available only for salt affected land, which has grown from 7.18 million hectares in 1987 to over 10 million in 1993 (Annon, 2002). According to NRSA / DOS project on Mapping of salt affected soils of India on 1:250,000 scale, the area under salt affected soils in the country is million hectares. An estimated area of 2.46 million ha land is suffering from water logging in irrigation commands in India (Anonymous, 1991). An area is said to be waterlogged when the water table rises to an extent that soil pores in the root zone of a crop become saturated, resulting in restriction of normal circulation of the air, decline in the level of oxygen and increase in the level of carbon dioxide. It 2 may result in various types of soil degradation like physical degradation or chemical degradation or salinity. Satellite data are being used regularly for mapping and monitoring of waterlogged Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 1

7 areas. Salt affected areas are one of the most important degraded areas where soil productivity is reduced due to either salinization( EC > 4 ds/m) or sodicity (ESP > 15) or both. The soils with EC more than 2 ds/m in black soils and >4 ds/m in non-black soils was considered as saline in the present project. Soils with soil ph more than 8.5 results in increase of exchangeable sodium percentage (ESP) in soils (> 15) and are termed as sodic. Based on the type of problem, it has been divided into saline, sodic and saline-sodic. Under NR Cenus project three types of salt affected soils viz., saline, sodic and saline-sodic are mapped using three seasons satellite data, field work and analysis of soil samples under three severity classes namely slight, moderate and strong. A.1 Saline soils These soils occurs in arid and semi-arid regions, coastal areas, irrigated commands and peripheries of streams in peninsular regions. The soil ph is usually less than 8.5 and EC is more than 4 ds/m. On satellite data it is seen in light grey to white with association of poor crop growth. In severe cases, there may not be any vegetal cover, even grass. A.2 Sodic soils Usually it occurs in the older alluvial plains. Because of high sodium content, soils will be moist during post-monsoon season which can be seen easily in the post-monsoon image. It appears on satellite data as grayish white / dull white discrete patchy. It occurs as 3 contiguous patches with smooth texture on the image. Multi temporal data set will help in delineation of affected areas and to some extent severity classes. The soil ph values will be more than > 8.5, and EC will be < 4 ds/m and ESP is greater than 15. A.3 Saline Sodic Soils The Saline-Sodic soils occur in arid and semiarid regions. It appears as grayish white with red and white mottle color on the image. Bright white tone, dominantly in Indo-Gangetic alluvial plains. In coastal plain it is creamy white color with mottle tone. The soil ph is greater than or equal to 8.5 and EC is greater than or equal to 4 ds/m. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 2

8 Chapter 2 Materials and Methods MYCORRHIZAL TECHNIQUES Collection Isolation of Spores Identification of Spores Staining Procedure for Root Percentage of Root Colonization Mass Multiplication of Inoculum Inoculation of Mycorrhizae in Seed Lings of Tree Species PLANT ANALYSIS Plant Height and Biomass Dry Weight Phosphorus Nitrogen Acid / Alkaline Phosphatase Nitrate Reductase Total Phenol Peroxidase & Polyohenol Oxidase MYCORRHIZAL TECHNIQUES Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 3

9 Collection During the present research investigation work rhizospheric soil of the Casuariana spp. were collected from various localities of Western Rajasthan namely Jodhpur Region Pali Region Udaipur Region Mount Abu Region The periodical survey of Western Rajasthan was undertaken in order to collect the rhizospheric soil as well as root samples. Root and Rhizosphere soil samples for plant species were collected from five individuals at different stages of growth (vegetative and reproductive). Care was taken during collection that roots of shrubs and tree species could be positively identified, so take them carefully without sample mixing. Root samples were washed thoroughly free of attached soil particles and cut into several small segments and stained within 24 hours or preserved in formalin-acetic acid alcohol upto six months before staining. Rhizosphere soil from roots and adjacent to plants were collected. Soil samples collected from different indivisuals of a species were mixed to form a composite sample. These composite soil samples were used for the isolation of VAM fungal spores and for soil chemistry. Isolation of Spores To Isolate Mycorrhizal spores from the soil many methods can be employed. Out of these, techniques three has been used in present investigation. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 4

10 1. Wet sieving and decanting technique: The soil samples collected were processed by wet sieving and decanting technique of Gerdemann and Nicolson (1963) to obtain spores. The details are as follows- 100 g soil was taken and mixed in luke warm water in a large beaker and the heavier particles were allowed to settle down. The suspension was then poured through a coarse sieve (710 µm) to remove large pieces of organic matter. The roots and organic matter on the sieve were washed with a fine jet of water from a squeeze bottle to ensure that all the small particles have passed through. The washings which have passed through the sieve were collected. The particles were resuspended by stirring several times and this suspension was decanted through 500 µm, 250 µm, 125 µm, 105 µm and 53 µm sieves respectively to retain the desiring spores. Each sieve was washed into separate small beakers and was examined in turn. Root pieces retained on the 710 µm sieves were examined for attached hyphae, spores and sporocarps under stereomicroscope. The organic matter from 250 µm sieve was examined for sporocarps and large spores. The 105 µm sieving yield most spores since their size range was between 100 µm and 250 µm and spores smaller than 100 µm often occurred in moths, trapped on the 105 µm sieve. Small detached spores were found on the 53 µm sieve. Spores were picked up with the help of plastic syringe and were mounted in polyvinyl alcohol lacto-glycerol (PVLG) (Koske and Tessier, 1983) and observed under stereo microscope for identification. The fungal propagules were used as primary inoculum for the pot culturing of different species. Spore Number and variability are counted by using grid-line intersect method, whatman filter paper No. 1(size, 11cm diameter) (By Gaur and Adholeya, 1994). Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 5

11 For clayey soil, which blocks the sieve by forming suspension, precipitate the particles in 0.1M sodium pyrophosphate. If the spore number is low, host baiting technique or trap pot culturing may be employed. A soil sample from the test soils are added to a sterilized greenhouse potting mix and planted with a suitable trap or bait crop. A mixture of a perennial grass and a legume is preferable. After 3 to 5 months the potting mix can be used for enumerating AM spores. 2. Sucrose Centrifugation: Wet sieved material processed with Sucrose Centrifugation method of Smith and Skipper, to obtain spores. The details are as follows- Take a suspension of spores with debries collected from the sieving in a 50ml centrifuge tube and make upto 35ml with distilled water. Centrifuge at 2000 rpm for 10 min. Filter the supernatant Suspend the pellet remaining after the first centrifugation in enough 2M sucrose solution and bring the volume to 35ml Stir it vigorously, centrifuge at 200 rpm for 10min., filter the supernatant and collect the spores. This technique gives a suitable and easy way to collect spores from soil sample even they present in low quantity in soil. 3. Alternative method for Ohm s technique: Wet sieved material processed with Alternative method for Ohm s technique of Menge, to obtain spores. The details are as follows- Transfer soil sieving to a blender and at high speed for 1 or2 min. This frees any spores attached to the roots, or in sporocarps or in the clay particles. Pass contents of blender through a fine sieve and wash the colloidal material thoroughly with a strong stream of water. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 6

12 Add 10 ml of 20% sucrose into a clean 50ml centrifuge tube, followed by 10ml of 40% and then 10ml of 60% sucrose into the bottom of the tube. Add ml of blended sieving onto the surface of the 20% sucrose layer. Centrifuge for 3 min. at 300 rpm. Remove debris which gathers at the 20-40% and / or 40-60% interfaces. Often the layer of spores is visible and can be removed without taking any of the debris which remained in solution. Rinse spores on a fine sieve with a strong stream of water to remove sucrose and collect the spores. Identification of Spores In the present investigation, different species of AM fungi were identified with the help of synoptic key of Trappe (1982), manual of Schenck and Perez (1987) and Marton (1988). For that species and genera of AM fungi were identified on the basis of morphology of their resting spores i.e. chlamydospores. Staining Procedure for Root VAM root infection consists of intra and intercellular hyphae and vesicals together with finely branched Arbuscules within the host cortical tissue. The anatomical feature characteristic of VAM infection cannot be seen unless the infected roots are suitably stained. To observe the VAM infection, two technique used. The details are as follows- 1. Method of Philips and Hayman (1970) Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 7

13 Roots are washed in tap water, but not vigorously enough to detach the external mycelium. Cut root into 2 cm segments Then root pieces simmered at about 90 0 C for minutes (depending upon hardness of the roots) in 10% KOH. KOH solution clears host cytoplasm and nuclei and readily allows stain penetration. Rinse root segments 4-5 times in tap water. After that, acidified root segments by immersing them in 2% HCl for 5minutes. Acid is poured off and stain is added viz. 0.05% trypan blue in lactophenol. Root segments are kept in stain overnight (covered). Stain was poured off, lactic acid: glycerol (1:1) was added and roots were kept over night in this liquid to destain the host tissue. The squashed roots were examined under the microscope. To observe hyphae, vesicles and arbuscules under light microscope the root pieces were mounted sealed with D.P.X. on glass slide temporarily in lactophenol or permanently in poly vinyl alcohol. The coverslip was pressed gently to make the roots flattened and observe under microscope for the infection. 2. Method of Brundrett et al., (1984) observation of arbuscules: For study of arbuscules in more clear way Brundrett et al., (1984) proposed a new method, which involves staining by chlorazol black dye. The detailed procedure of this method is as follows- Reagents- FAA, 10% KOH, 80% Lactic acid, Glycerin, 95% ethanol, fuchsin, Chloral hydrate. Chlorazol black E, Basic Preparation of mounting fluid- 20 g Chloral hydrate + 20 g Gum arabic + 20 ml Glycerine + 3 ml distilled water + 10 drops of basic fuchsin (0.3 g/10 ml 95% ethanol). Roots were washed in tap water and fixed overnight or stored in Formalin-Acetic Acid- Alcohol (FAA). Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 8

14 Rinsed with several changes of tap water to remove FAA. These were transferred into a 10% KOH solution in autoclave resistant jars. Roots were then sterilized in autoclave for 15 minutes at C. Samples with delicate roots may require shorter sterilizing time. They were rinsed with several changes of tap water followed by deionised water. Then, the roots were transferred into a staining solution consisting of equal volumes of 80% lactic acid, glycerin and distilled water with 0.1% chlorazol black E. Stain for 1 hour or longer at approximately 90 0 C. Staining solution was prepared several hours before use and undissolved particles were allowed to settle down. After decanting overnight in glycerin roots were mounted on slides using mounting fluid. Roots were examined under a light microscope. Percentage of Root Colonization The percentage of root colonization of AM fungi in the roots was calculated by Gridline intersect method of Giovannetti and Mosse (1980). The stained root pieces were spread evenly on a plastic petridish.. A grid of line was marked on the bottom of the dish to form 1 cm squares. To facilitate the observations, the roots were immersed in a solution of glycerol and water (1:8 v/v). The glycerol increases the viscosity of the medium and prevents excessive movements of roots. The roots were than observed under the microscope. The petri plates were moved first horizontally and than vertically along the grid line. Two observations were recorded simultaneously: (a) (b) Total number of roots intersecting grid lines. Total number of infected roots intersecting grid lines. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 9

15 From this the percentage of root colonization was calculated by using the following formula- Total Number of infected rootsintersecting grid line % Root Colonization x100 Total Number of Roots interesecting grid line When sufficient root pieces were not available, the slide method was followed. Root pieces 1 cm long were selected at random from a stained sample and mounted on microscopic slides in groups of ten. Presence of infection was recorded and percentage of infection was calculated. Mass Multiplication of Inoculum The pot trial was conducted at the Department of Botany, JNVU, and Jodhpur during Rain spring season of PVC pots of 18 cm diameter were filled (sterilized with alcohol) with sterile sand : sandy loam (1: 1 by volume) 3 kg/pot. Soil sterilized by autoclaving at 15-lbs/sq inch pressure, C for 40 minutes. (It was done twice with a day interval in between). The soil had 20 kg P 2 O 5 /ha (NH 4 F + HCl extractable) with a ph of 7.2 surface sterilized seeds of Cenchrus ciliaris and Sorghum vulgaris were grown in funnels and transplanted to pots after 30 days (Plate 2 b & c). Seven efficient strains of VAM fungi isolated from soil of different sites were separately placed 3 cm below the soil surface before sowing the seeds in funnel culture. Pots were watered regularly. They were neither allowed to dry nor were flooded. Pots were examined regularly for purity of the inoculum and were maintained through out the course of investigation. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 10

16 Since Cenchrus ciliaris and Sorghum bicolor is a perennial grass, so it was possible to maintain pot cultures regularly by cutting the aerial part of the plant time to time. Uninoculated plants were kept as control. The plants received 50 ml per pot of Ruakura nutrient solution (Smith et al., 1983) without P once in 30 days. The plants were harvested after 90 days. After harvesting, shoot and root s fresh weight and dry weight were recorded. Soil of pot culture is used for spore source for inoculum and further physiological and biochemical studies. Inoculation of Mycorrhizae in Seed Lings of Tree Species Inoculation of Mycorhizal spores done by three methods 1. Pellates mathods: (Menge and Timmer, 1982.) Use inoculum of VAM fungi consisting of ground granular crudely produced pot culture inoculums containing plant roots, mycorhizal spores and growth media such as perlite, peat mass, vermiculate, sand or soil for field inoculation. Air dry this inoculums to about 5-20% moisture. Prepare mycorhizal pellets by mixing 20 parts mycorhizal inoculums, 1 part autoclaved sedimentary clay (mean particle size, 16 um) and 1 part autoclaved tertiary sedimentary clay (mean particle 2-6 um) Add water until the mixture is malleable and could be rolled into pellets. Make pellats each weighing 1.4 g and use them before 28 days of storage. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 11

17 Prune the host plants to soil level. Remove the compacted soil mass from the pot and plunge it in water to save even the very fine roots. Chop the recovered roots and homogenize the roots and soil in a sterile blender. Examine for the presence of plants pathogens, mycorrhizal hyperparasites, cultural purity, spore number and spore maturation. Air dry the soil mixture to the point at which there is no free water. After drying, pack the culture in plastic bags and seal to prevent further drying. Store at 5 C o. 2. Direct Inoculation from Pot culture Inoculum: The inoculum in form of pot soils containing extrametrical chlamydospores and AMF infected roots pieces of Cenchrus ciliaris and Sorghum vulgaris was placed 4-5 cm below the soil surface before sowing. The seeds were sown and were kept in glass house under temperatures C. The seedlings were regularly examined for the mycorrhizal development. The samples were harvested on the requirement for further studies. 3. Production of alginate entrapped VAM inoculum: Sand and soil mixture containing azygospores and infected root segments (chopped) of Cenchrus ciliaris and Sorghum bicolor infected with VAm fungi grown for 90 days served as the mycorhizal inoculums. The inoculums were air dried and passed through 400 um sieve. To an aqueous suspension of sodium alginate (2%), 10% of the sieved sand: soil inoculums of the VAM fungus plus 2% of the carrier material (perlite, sorlite, talc, Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 12

18 vermiculite, kaolinite and bentonite were added separately and mixed using a magnetic stirrer. This mixture was passed through a sieve onto 0.1 M sterile calcium chloride solution to form beads (Plate 4 d) After 30 minutes the beaded inoculums was rinsed with tap water. Aportion of the alginate entrapped wet VAM inoculums was dried to surface dryness and stored at 4 o C. This formed the carrier based alginate entrapped wet VAM inoculums (Kropacek et al., 1989; Strllu and Plenchette, 1991). A portion of the beads were air dried for 5 days to form dry VAM alginate inoculums packed in polythene bags and stored at room temperature (32 ± 5 o ). The ph of the carrier materials used in the study was estimated by using a digital ph meter (substrate: water ratio = 1:10 w/v). The moisture content of the wet VAM beads was determined after drying to a constant weight. The number of propagules in the different carrier based alginate entrapped VAM inoculum was determined by the MPN method using four-fold dilution (Sieverding, 1991). The alginate beads were solubilized in 0.2 M sodium citrate solution (ph adjusted to 7.2) prior to carrying out the MPN test. The Propagule numbers in the dry VAM alginate beads was computed from the propagule numbers of the wet VAM alginate beads and its moisture content. A pot culture experiment was also conducted to know the effect of alginate entrapped VAM inoculums on the colonization of roots and growth of wheat as the host plant. The soil used for this study was an alfisol (Fine, Kaolinthic, isohyperthermic type, Kanhaplustalfs) of ph 7.2 with 2.4 mg available P/g (NH 4 F + HCl extractable) and an indigenous VAM population of 0.31 propagules/g of soil. Earthernware pots (18 cm deep x 18 cm diameter) were filled Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 13

19 with 3.5 kg of soil and inoculated with alginate entrapped wet and dry VAM inoculums (with perlite, soilrite, talc and vermiculite as carriers) and sand : soil inoculum of Glomus mossae at the rate of 300 prpagules per pot. Four Two seedlings were planted per pot. Each treatment had three replicates. The pots were arranged in a glass house (temperature 29 ± 2 o C) in randomized complete block design and watered whenever necessary. Fifty ml Rkura nutrient solution was added thrice (first application with P, 20 days after planting and the other two later applications without P, on 40 and 60 days after planting.) Observation on plant height, fresh weight and dry weight of shoot and bulb 90 days after planting. The plants were harvested 90 days after planting. Plant samples were oven dried at 60 o C to a constant weight to get plant biomass. Phosphorus and potassium content of the shoot and leaf samples were determined, by the Vanado molybdate phosphoric yellow colour method (Jackson, 1973) and flame photometric method respectively. Mycorhizal colonization pf the root was determined by the grid line- intersect method (Giovannetti and Mosse, 1980) after staining the roots with trypan blue ( Phillips and Hayman, 1970). The data obtained from the pot expermint was subjected to analysis of varience by randomized complete block design and treatment means were seprated by Duncan s multiple range (DMR) test (Little and Hills, 1978). PLANT ANALYSIS Plant Height and Biomass Dry Weight Plant heights were recorded in cm and plant dry weights were recorded after drying them in hot air oven at 80 0 C for 48 hours. Phosphorus Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 14

20 Total phosphorus was estimated by Vanadomolybdate method of Jackson (1973). Following reagents were prepared- Reagents: a. Ammonium molybdate - This solution was prepared by dissolving 6.25 g ammonium molybdate in 250 ml of distilled water. b. 5N Sulphuric acid ml of conc. sulphuric acid was taken and final volume was made up to 100 ml by adding distilled water. c. Stannous chloride mg of SnCl 2 was dissolved in 10 ml of conc. HCl by heating upto boiling. Volume was made up-to 25 ml by addition of distilled water. This solution was prepared freshly while performing experiments. d. Standard solution mg of KH 2 PO 4 was dissolved in 100 ml of distilled water. This was 100-ppm solution. From this stock solution of 100 ppm the stock was diluted to 10 ppm by adding distilled water in the ratio of (1:9) 1 ml stock and 9 ml distilled water. e. Triacid mixture - HNO 3, perchloric acid and H 2 SO 4 were mixed in the ratio of 10:3:1 respectively following the method of Krishna and Dart (1984). Preparation of standard curve: The stock solution was pippetted out in 11 test tubes ranging from 0.1 ml to 1.0 ml. The volume was raised to 1 ml by adding respective quantities of distilled water. 0.4 ml of ammonium molybdate, 0.4 ml of H 2 SO 4, 0.25 ml of SnCl 2 (freshly prepared) were added to each test tubes. O. D. was read at 700 nm. Extraction of plant material: 50 mg of dried plant material was taken in digestion tube and 3 ml of triacid mixture was added. Plant material was digested for one hour and then allowed to cool down. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 15

21 Final volume of digested solution was made up to 25 ml, 0.2 or 0.3 ml of plant extracts were raised to 1 ml by adding respective quantities of distilled water to it 0.4 ml of H 2 SO 4, 0.25 ml of SnCl 2 (freshly prepared) and 0.4 ml ammonium molybdate solution were added. It was incubated for 10 minutes and then 2 ml of distilled water was added. The O. D. was taken at 700 nm. Nitrogen Reagents: a. Sodium thiosulphate b. Sodium hydroxide pellets c. 0.7 g of mercuric oxide d. Potassium sulphate e. Salicylic acid f g of methyl red and 0.02 g of methylene blue in 50 ml of ehanol g. Boric acid h N NaOH Procedure: Weigh 2 g of sample and transfer to a Kjeldhal flask. Add 40 ml of concentrated sulfuric acid containing 2 g of salicylic acid and mix well. Allow standing for 1 hour with occasional shaking. Add 5 g sodium thiosulphate, shake, let stand 5 min, and then heat until forthing ceases. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 16

22 Turn off the heat, add 0.7 g of mercuric oxide and 15 g of potassium sulphate, Cool, add about 200 ml of water, wait until it cools to room temperature, and then add a few zinc granules. Tilt the flask and carefully add without agitation, 50 ml of a solution containing 500 g of sodium hydroxide pellets and 40g of sodium thiosulphate, dissolved in 1 litre of water. Immediately connect the flask to the distillation system with the condenser tip immersed in 50 ml standardized 0.1 N boric acid in a receiving flask. Then rotate the digestion flask slowly to mix the contents, and heat to collect 200 ml of distillate. Acid / Alkaline Phosphatase Reagents: a. Acetate buffer (ph-5.0) ( For acid phosphatase) b. 0.1M Tris-HCl buffer (ph-8.0) (For alkaline phosphatase) c. 50 mm p-nitrophenyl phosphate (p-npp) (0.18 g/10 ml in d.water) d. 0.5 M KOH Procedure: Take 2.5 ml soil suspension into a 10 ml test tube. Add 0.5 ml of 0.1 M acetate buffers and 3.3 ml d.water. Add 0.5 ml of 50 mm p-nitrophenylphosphate solutions. Incubate ona shaker, in the dark, at 40 o C for 1 hour. Add 2.5 ml of 0.5 M KOH to termination of enzyme reaction in the above mixture. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 17

23 Centrifuge at 1500 rpm for 10 min to removeal of precipitate, if any. Collect the supernatant and measure the OD at 450nm. Calculation: One unit of enzyme activity is expressed as the amount of enzyme required to liberate1umol p-nitro phenol produced g -1 wet soil h -1 Enzyme activity (U/h/g wet wt) = Molar extinction coefficient of p-nitro phenol = 18.8 M/LPath length = 1.0 cm = 18.8 x OD at 405 x 1.0 x DF x Total volume of water added (ml) Incubation time (h) x Initial wet wt of soil, in g Nitrate Reductase The assimilatory reduction of nitrate by plants is a fundamental biological process in which highly oxidized form of inorganic nitrogen is reduced to nitrite and then to ammonia (Plummer, 1988). Nitrate reductase is a substrate inducible enzyme of high molecular weight containing FAD, cytochrome and molybdenum as prosthetic groups. Depending upon the electron donor two major types of nitrate reductase occurs- (a) Ferredoxin dependent nitrate reductase (blue green algae) (b) Pyridine-nucleotide dependent nitrate reductase (higher plants) For the assay of nitrate reductase in-vitro, Wray and Filner s method (1970) was used. 500 mg of plant material was homogenized in 5 ml of extraction media containing 0.1 mol/l phosphate buffer (ph 7.5) and 1 mm cysteine. The reaction mixture containing 0.5 ml of enzyme extract, 0.01 ml of KNO 3 (0.1 M), 0.5 ml of phosphate buffer (0.1 M ph 7.5), 0.1 ml of NADH (1mM and 0.1 ml of double distilled water was incubated for 15 minutes at 30 o C). The Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 18

24 reaction was terminated adding 1 ml of sulphanilamide (1% in 3 N HCl) and 1 ml of 2.02% N- Naphthylene diamine dihydrochloride (NEDD). The reaction mixture was centrifuged to discard proteins and absorbance was recorded at 540 nm against blank in which enzyme was added after sulphanilamide. The standard curve was prepared by using 1 µg/ml of NaNO 2. Total Phenol Total phenol was determined following Mahadevan s method (1975) using folinciocalteu reagent. Estimation of the total phenols with folin-ciocalteu reagent is based on the reaction between phenols and an oxidizing agent phosphomolybdate that results in the formation of a blue complex (Bray and Thorpe, 1954). Fresh plant materials were extracted in 80% ethanol in soxhlet apparatus. The alcohol extract was evaporated to dryness and was redissolved in 30% methanol. The methanolic extract was used for calorimetric estimation of total phenols. Suitably diluted methanolic extract of the plant material was taken in test tubes. To it 1 ml of folin-ciocalteu reagent (diluted with equal volume of double distilled water) was added followed by 2 ml of Na 2 CO 3 (20% W/V) solution. The test tubes were shaken gently and then heated in a boiling water bath for exactly one minute. It was then cooled down under running tap water. The blue colored solution was suitably diluted with distilled water and the absorbance was measured at 650 nm in a spectrophotometer. Qualitative estimation of total phenols was attempted with the help of standard curve prepared from different concentrations of catechol (5 to 50 µg). The polyphenolic contents are expressed as mg/g fresh weight. All the experiments were performed in triplicates to avoid errors. Average value of five observations was considered for final calculations of total phenols. Peroxidase & Polyohenol Oxidase Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 19

25 (a) Peroxidase and Polyphenol oxidase (PRO and PPO) Root pieces were homogenized in 0.1 M phosphate buffer (ph 7.0), with a pre-chilled mortar and pestle at 4 0 C. The homogenate was centrifuged at 5,000 rpm for 15 minutes and the supernatant was used for enzyme assay. Peroxidase activity was measured by incubating the enzyme with guaiacol and hydrogen peroxide (Racusen and Foote, 1965). The arbitrary unit of enzyme activity chosen was change in absorbance of 0.001/sec. Polyphenol oxidase activity was measured at 420 nm, using the method of Mahadevan (1975). The activity is presented in terms of absorbance of 100 mg/g fresh weight of tissues. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 20

26 Chapter 3 Results It is well recognized that AM fungi helps in improving plant biomass production and nutrient uptake under different climatic condition. In order to evaluate potentiality of different AM fungi on biomass production and nutrient uptake in two species of Acacia s (namely A.tortilis and A. nilotica). After collection of plant species the studies were further carried out to find out the effect of abiotic factors viz. soil ph, moisture organic carbon, phosphorous and nitrogen on spore population and percentage root colonization by AM fungi. For this purpose soil samples along with roots were collected from rhizosphere soil of the Acacia from various localities viz.pachpadara, Balotara, Luni and Phalodi.Respective soil of all the four localities was sandy (Table 1, Histogram 1), and per sent sand particles present in sampled rhizospheric soil. Mycorrhizal spore population at various localities varied from spores per gram of soil. Soil sample of Pachpadara showed minimum spore population i.e. 90 spores per g of soil while sample from Balotara showed maximum spore population i.e. 150 spores per g of soil. Percentage root colonization by different AM fungi at various localities varied from per cent. Soil sample collected from Phalodi showed minimum percentage of root colonization i.e. 48 per cent and maximum is observed in the sample collected from Luni i.e. 68 per cent. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 21

27 During the present study mycorrhizal spore population was not found to be correlated with percentage root colonization in rhizosphere of Acacia spp., collected from various localities of the region. This suggests that it is not the quantity of mycorrhizal spores which affects the root colonization, but it is the potentiality of AM spore which decides the rate of colonization. Since the rhizosphere soil samples of the Acacia spp. at one locality were almost similar in various physical factors only one observation of each locality is represented in the table (Table 2a and 2b). It is clear from the observations that soil ph at various localities varied from , while soil moisture varied from per cent, organic carbon ranged from , soil phosphorus was k/ha and soil nitrogen k/ha at different localities. While correlating mycorrhizal root colonization with abiotic factors, it was observed that increase in soil ph with decrease in soil phosphorus and soil nitrogen resulted in increased percentage root colonization as the sample of Pali showed. However soil moisture and soil organic carbon level could not be correlated with percentage root colonization by AM fungi during the present study. Possible reason for this might be almost similar level of soil moisture ( ) and organic carbon ( per cent). Observation further reveal that all the ten species were found distributed in all the four localities. Among the five Acaulospora morrowae,glomus constrictum,gigaspora gigantean, Sclerocystis rubiformis and Scutelospora nigra,were found in lesser number at various localities, while Glomus deserticola,g.faciculatum, G. mossae, Gigaspora margarita and Sc. calospora were equally distributed at all the places with maximum in number. Hence the observation suggests that AM fungi belonging to ten species at various localities, surveyed during the present study. However their number varies from different localities. Selection of suitable genera and efficient strain of AM fungi: During the first phage of study different genotypes of AM fungi were studied for selection of suitable genera and efficient strain of AM fungi.to this region. It was observed Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 22

28 that plant height was ranged between cm at 60 days, cm at 90 days and cm at 120 days of AM inoculation for A.tortilis and cm at 60 days, cm at 90 days and cm at 120 days for A.nilotica. Plant dry weight ranged between g at 60 days, g at 90 days and g at 120 days, presented in Table 2a and 2b. The most efficient 6 native genotypes were further studied with inoculation of different AM fungi. It is observed that most efficient genera for each genotype isglomusmossae and Glomusdeserticola. These results were followed by genotype G. faciculatum, Gigaspora margarita Sc. calospora and A.morrovaeSo for further study these six genotypes were used. Biomass production and nutrient uptake. Influence of different AM fungi on nutrient uptake (phosphorus and nitrogen) and productivity of the Cowpea was presented in Table 6a and 6b and Histogram 6a and 6b. This was studied by sowing these two cultivars with treatments of six suitable and efficient species of AM fungi. Arbuscular mycorrhizal inoculation resulted in increased biomass production and productivity (height and plant dry weight) in Acacia spp. irrespective of the mycorrhizal species as compared with non-mycorrhizal plants. The height of A.tortilis ranged from 60 cm cm of different mycorrhiza treated plants, as compared with 55 cm. of nonmycorrhizal ones. The most efficient response was observed by Glomus mossae which resulted in almost two fold increase in height followed by Gigaspora margarita while least response was observed in Acaulospora morrowae treated plants. Where as the height of A. nilotica ranged from 64 cm cm of different mycorrhiza treated plants, as compared with 52 cm. of non-mycorrhizal ones. The most efficient response was observed by Glomus deserticola which resulted in almost two fold increase in height here too followed by G. mossae and Gigaspora margarita while least response was observed in Acaulospora morrowae treated plants. Similar trend in the efficacy of different AM fungi towards increase in productivity of the Acacia spp. was also observed during the present studies. In A.tortilis per sent root AM colonization were observed in different AM treated plants as compared with no association present in non-mycorrhizal ones and least 34 per sent is in Gigaspora margarita and in A. nilotica per sent root AM colonization were observed in Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 23

29 different AM treated plants as compared with no association present in non-mycorrhizal ones and least 31 per sent is in Gigaspora margarita. It is clear from the observation that inoculation of Glomus mossae and Glomus deserticola in A.tortilis and A. nilotica respectively resulted in 100 per cent increase in productivity followed by G. facciculatum while least response was observed in Acaulospora morrowae treated plants as compared with nonmycorrhizal plants. The phosphorus uptake of the host plant ranged from mg/g dry weight in A.tortilis and in A. nilotica as they are different mycorrhiza treated plants as compared with mg/g dry weight of non-mycorrhizal ones. The most efficient response was observed by G. mossae and G. deserticola which resulted in more than two fold increase in phosphorus uptake followed by G. facciculatum and Gi. margaritawhile least response was observed in Acaulospora morrowae treated plants. Similar trend in the efficacy of different AM fungi towards increase in nitrogen uptake of the Cowpea was also observed during the present studies. In case of A.tortilis mg/g dry weight and in A.nilotica mg/g dry weight nitrogen was observed in different AM treated plants as compared with 3.7 and 4.0 mg/g dry weight of non-mycorrhizal ones. It is clear from the observation that inoculation of Glomus deserticola resulted in 100per cent increase in nitrogen uptake followed by G. facciculatum and Gigaspora margarita while least response was observed in Acaulospora morrowae treated plants as compared with non-mycorrhizal plants. Influence of AM fungi on enzymatic changes: AM fungi are well known to bring about physiological changes in plants via increasing enzymatic activities i.e. acid and alkaline phosphatases, nitrate reductase, peroxidase and polyphenol oxidase etc. Among these enzymes phosphatases are important enzymes of phosphorus metabolism while nitrate reductase is important enzyme of nitrogen metabolism, peroxidase and polyphenol oxidase are the two important enzymes of defense mechanism of plants. Keeping all these facts in mind another sets of experiments were designed to evaluate potentiality of different microorganisms towards increasing activities of these enzymes in the Acacia plants. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 24

30 The results of influence of symbiotic relationship towards acid phosphates, alkaline phosphatase and nitrate reductase of the plant are presented in histograms. It is clear from the observations that in Acacia tortilis inoculated by different AM fungi resulted in (M mol -1 h -1 kg -1 Fr. wt) nitrate reductase activity as compared with 0.06 (M mol -1 h -1 kg -1 Fr. wt) of non-inoculated ones. In Acacia tortilis (M mol -1 h -1 kg -1 Fr. wt) nitrate reductase activity present when they inoculated with AM fungi, where as 0.06 (M mol -1 h -1 kg -1 Fr. wt) nitrate activity present in non-inoculated ones. Glomus mossae and Glomus deserticola inoculation resulted in more than two fold increases in nitrate reductase activity followed by Glomus facciculatum while least response was observed in A. morrovae treated plants as compared with the noninoculated plant species. Similarly acid phosphatase activity in different AM fungi treated plants varied from (X10 4 n mol PNP hydro S -1 g -1 ) as compare with 1.2 (X10 4 n mol PNP hydro S -1 g -1 ) of non-inoculated plant species. The acid phosphatase activity was increased up to more than 83 per cent due to Glomus deserticola and Glomus mossaeinoculation as compared with non-inoculated ones. Plant resistance and servility improvement: When plants are exposed to open fields, there are several factors, which results in destruction of the plant species. Among these factors soil microorganisms particularly soil borne plant pathogens plays a vital role in managing the plants growing in natural habitats. In order to save the plants from attacking soil borne pathogens, some technology should be applied by which plants can develop resistance against attacking pathogens. Arbuscular mycorrhizae are now days well recognized as biocontrol agents, some rhizobacteria are also helpful in this respect. In view of all these facts experiments were designed to find out potentiality of different AM fungi towards biological control of soil borne plant pathogens. For this purpose two enzymatic estimations were done namely peroxidase and polyphenol oxidase. Since these two enzymes are important enzymes of phenolic metabolism of the plant, as they bring about oxidation of phenols into quinones, which are well known to be toxic to the attacking plant pathogens, the experimental studies carried out during the Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 25

31 Soil % present study can be useful. The results of these experiments are presented in Table: 10a and 10b.In Acacia tortilis peroxidase activity due to inoculation of different microorganisms ranged from (unit mg -1 Protein) as compared with 95.5 (unit mg -1 Protein) of noninoculated plants. Same experiment resulted into (unit mg -1 Protein) in AM fungi inculated and 97.0 (unit mg -1 Protein) in non-inoculated. As observed in previous experiments Glomus deserticola and G. mossae responded most efficiently by increasing more than 57 per cent activity of this enzyme in thehostplantas compared with controls after 120 days of inoculation. Similarly polyphenol oxidase activity and total phenolic contents were also increased due to different AM fungi inoculation. Polyphenol oxidase activity ranged from units in different microbial treated plants as compared with of control in A.tortilis.Glomus mossae resulted in almost 43 per cent increase in polyphenol oxidase activity with 44 per cent increase in total phenolic contents. As per in A.senegal Polyphenol oxidase activity ranged from units, Glomus desrticola in different microbial treated plants as compared with of control. Histogram 1: Physical characterstic of rhizosphere soil of Various Locations 100% 80% 60% 40% 20% 0% Locations of Soil Collection Luni,Pachpadara,Balotara, Phalodi Clay (%) Silt (%) Fine sand (%) Coarse sand (%) Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 26

32 Plant Height (in cm) Plant Height (in cm) Histogram 2a: Influence of different AM fungi on biomass production (plant height) of Acacia nilotica Days 90 Days 120Days 0 Control Gi. margarita Glomus deserticola Treaments G. mossae Histogram 2b: Influence of different AM fungi on biomass production (plant height) of Acacia tortilis Days 90 Days 120Days 0 Control Gi. margarita Glomus deserticola Treaments G. mossae Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 27

33 Plant AM colonization in percent Plant AM colonization in percent Histogram 3a: Influence of different AM fungi on plant AM colonization of Acacia nilotica Control Gi. margarita Glomus deserticola Treatment G. mossae 60 Days 90Days 120Days Histogram 3b: Influence of different AM fungi on plant AM colonization of Acacia tortilis Control Gi. margarita Glomus deserticola Treatment G. mossae 60 Days 90Days 120Days Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 28

34 mg per plant mg per plant 9 Histogram 4a : Influence of Arbuscular Mycorrhizae on nutrient uptake in Acacia nilotica after six months of inoculation Total phosphorus (mg pt-1) Control Gi. margarita Glomus Treatment deserticola G. mossae 8 Histogram 4b : Influence of Arbuscular Mycorrhizae on nutrient uptake in Acacia tortilis, After six months of inoculation Total phosphoru Control A. morrovaegi. margarita Scutelospora calospora Glomus deserticola G. faciculatumg. mossae Treatment Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 29

35 Concentration Concentration Histogram 5a : Changes in nitrate reductase, acid and alkaline phosphatase activities in Acacia nilotica after six months of AM inoculation Nitrate reductase (M mol-1 h-1 kg- 1 Fr. Wt.) Control Gi. margarita Glomus deserticola G. mossae Treatment Histogram 5b : Changes in nitrate reductase, acid and alkaline phosphatase activities in Acacia tortilis after six months of AM inoculation Nitrate reductase (M mol-1 h- 1 kg-1 Fr. Wt.) Control Gi. margarita Glomus deserticola G. mossae Treatment Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 30

36 Concentration Concentration Histogram 6a : Changes in total phenol, peroxidase and polyphenol oxidase activity in root of Acacia nilotica after six months of AM inoculation Total Phenol (% dry wt.) Control Gi. margarita Glomus deserticola G. mossae Treatment Histogram 6b : Changes in total phenol peroxidase and polyphenol oxidase activity in root of Acacia tortilis after six months of AM inoculation Total Phenol (% dry wt.) PRO activity (unit mg-1 Protein) Control Gi. margarita Glomus deserticola G. mossae Treatment Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 31

37 DESCRIPTION OF GENERA Periodical survey of different parts of Western Rajasthan was undertaken to collect and identify different AM species associated withacacia sp. Rhizosphere soil samples collected from various localities revealed presence of eighteen arbuscular mycorrhizal species belonging to the five genera viz. Acaulospora, Gigaspora, Glomus, Sclerocystis and Scutellospora associated with these plant species. The detailed description of these AM species is as follows- Acaulospora Gerd. & Trappe emend Berch. Spores produced singly in soil or in sporocarp that may attain several cm in length, spores globose, subglobose, ellipsoid with oily content; borne laterally on the subtending hyphae of large, terminal relatively thin walled, sporogenous saccule. Spore composed of essentially two distinct, separable wall groups. Gigaspora Gerdemann & Trappe Spores produced singly in soil, large, variable in shape, usually globose to subglobose, often ovoid, obovoid, pyriform or irregular, borne on a bulbous suspensor like cell, usually with narrow hyphae. Spore wall structure of a single wall group, lacking flexible walls. Thin walled, echinulate auxillary cells borne in soil on straight or coiled hyphae, formed singly or in clusters. Glomus Tul. & Tul. Chlamydospores borne terminally on single (rarely two) undifferentiated, non gametangial hyphae in sporocarps or individually in soil. Spore contents at maturity separated from attached hyphae by a septum or occluded by spore wall thickening. Spores of Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 32

38 most Glomus species are borne singly. A few species are known only from sporocarps. Glomus species are very common in cultivated soils and widespread in native grasslands and forests. Sclerocystis Berk. & Broome Chlamydospores form in sporocarps or single, crowded layer of erect spores that surrounds the side and top of a spore-free, control mass of tightly interwoven hyphae. The sporocarps may be borne singly in soil or fused together with organic debris. Scutellospora Walker & Sanders Spores produced singly in soil, large, variable in shape, usually globose or subglobose, but often ovoid, obovid, pyriform or irregular especially when constrained during formation; borne on a bulbous suspensor like cell, usually with a narrow hyphae. Spore wall structures of at least two wall groups. Germination by means of one or more germ tubes produced near the spore base from a germination shield formed upon or within a flexible inner wall. DETAILED DESCRIPTION OF VARIOUS SPECIES Acaulospora morrowae Spain & Schenck Azygospores formed singly in the soil, borne laterally on hyphae ending in a globose hyphal terminus (58-) 79 (-94) µm diameter with walls µm thick: hyphae at the point of spore attachment µm wide; hyphal terminus contents subhyaline to white; distance between the hyphal terminus and the developing azygospore µm; terminus contents emptying to form the spore, leaving a hyaline, thin walled, empty terminus that readily collapses and detaches from the spore; spores rarely found with an attached terminus. Young Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 33

39 azygospores with light yellow walls and white contents, becoming light yellow with globular, transparent contents in reflected light. Spores predominately globose or subglobose, (63-) (-120) µm diameter, but also lacrimoid to irregular, X µm diameter; spore wall 2-4 (-6) µm thick consisting of several wall layers, readily apparent on broken spores; outer wall µm thick, hyaline in water, adhering to wall two but swelling in lactophenol, separating and sometimes with adhering debris; wall two light yellow, µm thick; wall three brittle, hyaline 0.5 µm thick; wall four membranous, 0.5 µm thick, usually adhering to wall five; wall five membranous, 0.5 µm thick forming vesicular-arbuscular mycorrhizae. Acaulospora laevis Gerdmann & Trappe Sporocarps unknown.spores forms singly in soil, sessile, born laterally on a wide, thinwalled hypha 30-40µ diameter. that terminates near by in a globose, thin walled vesicle. Vesicle approximately the same size as the spore, developing to full size prior to spore formation, with dense, white contents, becoming empty and shrunken at spore maturity and then usually lost in sieving. Spores smooth, x µ, globose to sub-globose, ellipsoid or occasionally reniform to irregular, dull yellow in youth, becoming deep yellow brown to red-brown or dark olive brown at maturity. Spore wall continuous except for the occluded opening consisting of three layers : a rigid, yellow-brown to red-brown outer wall 2-4µ thick,and two hyaline inner membranes,the inner most sometimes minutely roughened: in older specimens wall at times becoming minutely perforate and the outer surface sloughing away. Spore contents globose to somewhat polygonal (reticulate in optical section). Hypha below spore attachment giving rise to many slender branches µ diameter. Vesicles in vesicular-arbuscular mycorrhizae thin walled and lobed. Acaulospora mellea Spain & Schenck Azygospores formed singly in soil;borne laterally on hyphae tapering to a globose to sub-globose swollen hyphal terminus µ diam. Azygospores honey coloured to yellow brown, globose to sub-globose,95-105µ diameter, ellipsoidal or irregular, x µ Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 34

40 ,spore wall 4-8µ thick. Hyphal terminus contents white, emptying during spore formation, resulting in a transparent to sub- hyaline receptacle attached to the azygospores : hyphal terminus remains attached to young spores: old spores in soil usually devoid of hyphal terminus. Gigaspora gigantea (Nicol. & Gerd.) Gerd. & Trappe Azygospores formed singly in the soil, X µ, globose to ellipsoid, greenish yellow, with a thin outer wall tightly covering an inner wall 5-7 µ thick and continuous, except for an occluded pore at the attachment. Suspensor like cells bulbous, 42-48µ diameters, giving rise to slender hyphae that project to the spore.spherical to clavate vesicles formed in soil X µ, in clusters of 1-16 on complex system of inter-coiled hyphae. Diagnostic feature: Mature spore bright yellow with greenish tinge. Germ tubes produce directly through the spore wall in the base region. Vesicles formed in soil have septate echinulation at apices. Vesicles lacking in roots. Spores with two-layered wall, which is rarely 7 μm thick. Gigaspora margarita Becker & Hall Azygospores formed singly in the soil, dull white with a light greenish-yellow tint; mostly spherical µ diameter, averaging 265 µ, occasionally ellipsoidal X µ; spore walls continuous, except for an occluded pore from 4-12 µ thick, with 1-6 walls; outer wall thin, smooth, 1-2 µ thick, readily cracking under light pressure. Germ tubes produced directly through the spore wall near the bulbous suspensor without forming an enclosed compartment separating it from the spore contents. Azygospores attached to a single, hyaline to yellow bulbous suspensor µ attached to separate hyphae with hyphal branches. Extrametrical vesicles hyaline to yellow, turbinate, obovate or clavate, µ diameters formed in clusters of 5-14 on coiled hyphae in the soil. Diagnostic feature: Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 35

41 This is readily distinguished from other member of the genus in having white spores with laminated wall and white clustered, varty vesicles. Spores white, having a spore wall consisting of several laminations. Spore wall with up to 10 fused laminations; μm thick, which do not readily separate, when spores are crushed. Gigaspora roseanicol & Schenk Azygospores produced signally in soil, predominantly globose, μm diam, occasionally sub globose, white to cream in color with a rose-pink tint on the azygospore wall near the hyphal attachment encompassing up to half the spore. Pink coloration variable from distinctly rose pink to barely detectable layers 1-2 μm thick.outer wall layer smooth. Suspensor like cell attachment to azygospore usually spherical, occasionally sub globose, subtending hyphae, 7-14 μm wide, hyphal walls 1-2 μm thick, septate. Soil born vesicles in clusters of 5-12 on coiled hyphae, individual vesicles μm wide, and echinulate with spines up to 5.0 μm long and 2.5 μm wide.forming mycorrhizae with arbuscule. Diagnostic features: G. rosea can be distinguished from other light spored species of Gigasporaby the rose pink tint associated with the wall of the azygospore near the wall of the attachment. Soil borne vesicles echinulate, with spines (5 μm long and 2.5 μm wide). Spores white to cream. Spore wall consist of 2-5 inseparable layers. Glomus deserticola Trappe, Bloss & Menge Spores borne singly or in loose clusters in soil or within roots, globose to subglobose (47-) X (38-) µ, shiny, smooth, reddish brown, with a single, sometimes laminated wall (1.5-) (-4) µ thick. Attached hypha 6-12 µ in diameter, cylindrical, the walls thickened and reddish brown, especially thick adjacent to the spore but not occluding Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 36

42 the hypha. Interior of the spore wall at the hyphal attachment thickened at maturity to form an inner-mounded collar, which appear to be closed by a membranous septum. Diagnostic feature: Interior of the spore wall at the hyphal attachment thickened at maturity to form an inner-mounded collar, which appears to be closed by a membranous septum. Spore walls deep reddish brown. Wall diameter 1-4 μm. Reddish brown hyphal attachment not occluded by wall thickening. Glomus fasciculatum (Thaxter sensu Gerd.) Gerd. & Trappe Chlamydospores borne free in soil, in dead root lets, in loose aggregations, in small compact clusters and in sporocarps. Sporocarps up-to 8 X 5 X 5 mm, irregularly globose or flattened, tuberculate grayish brown. Peridium absent. Chlamydospores µ diameter when globose, X µ when subglobose to obovate ellipsoid, sublenticular, cylindrical or irregular; smooth or seeming roughened from adherent debris. Spore walls highly variable in thickness (3-17 µ), hyaline to yellow or yellow brown, the thicker wall often minutely perforate with thickened inward projections. Hyphal attachments 4-15 µ diameter, occluded at maturity. Walls of attached hypha often thickened to 1-4 µ near the spore. Diagnostic feature: Thicker walls of the spore often minutely perforated with thickened invert projections. Chlamydospores borne free in soil in aggregates, in small compact clusters and in sporocarps; peridium absent. Chlamydospores tightly packed together. Glomus geosporum(nicol. & Gerd.) Walker Sporocarps unknown. chlamydospores formed singly in soil, globose to subglobose or broadly ellipsoid, μm, smooth and shiny or with a dull appearance, or roughened from adherent debris; light yellow-brown and transparent to translucent when young, becoming dark yellow-brown to dark red-brown at maturity. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 37

43 Spore walls 4-18μm thick, 3 layered, with a thin, hyaline, tightly adherent outer wall (<1μm), a yellow brown to red brown laminated middle wall (316 μm); and a yellow to yellow brown inner wall (< 1μm) that appears membranous and that forms a septum separating the spore contents from the lumen of the subtending hypha. Spores with straight to recurved, simple to slightly funnel shaped subtending hypha up to 200 μm long.occasional spore lacking a subtending hypha due to breakage close to the spore base. Spore contents uniform oil droplets when young, becoming increasingly granular in appearance with age; cut off by a thick septum that protrudes slightly into the subtending hypha after rupture of the septum. Diagnostic feature: Spore cut of by a septum that protrudes slightly into the subtending hypha. Spores red brown to opaque at maturity. Glomus macrocarpum Tul. & Tul Sporocaps are fragmentary, non of the pieces more than 5 mm diameter.spores are usually slightly longer than wide, sub-globose or globose, to irregular, (90-)120(-140) x (70- )110(-130) μm. Spore wall is composed of two distinct layers :outer layer is thin (1-2 μm) and hyaline when mounted in water or glycerol, usually swelling to at least twice its original thickness in lactic acid : inner wall layer is yellow in section,6-12 μm thick,with a series of laminations occasionally visible or rarely appearing as two distinct layers, swelling relatively little in lactic acid. Spores taper to the point of attachment of the single persistent hypha. The average diameter of the hyphae at this point is 16 μm. The inner wall at maturity thickens to occlude.the pore of the attached hyphae and the wall thickening continues into the subtending hypha for up to 90 μm from the spore.infrequently the pore seems to be closed by septum that is thinner than the normal occluding wall thickening. Spores characteristically bear a straight, long subtending hypha which may extend up to 100 μm before branching or breaking. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 38

44 Glomus mosseae (Nicol.& Gerd.)Gerd.& Trappe Sporocarps 1-10 spored, globose to elliposoid,up to 1mm diameter.peridium of loosely interwoven, irreguraly branched,hyaline, septate hyphae 2-12 μm diameter., the walls upto 0.5 μm thick,frequently anastomosing to form a thin network, enclosing the chlamydospores entirely, incompletely or with some spores unenclosed.endocarpic and ectocarpic spores similar. Chlamydospores yellow to brown, globose to ovoide, obovoid or somewhat irregular, x μm, with one or occasionally to funnel shaped bases 20-30(-15) μm diameter, divided from subtending hyphae by a curved septum ;walls 2-7 μ thick,with a thin often barely perceptible hyaline outer membrane,and a thick, brownish-yellow inner wall. Sclerocystis rubiformis Gerd. & Trappe Sporocarps dark brown, subglobose to ellipsoid, 180 X X 675 µ, consisting of a single layer of chlamydospores surrounding a central plexus of hyphae, resembling a miniature black berry.peridium nearly absent, individual spores at times partially enclosed in a thin network of tightly appressed hyphae. Chlamydospores dark brown, obovoid to ellipsoid or subglobose, X µ, with a small pore opening into the thick walled subtending hypha. Spore wall laminated, µ thick, often perforated. A variable stalk like projection protrudes near the base of some spores. Diagnostic feature: Spore wall often perforated, and often with thick, perforated projections on the inner surface. Peridium nearly absent. Sclerocystis corieomoidesberk. & Broome Sporocarps μ broad, subglobose to pulvinate, flattened at base, at times borne on a short stalk up to 100µ broad, white when immature, becoming tan to dull brown when fully mature, gregarious in mats containing large number of sporocarps fused together laterally and one above the other to about 4 sporocarps thick. Peridium μ thick, of Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 39

45 interwoven hyphae. Chlamydospores (-102) X (-82) μ, obovoid-ellipsoid to oblong-ellipsoid, often but not always cut off from subtending hyphae by septa just below spore base, arranged in a single layer, tightly grouped in a hemisphere around a central plexus of hyphae. Spore absent at base of sporocarp. Chlamydospore wall up to 4 μ thick at base and 2 μ thick at apex, brown.forming vesicular arbuscular mycorrhizae. Diagnostic feature: Young sporocarps wide enclosed in a peridium μ thick of interwoven hyphae. Sporocarps fused together, laterally and vertically to about 4 sporocarps thick. Sclerocystis microcarpus Iqbal and Bushra Sporocarps dark brown, µ in diam, globose to subglobose, minutely verrucose from exposed tips of spores formed radially in a single, tightly packed layer around a central plexus of hyphae; peridium lacking.chlamydospores clavate, cylindrical clavate with a small pore opening into the thick walled subtending hyphae. Chlamydospores walls laminate, brown, generally thickest at apex. Diagnostic features: Sporocarps minutely verrucose from exposed tips of spores. Chlamydospores broader at the upper end.sporocarp µ in diam. Scutellospora calospora (Nicol. & Gerd.) Walker & Sanders Spores formed in the soil, terminally on a bulbous suspensor like cell; translucent, hyaline to pale greenish-yellow, globose, ellipsoidal or cylindrical, occasionally broader than long; 114 X 285 X µ. spore wall structure of four walls in two groups (group A and B). Group A consisting of an inner, brittle, hyaline to pale yellow, very finely laminated wall 3-5 µ thick that may be surrounding by a thin very closely appressed hyaline unit wall, µ thick. Group B of two hyaline membranous walls.wall 3, µ thick often wrinkling in crushed Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 40

46 spores.wall 4, µ thick.suspensor like cell borne terminally on a septate subtending hypha; µ broad. Diagnostic features: Spores translucent hyaline to pale greenish yellow. Smooth knobby vesicles borne singly on coiled hyphae in the soil. The oval germination shields often with invagination along the margin. Pore at the attachment occluded. Inner wall group consist of 2 juxtaposed membranous walls. Scutellospora nigra (Red head) Walker & Sanders Azygospores formed singly in the soil, dark brown to black spherical, and µ diameters with an inner and outer wall. Outer wall black to dark brown, pitted with larger pores, 7-10 µ diameter; inner wall light brown, transparent. Suspensor like hyphal attachment light brown attached laterally, X µ. accessory soil borne vesicles dark brown, globose to subglobose, smooth to knobby, in usually tight clusters of 3-12 on coiled or twisted hyphae arising from straight hyphae µ in width. Diagnostic feature: This can be readily separated by its large black, shiny spores, with pores in the outer wall. Suspensor-like cells μm diameter.spores with 2 walls. Scutellospora heterogama (Nicol. & Gerd.) Walker & Sanders Spores borne singly in the soil, terminally, subterminally, or laterally on a bulbous suspensor-like cell; globose to subglobose or irregular; μm, ellipsoidal specimens up to 210 X 230 μm; pale yellow-brown to red-brown. Spore wall structure of four walls in two groups (A and B). Group A with an outer ornamented unit wall (1) tightly adherent to an inner wall (2).Wall 1 brittle, pale yellow to pale brown.1-1.5μm thick, excluding the hyaline warts (papillae). Warts very densely crowded, usually touching or less than 0.5 μm + apart at the base, μm high, μm diameter. Wall 2 yellow-brown, finely laminated, 4-7μm thick. Group B of two membranous walls (3 and 4) separated by an apparent amorphous cementing Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 41

47 layer. Each wall hyaline, <1 μm thick; total thickness of walls and separating material μm. Suspensor like cell borne terminally on a coenocytic to sparsely septate subtending hypha; μm wide; yellow-brown. Wall suspensor like cell μm thick distally, thickening to μm at the spore base. One or two peg-like hyphal projections present or lacking; when present X 5-9 μm, arising from the suspensor like cell and projecting toward the spore. Diagnostic features The small, closely crowded warts on the spore surface and the wall surface differentiate S. heterogama from other species. Wall group B of 2 membranous walls separated from an apparent amorphous cementing layer, each wall less than 1μm thick. Scutellospora aurigloba (Hall) Walker & Sanders Spores ectocarpic, globose or more rarely polymorphic, x x μm diameter, pale yellow, transparent and shining when yellow and becoming dull at maturity. Spore wall 2-4 layered, outer wall coloured 6-16 μ thick, inner walls approx. 1 μm thick, colourless to yellow.spores formed on a bulbous suspensor μm diameter.walls of subtending hypha 3-10 μm thick, yellow to light brown. Subtending hypha sometimes with a well to poorly developed lateral projections. Pore approx. 4 μm diameter without a septum cup or dome shaped septa often form in the subtending hypha. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 42

48 Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 43

49 Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 44

50 Chapter 4 Development of arbuscular mycorrhizae Arbuscular mycorrhizae are well recognized as biofertilizers now days. Since the plantfungus relationship in this case is of symbiotic type, both the organisms get benefited from each other. The fungus produces external as well as internal structures to establish symbiotic relationship with the host. The infection of the roots by an arbuscular mycorrhizal fungus and subsequent development of the AM could be categorized into following four stages- (a) (b) (c) (d) Spore germination or initiation of hyphal growth from the infective propagules. Growth of hyphae through the soil to the host roots. Penetration and successful initiation of infection in roots and, Spread of infection development of a mycorrhizal relationship with root and spore production. Brundrett et al., (1985) stated that the application of VA mycorrhizal symbiosis to agriculture and forestry requires an understanding of the events that occur during the establishment of this association. The present investigation was undertaken to study the sequence of events in the colonization process and the time required for the formation of each stage in the Cowpea. The plant roots of Cowpea collected from the fields as well as inoculated in the pots were regularly examined. A series of squash preparation reveals the presence of different stages in the development of arbuscular mycorrhizal infection in the roots of Cowpea. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 45

51 The infective propagules of different AM fungi exist in the form of penetrating chlamydospores in the fields. The pot experiments have shown that the infection in plant roots was initiated either by the germ tube formed on the germination of chlamydospores or by young juvenile hyphae. It was observed that the young developing feeder roots come in contact with the germ tube of germinating spore on the third or fourth day of inoculation. The infecting hyphae developed close contact and adhered strongly to the root surface. This adherence leads to appressoria formation or direct penetration of the epidermal cells by the rupturing of outer cell wall. The penetration was rarely without appressorium formation. The penetration of the host was a continuous process and could be seen even at later stages, when endophyte had already established itself in the cortical cells. This was followed by the development and ramification of fungal hyphae, which grows, inter as well as intracellularly. The arbuscule formation.was observed on the seventh day of inoculation. Arbuscules development started after the penetration of the host cell wall by a lateral branch produced from hyphae of the adjoining cell.these hyphae became the arbuscular trunk and showed repeated dichotomous branching in the cell. The arbuscule occupy a major portion of the host cell and can be seen in the various stages of development. The arbuscules were ephemeral structures and remain active only for four to fifteen days. During degeneration the finer hyphae of arbuscular were the first to collapse into a dense residual mass. The arbuscular trunk was quite apparent in the centre of the cells and was the last hyphal elements to collapse. It was of interest to notice that young arbuscule was present in the cell adjacent to a cortical cell containing collapsed arbuscule. It was observed that the endophyte remains confined to the cortical region of the host. The vesicle formation was noticed on the eighth day onwards, after the penetration of the host cell. The vesicles were oval spherical or irregularly lobed. These are thick walled structures formed terminally in the inter or intracellular spaces, with their size ranging between 9-45 µm in diameter. The vesicles were usually multinucleate having open Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 46

52 connections with parent hyphae. Vesicles have been considered to function as temporary storage organs. They also serve as propagules as such as roots decay or develop into thick walled chlamydospores functioning as reproductive structures. Due to these properties the so-called vesicles are now termed as chlamydospores (Mehrotra, 1997). In addition to this extramatrical hyphae commonly developed into the soil up to some distance around the roots. The mature extramatrical hyphae also bear the resting spores. The chlamydospore formation was observed in the host tissue on the surface of rootlets and also in the rhizosphere. The process of development of mycorrhizal infection in the roots of the Cowpea plant is consistent with observations made on other endomycorrhizae (Reddy, 1996; Kumar et. al, 1997 and Chandra and Jamaluddin, 1999). The infection of the roots was possible through the germ tube formed on the germination of chlamydospores or through the young fungal hyphae. The roots were penetrated either directly or was accompanied with the formation of appressoria. The penetration of host tissue was a continuous process and was seen at later stage when the endophyte had already established itself inter as well as intracellularly. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 47

53 Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 48

54 Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 49

55 Chapter 5 Discussion Periodical survey of four regions of Western Rajasthan namely, Jodhpur, Pali, Udaipur, and Mount Abu revealed that nearly eighteen AM fungi were found associated with the two Acacia species namely, Acacia tortilisand Acacia nilotica. The frequency of occurrence of mycorrhizal spores in rhizosphere soil and root colonization of the two host plant was found to be affected by abiotic factors like soil ph, soil phosphorus and soil nitrogen at all the above localities. Different abiotic factors and their influence on spore population and percentage of root colonization are presented in Tables 1 & 2. During the present study increase in soil ph with decrease in soil phosphorus and nitrogen was found to be correlated with increasing colonization of host root by the AM fungi at all the localities irrespective of host plant. Edaphic factors such as soil texture, soil fertility, soil moisture, soil temperature and soil ph may effect the composition, distribution and efficacy of AM fungi in the natural habitat (Singh, 1999). Singh and Tewari (1999) reported seasonal fluctuation in number of VAM spores in soils of sand dunes. Pavan Kumar et. al., (1999) reported that the percentage of infection was suppressed under the influence of effluence. They observed that the spore population in the rhizosphere soil varied both with the plant and also with the type of pollutants. Soil ph is the major edaphic factor, which effect the establishment and efficiency of mycorrhizal fungi in natural vegetation. Siddu and Behl (1997) observed relative tolerance of VAM fungi to graded level of ph ( ) and there influence on P uptake in Prosopis juliflora. They showed that increase in Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 50

56 ph adversely effect growth, biomass and P concentration in seedlings of Prosopis. Chlamydospore formation in the rhizosphere soil by all three VAM fungi decreased with increase in soil ph. Soil ph and available soil nutrient have a cumulative effect on the efficiency of VAM on different plant species (Singh, 2000). Domisch et. al., (2002) reported effect of soil temperature on root colonization by AM fungi in Scot pine seedlings. The observed increase in AM sporulation and rate of colonization of the two Acacia species due to increase in soil ph was found to be correlated to the distribution of different AM species in these arid and semi arid areas. The concentration of K, soil moisture and organic carbon could not be correlated with AM spore population and root colonization of the two plant species in this region. The reason for such observation could be very low soil moisture level, almost similar quantitative occurrence of K at different localities and very low organic carbon level of the soils of this region. AM fungi and its potentiality to establish symbiotic relationship with the host plant is affected most severely by the nutrient status of the host plant as well as the rhizosphere soil. Since AM fungi compensate to nutrient deficiency of the host plant, its potentiality to colonize the root is likely to be decrease with increase in nutrient status of both the rhizosphere soil and the host plant (Mathur and Vyas, 2000). Arbuscular mycorrhizae are well known to be of ubiquitous occurrence. Its distribution in Indian Thar Desert has been reported (Mathur and Vyas, 1995 I). However, occurrence of AM fungi inassociation with Acacia tortilisand Acacia niloticahas not been studied properly. The present study reveal occurrence of eighteen species of AM fungi in different arid and semi arid regions of Western Rajasthan. The type of plant species had almost no effect on sporulation of a particular AM species in this region. There has been a phenomenal increase of interest on AM fungi in recent years leading to numerous surveys for enumerating and accessing AM fungal species and their colonization of host plants in different regions of this country (Muthukumar and Udaiyan, 2000). The significance of AM fungi is based on its wild spread occurrence in natural ecosystems. Until now, there has been a paucity of information on the mycorrhizal status of Acacia species of this region. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 51

57 The present study revealed association of eighteen species of five genera of AM fungi with the two Acacia species. These genera and species were invariably present throughout the region irrespective of the host plant type present at particular locality. Among the five genera the species belonging to genera, Gigaspora, Glomus and Scutellospora was found very common while the species belonging to genera Acaulospora and Sclerocystis were found comparatively at lower rate in distribution. Our results support the previous observations about frequent occurrence of Glomus in different regions of Indian Thar Desert (Mathur and Vyas, 1995 I). Though AM fungal species were not found to be effected by host specificity for its distribution in this region however, some species were found to be more abundant in its occurrence as compare with the others. All the eighteen AM fungal species were successfully cultured on Cenchrus ciliaris and Sorghum bicolor forpreparation of pure pot cultures. The inoculum from these pot cultures was used to inoculate the two Acacia species. Both the plant species viz. Acacia tortilisand Acacia niloticawere successfully colonized by different AM fungal species. The symbiotic relationship was established very well between AM fungal species and the host plant species. All the stages of symbiotic relationship i.e. appresoria formation, hyphal penetration to the cortical region, intra-cellular penetration and formation of Arbuscules and formation of vesicles of different size and shape at both inter as well as intra cellular level was observed during the present study. Seedlings of the two Acacia species were inoculated with commonly found eight arbuscular mycorrhizal species for the further studies. In this phase of experiment efforts were made to exploit the potentiality of different arbuscular mycorrhizae on biomass production and nutrient uptake in the two Acacia species namely, Acacia tortilisand Acacia nilotica. Observations revealed that different arbuscular mycorrhizal species varied in their efficacy to improve biomass production and nutrient uptake in the two Acacia species. Scutellospora nigra was found to be most efficient in increasing biomass production and nutrient uptake of Acacia tortiliswhilegigaspora gigantea was proved most efficient for Acacia nilotica. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 52

58 The improved biomass production of the two Acacia species by AM fungi during present study could be due to improved nutrient status of the host plant provided by efficiently root colonization by the particular AM endophytes. Phosphorus nutrient exerts a significant influence on plant growth and development.arbuscular mycorrhizae acts as biofetilizer for the host plant having attachment of their hyphal system with root s of the plant acting as extension to the root system. Thus, external hyphae of the mycorrhizal plant more rapidly exploit a given volume of soil for available P then roots of non-mycorrhiza plant. Under nutritional deficient conditions AM fungi increases mobility of P (which is very less mobile under natural conditions) thereby increase in the availability of the nutrients for the host plants. The bidirectional exchange of nutrients is the basis of the arbuscular mycorrhizal symbiosis; in this way, the fungus interacts with host plant roots to increase their absorption of water, phosphate, and other nutrients from the soil. In turn, the plant provides photosynthesized sugars to the fungus, a phenomenon that provokes many cellular, physiological, and energetic changes in the host roots (Ramos et al. 2009). Linderman (1999) reported that the response of plant to VAM fungi is highly variable, being influenced by host plant physiology, genotype, environmental conditions and root excretions. In order to have a good plant growth effective mycorrhizal symbiosis is most essential. Hence, screening for efficient VAM fungi for a particular plant suitable to a particular agro-climatic region is needed. Al-Karaki (2000) reported improved growth and nutrient uptake of tomato plants under slat stress conditions. Fidelibus et. al., (2000) reported variation in efficacy of different AM fungi to improve growth of lemon under dry soil conditions. Bhattacharya and Bagyaraj (2002) reported variations in effectiveness of different AM fungal isolates on coffee. They suggested that extent of growth and nutritional status enhanced by AM fungi varied with the isolates of AM fungi inhabiting the roots of coffee seedlings. Arbuscular mycorrhizal (AM) fungi facilitate inorganic N (NH4 + or NO3 )uptake by plants, but their role in N mobilization from organic sources is unclear. They hypothesized that arbuscular Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 53

59 mycorrhizae enhance the ability of a plant to use organic residues (ORs) as a source of N.This was tested under controlled glasshouse conditions by burying a patch of OR in soil separated by 20-μm nylon mesh so that only fungal hyphae can pass through it. The fate of the N contained in the OR patch, as influenced by Glomus claroideum, Glomus clarum, or Glomus intraradices over 24 weeks (Atul-Nayyar. et al,2009). Nikolau et. al., (2002) reported variation in different mycorrhizal species on biomass production and mineral uptake of Vitis venifera. Hart and Reader (2002) reported the variation in efficacy of different AM fungi for biomass production and nutrient uptake could be due to difference in the size of the mycelium of the AM fungi. Allison (2002) also observed similar type of results in Achillia millefolium and suggested that the variation could be due to difference in nutritional status of the host plant. Cavagnaro et. al., (2003) reported relative variation in effectiveness of different AM fungi on growth and P nutrition of a Paris type arbuscular mycorrhizal. Scagel (2004 a) reported increased nutrient uptake and biomass production of harlequin flower due to AM fungi. Linderman and Davis (2004) reported varied response of marigold genotypes to inoculation with different AM fungi. Jamalluddin and Chandra (1995) reported improved growth performance of Eucalyptus by VAM fungi in the coalmine spoils of Korba due to improved nutrient uptake. Verma and Jamalluddin (1995) reported mycorrhiza mediated improved biomass production of Tectona grandis due to improved nutrient uptake by the endophytes.jamalluddin et al., (1997) reported symbiotic relationship of VAM fungi in different bamboos. Chandra and Jamalluddin (1999) reported variation in percentage of root colonization by VAM fungi in different plant species. Bhattacharya et. al., (1999) reported improved biomass production and nutrient uptake in bamboos in wasteland soils. Mathur and Vyas (2000) reported improved biomass production and nutrient uptake of Ziziphus mauritiana by different AM fungi under drought stress conditions. Different species and even geographic isolates of the same species of AM fungi might vary with respect to their ability to colonize roots and improve plant growth (Graham et. al., 1996; Pelletier and Dionne, 2004). Relatively high water and nutrient soil inputs might, Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 54

60 overtime favour proliferation of species or strain of AM fungi colonizing Citrus roots (Fidelibus et. al., 2000). Hence, from the above discussion it is clear that different arbuscular mycorrhizal fungi vary in their efficacy to improve biomass production and nutrient uptake of the host plant. However, the important factors that determine the potential benefits of a particular mycorrhizal species to a host plant are the nutritional level of the host plant, availability of nutrient in the rhizosphere soil of the particular plant, the root system of the plant and efficiency of the particular mycorrhizal species to compensate the nutritional requirement of the host plant. In order to further understand the physiology of symbiosis experiment were also conducted to evaluated the potentiality of different AM fungi towards uptake of micronutrients, Zinc (Zn), Iron (Fe), Copper (Cu) and Manganese (Mn) as well as chlorophyll, sugars, starch, protein, carotenoids and phenolic contents in the two Acacia species. The observations revealed considerable increase in all the parameters due to different AM fungi in both these Acacia species. Adriaensen et. al., (2004) reported increased uptake of zinc by pines inoculated with mycorrhizal fungi. Bi et. al., (2003 a) reported increase uptake of Zn by red clover at early stages of arbuscular mycorrhizal development. Jamal et. al., (2002) reported increase uptake of Zn and Nickel (Ni) form contaminated soil by soybean due to arbuscular mycorrhizal association. Liao et. al., (2003) reported increased uptake of heavy metals by arbuscular mycorrhizae under different soil types. Mogueira et al., (2002) reported removal of Mn toxicity by soybean due to mycorrhizal symbiosis. Chen et. al., (2003) reported increased Zn uptake by red clover growing in a calcareous soil by arbuscular mycorrhizae. Al- Karaki et. al., (2000) reported increased uptake of Zn, Cu and Fe in tomato by arbuscular mycorrhizae under salt stress conditions. Schubert et. al., (2004) reported increase in sucrose content in roots of soybean colonized by different AM fungi. Mathur and Vyas (1995 I) reported increased chlorophyll, carotenoids, sugar and protein content of Ziziphus zylopyrus by different VAM species. Joseph et. al., (1999) reported increase sugar and starch contents in Pueraria Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 55

61 phaseolidesinoculated with 11 different mycorrhizal species. Prasad and Bilgrami (1999) reported increased chlorophyll and sugar content in saccharum officinarum due to VAM inoculation. Prasad (1999) reported increased chlorophyll, sugar and protein content in Acacia nilotica due to inoculation with indigenous fungi. Auge (2001) reported increased chlorophyll content of different host plants due to VA mycorrhizal symbiosis. Declerck et al., (2002) reported increase chlorophyll and sugar contents in micropropagated bananas by in vitro monoaxenically produced arbuscular fungi. Mathur and Vyas (2000) reported increased biomass production, nutrient uptake, protein, chlorophyll, and sugar contents of Ziziphus mauritiana by different VAM fungi under water stress conditions. From the above discussions it is clear that arbuscular mycorrhizae brings about certain physiological changes of the host plants by improving carbohydrates, protein and photosynthetic pigments in different plant species. This beneficial effect of the endophytes could be attributed to either improved nutrient uptake which resulted in over all change in the metabolism of the host plant or it can be due to improved leaf surface area which resulted in increase photosynthetic rate thereby increasing the carbohydrates contents of the host plants. The above physiological changes in the two Acacia species could also be due to increasing various enzymatic activities like phosphatases, nitrate reductase, peroxidase, poly phenol oxidase etc. In view of these facts experiments were conducted to find out application of different AM fungi towards biochemical changes of the two Acacia species. Observations revealed that significant increase in activities of all the four enzymes were recorded due to different arbuscular mycorrhizal species in both Acacia species. Pearson et. al., (1991) reported that increased phosphates activity (both acid and alkaline phosphatases) by VAM fungi is due to presence of specific isozymes of the two enzymes in AM colonized plants. Zhu and Smith (2001) reported increased phosphatases of wheat plant by arbuscular mycorrhizal plant under field condition. Buscot et. al., (2000) reported changes in various enzymatic activities by mycorrhizal symbiosis in natural ecosystem. Mathur and Vyas (2000) reported biochemical changes in Tamarix aphylla by different VAM fungi. This Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 56

62 phosphatases activity (increased) due to above reasons might also be responsible for P uptake in mycorrhizal colonized plants. Similarly increase in nitrate reductase activity in the two Acacia species was observed irrespective of the mycorrhizal treatment. Nitrate reductase is one of the important enzymes of nitrogen metabolism in all the plant. This increased NR activity in roots and leaves of mycorrhizal colonized clover was attributed to improved P nutrition provided by the mycorrhizal symbiosis. Similarly, Mathur and Vyas (1995 II) reported increase in NR activity in Ziziphus nummularia by different VA mycorrhizal fungi. Endomycorrhizal fungal species like Glomus macrocarpum and Glomus mossae have also been shown to reduce nitrate ions. Mc Farlend et. al., (2002) reported increased nitrate reductase activity of the plants of a deciduous forest ecosystem by arbuscular mycorrhizae. Hobbi and Colpaert (2003) reported increased nitrate reductase and Glutamine synthetase activity in different plant species by arbuscular mycorrhizae. The results in present study suggest that with a capacity for reducing nitrate, it is likely that the symbiotic effectiveness of the arbuscular mycorrhizal fungi is enhanced in terms of nitrogen acclimation and translocation to the host plant. The increased peroxidase and polyphenol oxidase activities in the two Acacia species by different AM fungi during present study can be important. These two enzymes are of great importance in the defense mechanism of the plants against attacking pathogens. These two enzymes bring about oxidation of phenols into quinines, which are well to be toxic to the plant pathogens. The observe increase in peroxidase activities during the present study is indirect effect of the mycorrhizal symbiosis. Further, it is the P mediated effect on peroxidase activity (which was provided by the mycorrhizal symbiosis). Lower peroxidase activity is well known in low P roots than in high P roots (Mc Arthur and Knowels, 1992). Reduction in such activities may be indicative of a lower capacity for the induction of a defense response from the plant. Since low P roots have lower capacity for ethylene generation and thereby also have less peroxidase activity then high P roots which further suggests that high P roots have high capacity for ethylene generation which results in higher peroxidase activity hence increased P uptake by AM fungi might have resulted in increase P activity of the two Acacia species during the present study. Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 57

63 A positive correlation was observed between total phenolic accumulation and polyphenol oxidase activity in AM colonized both Acacia species in the present study. Accumulation of phenol in mycorrhizal colonized plant has been well recognized in plant. Mathur and Vyas (1995 III) reported changes in isozyme patterns of peroxidase and polyphenol oxidase in roots of Ziziphus species by different VAM fungi. They reported correlation between increase in isozyme numbers and activity of both these enzymes by the AM fungi. Hence during present study the increase peroxidase and polyphenol oxidase activities in the two Acacia species can also be due to mycorrhiza specific isozymes of the two enzymes in the roots of both the Acacia species. The present study clearly reveal application of arbuscular mycorrhizal fungi in improving status of arid and semi arid regions of western Rajasthan in various ways i. e. by improving nutrient status and biomass production of the Acacia species, by removing higher metals from the wastelands, by changing host plant physiology. Pelletier and Dionne (2004) reported improved survival and establishment of turf grass without irrigation and fertilizer inputs by inoculating with AM fungi. All these factors collectively would contribute for development of arid and semi arid wastelands. Chapter 6 Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 58

64 Summary Salinisation of soils and ground water is a serious land degradation problem in arid and semi arid areas, and is increasing steadily in many parts of India, causing major problems for land productivity. On a world scale there is an area of around 380 million hectares that is potentially usable for agriculture, but where production is severely restricted by salinity. These areas occur predominantly in regions where evaporation exceeds precipitation. The problem of saline soils is ever increasing, due to poor irrigation and drainage practices, expansion of irrigated agriculture into arid zones with high evapotranspiration rates, or land clearing, which leads to rising saline water tables i.e. dry land salinity. Dry land salinity is a major environmental problem in arid and semi-arid regions of India. The impact of agricultural clearing through salinisation extends across the country, but they are particularly severe in saline areas of Indian Thar Desert, which covers nearly 45% area of this region. The major parts of saline habitat includes Luni, Pachpadra, Balotra, Bikaner, Churu, Osian, Nagaur, Barmer and Jaisalmer to Kuchh of Ran. Physical, chemical and biological constrains in soil horizons impose an additional stress on plants in these habitats, restricting plant growth and development. Hard setting, crusting, compaction, acidity, alkalinity, nutrient deficiency and high temperature are major factors that cause these constrains. Productivity of many salt affected soils has declined due to inappropriate land use practice, over grazing or removal of trees for various purposes. In Indian Thar Desert irrigation by fresh water is not possible, due to severe scarcity of water. Thus exploiting the possibilities of using salt stress water for irrigation, especially drainage and underground water is of great importance. Thus, there is an urgent need to develop new technologies to cope with these adverse climatic conditions. Mycorrhizal fungi are well recognized as biofertilizers now a day. Due to their manifold benefits provided to the host plant, they are being frequently used in revegetation and reclamation programme worldwide. By improving nutrient uptake and water transport, they help the plants to survive more efficiently under adverse climatic conditions of drought prone areas. Mycorrhizal fungi have also been shown to reduce transpiration rate and increase water use efficiency of plants under arid and semiarid conditions, where water is the most important factor which determine plant growth. Under arid and semi arid conditions water Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 59

65 is the most important factor which determine plant growth.arpuscular mycorrhizae improve water economy of plants. Major constrains imposed by saline habitat are physical, chemical and biological; like structural decline in the form of compaction and crusting,high concentration of sodium salts, chloride,carbonate and bi carbonate and soil borne pathogens. Arbuscular mycorrhizal fungi by various mechanisms help the plant to over come these constrains thereby improving their survival and establishment under saline habitats. AM FUNGI BETTER EXCESS TO NUTRITIONAL STATUS MODIFICATION OF PLANT PHYSIOLOGY i.e.osmotic MODIFICATION PHOTOSYNTHESIS PROTECTION AGAINST SALT STRESS MYCORRHIZAL MECHANISM FOR SURVIVAL OF PLANTS UNDER SALINE HABITAT Mycorrhzal Technology for Reclanation of Saline Waste Land of Indian Thar Desert 60

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