A PROCEDURE FOR ISOLATION OF SINGLE-SPORE CULTURES OF CERTAIN ENDOMYCORRHIZAL FUNGI

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1 Aw Phytol. (1983) A PROCEDURE FOR ISOLATION OF SINGLE-SPORE CULTURES OF CERTAIN ENDOMYCORRHIZAL FUNGI BY YU-CHENG FANG«, A.-C. MCGRAW, HAKAM MODJO AND J.W. HENDRIX Department of Plant Pathologv, University of Kentucky, Lexington, KY 4546, USA (Accepted 19 April 1983} SUMMARY.\ procedure for the isolation of single-spore cultures of endomycorrhizal fungi is described. A single spore is placed on plant roots with a Pasteur pipette by use of a binocular microscope, and the plant is transplanted into sand in a 5 ml growth tube. Plants are incubated for 6 to 8 weeks in a greenhouse and are continuously subirrigated by capillarity with dilute (5 to 1%) Hoagland's solution. Forty to 8 o of plants inoculated by this technique with single spores of Gtomus macrocarpus, G. etunicatus and G. caledonius became infected. Cultures of G. constrictus and G. microcarpus were not obtained. Hoagland's solutions of up to 25,, were equally effective for isolating G. macrocarpus, but limiting plant size by using only 5 or 1% Hoagland's solution was advantageous. The presence of spores on living root systems was the most reliable indicator of fungal development. Non-inoculated control plants incubated in a common Hoagiand's solution reserv^oir with inoculated plants never became mycorrhizal. The procedure uses readily available materials, is easy to use in the laboratory- and conserves greenhouse space and labour. INTRODUCTION Arable soils contain a complex community of species of endomycorrhizal fungi (e.g. Mosse and Bowen, 1968; Schenck and Kinloch, 198; Schenck and Smith, 1981). In our studies, we have identified as many as 12 species in one agricultural soil (McGraw and Hendrix, unpublished). However, research on the influence of a community of mycorrhizal fungi on plants is lacking. Furthermore, most studies of the influence of a single mycorrhizal species on a plant species have utilized only a few commonly-used cultures (e.g. Glomus fasciculatus, Glomus mosseae, Gigaspora margarita and Glomus etunicatus). Cultures are usually established and maintained by transfer of a minimum of 25 spores. Mycorrhiza! fungi, like other microorganisms, can be expected to consist of a diversity of morphologic and biotypic variants. However, research on variability is impeded by cultural procedures now in use. The obligately symbiotic nature of endomycorrhizal fungi places a practical limit on the procedures which may be employed to establish genetically homogeneous cultures. We concluded that the best option is to produce cultures from single spores. Our objective was to develope a technique for isolating single-spore cultures which succeeds with all the species present in a soil, is reproducible and conserves time and greenhouse space. Permanent address: Agricultural College of Shandong, Taian, Shandong 27118, People's Republic of R.-'hina. IWI28-646X/83/9O17 + O8 J3./ 1983 The New Phytologist

2 io8 Y.-C. FANG et al. MATERIALS AND METHODS The species studied were those predominating in soils used for cultivating burley tobacco in central Kentucky: Glomus macrocarpus var, macrocarpus (Tul, & Tul.) Gerd & Trappe, G. constrictus Trappe, G. etunicatus Becker & Gerd., G. caledonius (Nicol. & Gerd,) Trappe & Gerd, and variants of G. microcarpus (Tul. & Tul,) Gerd. & Trappe which have red-brown or hyaline spores. Spores were collected by wet-sieving and decanting soil from one of two field locations (Franklin County and Scott County, KY) or from pot cultures originally obtained from one of the locations (Franklin County), (5 g) was dispersed in 1 1 of tap water, allowed to settle for 15 s, and sieved through 85 and 38 /im sieves, Sievings from the latter were centrifuged in deionized water in 5 ml tubes at 127 g for 3 min. The pellet was resuspended in 25 ml of 45 % (w/v) sucrose and centrifuged at 127g for 1-5 min with the centrifuge brake operating. The supernatant was sieved quickly on a 38 fim sieve, and the spores were rinsed with tap water, then rinsed with dionized water into a plastic Petri dish. Spores were sometimes stored for several days at 5 C. Spores of G macrocarpus stored in deionized water at 5 C had undiminished infectivity after 3 months. Tobacco {Nicotiana tabacum L, cv, Kentucky 15) was usually used as the host because of our interest in isolating cultures for use in tobacco research. Other hosts included tall fescue (Festuca arundinacea Schreb, cv, Kentucky 31), orchard grass {Dactylis glomerata L.), r\^e {Secale cereale L.), wheat {Triticum aestivum L,), sorghum-sudangrass hybrid [Sorghum bicolor (L.) Moench, x 5, sudanense (Piper) Stapf. cv. FFR-66] and red clover {Trifolium pratense L,). Seeds were sown in vermiculite. Seedlings were transplanted to sand in growth tubes (described below) and grown in full-strength Hoagland's solution (Hoagland and Amon, 195). Transplants of the following ages were used for experiments: tobacco, 6 weeks (four-leaf stage); sorghum Sudangrass hybrid, wheat and r\'e, 3 weeks; orchard grass, fescue and red clover, 4 weeks. Experiments were conducted in 5 ml growth tubes of black plastic, 2*5 cm diameter x 1 cm long. Plywood sheets (6 x 6 x 1 cm thick) with holes bored to support ten rows of ten tubes were suspended over nutrient solution reservoirs confined by black polyethylene film supported by lengths of timber (1 X 1 X 24 cm). Plants received Hoagland's solution continuously by capillarity through holes bored in the bottoms of the tubes. The growth medium (sand or sand-soil mixtures) was retained by glass marbles (1-7 cm diameter) in the bottoms of tubes. The sand was river-dredged commercial building sand. The soil was from the A horizon of a Maury silt loam, a highly productive agricultural soil. Analyses of the sand and soil are given in Table 1. Before inoculation, root systems of plants were carefully rinsed free of sand and cut to 4 cm in length. Plants were inoculated by transferring single spores with a Pasteur pipette to the root system 2 cm from the crown. The placement of the spore was observed with a dissecting microscope (25 x magnification) [Fig, l(a)] Growth medium was then carefully poured around the root system in the growth tubes. Plants were maintained in a greenhouse for 8 to 12 weeks. Roots growing into the nutrient reservoir were removed periodically. Plants were prepared for evaluation of development of cultures by immersing the root system in a container of water and gently agitating to remove the growth medium. The root system was examined for sporulation with a dissecting microscope (25 x magnification) [Fig. l(b)]. Colonization of roots was furtber

Single-Spore cultures of mycorrhizal fungi IOQ Table 1. Chemical analysis of autodaved sand and soil used as plant growth media Test Spurway and Lawton (1949). t Bray and Kurtz (1945).

3 Single-Spore cultures of mycorrhizal fungi IOQ Table 1. Chemical analysis of autodaved sand and soil used as plant growth media Test Spurway and Lawton (1949). t Bray and Kurtz (1945). Spurway P (jig ml ') Bray 1 P (kg ha^') K (kg ha"') Ca (kg ha') Mg (kg ha'') Zn (kg ha-') Cu (kg ha ') Mn (kg ha-') Organie matter (%) ph (water) g 281 S Fig. 1 (a) Placement of a single spore of Giomus macrocarpus on roots of a tobacco plant with a Pasteur pipette, (b) Sporulation of G. macrocarpus on roots of a tobacco plant inoculated with a single spore. Both photographs taken by use of a dissecting microscope-

4 no Y.-C. FANG et al. estimated with a no-heat method for clearing and staining with tr>'pan bluelactopheno] (Kormanik and McGraw, 1982). Root systems were categorized as follows: (1, < 5 o colonization; 2, 5 to 25 %; 3, 25 to SO %; 4, > 5 %. Plant roots were also categorized for the presence of several fungal structures. In some experiments, the percentage of root length colonized was determined by a grid-line intersect method (Giovannetti and Mosse, 198). Spores not adhering to roots were collected by sieving the plant growth medium. RESULTS Single-spore cultures were obtained on tobacco with G. macrocarpus, G. etunicatus and G. caledonius with 4% or higher success when 1% Hoagland's solution was used (Table 2). No cultures were obtained with G. constrictus or with the red-brown G. microcarpus. Sporulation by G. macrocarpus was observed as early as 4 weeks after inoculation. Sporulation occurred on 5 "o of inoculated plants at 4 weeks, and this percentage did not increase over an 18-week period. After 5 weeks, all spores observed were mature (pigmented), and sporulation was more profuse than after 4 weeks. Undiluted sand appeared to be as effective for obtaining pure cultures of mycorrhizal fungi as media with soil (Tables 2 and 3). The second experiment suggested that increasing the proportion of soil reduced fungal development Table 2. Influence of nutrient level and composition of plant growth medium on colonization of tobacco plants by single spores of five mycorrhizal fungi after 8 to U weeks Species Source of spores Medium Hoagland's solution (%) No. plants colonized/no. plants inoculated Frequency (no plants) amon^ root colonization classes* G. macrocarpus G. constrictus G. etunicatus G. caledonius G. microcarpus (red-browti) Control 1 (non-inoculated) Control 2t t f 3 : 1 soil 3 :l soil /3 37/5 1/2 11/5 /3 /6 /2 /5 21/26 /1 4/1 /7 /5 /1 / n Root colonization classes; 1, < 5 % of root system colonized; 2, 5 to 25%; 3, 25 to 5%; 4, >io,. t Natural soil was from the Franklin County source. The pure culture was originally isolated from soil from the same source. X Inoculated with 1 ml of a supernatant liquid prepared as follows; SO g soil was sieved through a 85 fi"' sieve. The material collected on a 38/tm sieve was brought to 3ml and centrifuged for 3 min at 127^

5 Single-Spore cultures of mycorrhizal fungi III Table 3. Influence of soil content of plant grotvth medium on culture development by single spores of a pure culture of G lomus macrocarpus and on growth of tobacco plants in 1 o Hoagland's solution for weeks Medium No. plants colonized/ no. plants inoculated No. plants with fungal structures External Vesicles Arbuscules hyphae Spores Plant height (cm)* Inoculated 3 sand; 1 soit 2 sand: 1 soil 8 sand ; 1 soil 32 sand:l soil 1 sand : soil \oti-inoculated 1 sand: soil 5/2 6/2 9/2 11/2 1/2 / I 7 g g S ± ± ± ±-8 Mean of heiffhts of all 2 plants + standard error. (Table 3). A white, quartz sand was not superior to the common construction grade used in these studies. As expected, high nutrient concentrations reduced development of G. macrocarpus (Tables 2 and 4). Similar percentages of plants were colonized at concentrations of Hoagland's solution of up to 25 ", but plants were much larger at the higher concentrations (Table 4). Occurrence of vesicles, arbuscules, external hyphae and spores was similar up to 25 "o Hoagland's solution, but 5 o Hoagland's solution reduced funga! colonization structures about tenfold and reduced the number of colonized plants to half. At loo*',, Hoagland's solution, only one plant became colonized (Tables 2 and 4) and then only lightly (Table 2). Differences in plant sizes suggest that the inhibition of plant colonization by higher proportions of soil may be a nutritional effect (Table 3). Inoculation had little influence on plant size (Table 4). After the initial failure to isolate single-spore cultures of G. constrictus and G. microcarpus (Table 2), further attempts were made. Both the red-brown and hyaline variants of G. microcarpus were included in the study of effects of nutrient concentrations on culture development (Table 4), but no cultures were obtained. ^ limited study on host range was conducted with G. constrictus and G. etunicatus. Often plants per host species inoculated with single spores, two cultures of G. etunicatus were obtained on fescue, red clover and wheat; one culture on orchard grass and tobacco, and none on rye and sorghum-sudangrass hybrid. If more plants had been inoculated, cultures would probably have been obtained at least with sorghum-sudangrass hybrid; inoculum of G. etunicatus isolates obtained from abandoned mine lands has been grown on this host in this laboratory (Kiernan, Hendrix and Manonek, 1983). Parallel inoculations often plants of each host species with G. constrictus did not yield cultures. Sporulation occurred on all plants colonized by G. macrocarpus (Tables 3 and "^j. External hyphae and spores usually occurred together. The degree of sporulation appeared related to the extent of external hyphae. This fungus produces few vesicles "1 tobacco, and arbuscules are considered transient; therefore, vesicles and arbuscules are less reliable indicators of colonization than are spores and external

6 112 Y.-C. FANG et al Table 4. Influence of nutrient level on colonization of tobacco plants by single spores of Glomus macrocarpus* isolated from the Scott County soil and on plant size after vieeks Paratneter 5 Hoagland s solution (" [ No. plants colonized/ no plants inoculatedf No of plants withj Vesicles.\rbuscuies External hyphae Spores Root length colonized ( o)t Vesicles Arbuscules External hyphae Spores Plant height (cm)fl inoculated Non-inoculated 31/ ±I /4 13 IS ±-2 24/ / ll-4± / / Forty- plants per treatment were also inoculated with the G. microcarpus red-brown and hyaline variants Roots were not colonized. Plant growth was sin^ilar to that with G. morrocarpuj-inoculated or non-inoculated plants. t Data are totals of three inoculation dates within 3 weeks in which 15 plants per treatment were inoculated on each of two dates and ten plants per treatment were inoculated on the third date. Spores were stored in deionized water at 5 C. t -25 g (fresh weight) root sample examined Means of all plants colonized + standard errors. fl Means of 4 inoculated or ten non-inoculated plants + standard errors. hyphae. With dilute nutrient solutions and sand as plant growth medium, single cultures often yielded more than 1 spores. Non-inoculated control plants were incubated with inoculated plants to test the possibility of contamination through the nutrient solution reservoir. Colonized non-inoculated plants were not found. DisccssroN This procedure mostly satisfies the objective of the research. The materials are both easily obtained and easily manipulated in the laboratory and in the greenhouse. The tube system is compact, permitting the incubation of more plants per unit area of greenhouse bench than any kind of pot system, and the inoculated plants are transported readily from laborator>' to greenhouse. In this system, 27 plants m~^ were incubated. Tube and rack systems which permit incubation of more than 1 plants m"^ are available commercially. Care of incubating plants is minimal because plants are not watered individually. If plants are kept small by using dilute nutrient solutions, water losses by transpiration are small, and nutrient solutions need be replenished only occasionally, perhaps weekly. Further, the control of insect pests, which can build up during the 6 to 8 weeks needed for culture development, is facilitated. The superiority of sand to mixtures of sand and soil in obtaining cultures is fortunate, for sand is readily washed from roots, whereas soil with large amounts

7 Single-Spore cultures of mycorrhizal fungi 113 of Slit and clay is not. also causes few problems in the sieving and centrifugation procedures. Although connmercial sand is probably variable, batches of sand obtained at different times are probably more uniform than are batches of soil. The sand we used contained a considerable amount of limestone, which probably is to be expected in river-dredged sand in this region of frequent limestone deposits. The contribution of the lime or Ca'^* to the success of the method is not known. The high ph of the sand may affect development of some fungi, and this factor should be considered if success is not obtained with a particular species. The reliability of sporutation as an indicator of colonization by G. macrocarpus is also fortunate. Entire living root systems can be examined readily for the presence of spores w ith a bmocular microscope. This procedure is non-destructive and permits culture identification, which is not possible if vesicles, arbuscules or hyphae are the diagnostic characters. The labour involved in clearing and staining roots also is not required. The objective of obtaining cultures of all species present in a soil was not attained. We have no explanation for our lack of success with G. constrictus and G. microcarpus. Failure of G. microcarpus to develop on tobacco was not due to nutrient concentration. Sporulation of G. microcarpus, along with G. macrocarpus and G. etunicatus, has been obtained on tobacco in this system when soil sievings, rather than isolated spores, were used as inoculum (Modjo and Hendrix, unpublished). The lack of success in obtaining cultures of G. constrictus on one of the host species tried is also not understood. This species must have occurred in the soils studied because it colonized and reproduced on a plant host which grew on the land. The hosts tested were selected because of their use in systems for tobacco production. Tobacco with winter cover crops of rye or wheat is rotated with turf crops of fescue or orchard grass, with or without red clover. When tobacco is grown, few weeds are allowed to develop. Weeds may develop in the turf crops, but the spores of G. constrictus were obtained from soil which had been in tobacco continuously for several years. Glomus microcarpus frequentiy occurs m high populations, and our inability to obtain single-spore cultures of this species and G. constrictus impedes our objective of studying experimentally the ecology- of synthesized communities of mycorrhizal fungi occurring in agricultural soils. ACKNOWLEDGEMENTS Financial support was provided by grants from R. J. Reynolds Tobacco Company and the US Department of Interior Office of Surface Mining. The assistance of two farmers, Sam Tracy and Victor Perkins, and greenhouse and laboratory assistance by George Braman, Janet Finley, Kelly Hobbs and Chris Hoskins, is appreciated. Photography was done by Keith Jones. REFERENCES BBAY. R. H. & Ki RTZ. L. T (1945). Determination of total, orranic and available forms of phosphorus in soils. Smi Science, 59, 39-t5. GIOVANNETTI, M. & MOS.SE, B. (198). An evaluation of techniques to measure ves.cul.r-arbuscular infections m roots. Nni- Phytologisl, 84, HOAGLAND, D R. & ARNON, D. I. (195). The uater culture method for growing plants without soil. taltjornta Agricultural Experiment Station Circular 347.

114 Y.-C. FANG et al. KIERNAN, j. M., HENDRIX, J. W. & MARONEK, D. M.( 1983). Fertilizer-inducedpathogenicity of mycorrhizal fungi to sweetgum seedlings..soii Biology and Biochemistry (in press).

8 114 Y.-C. FANG et al. KIERNAN, j. M., HENDRIX, J. W. & MARONEK, D. M.( 1983). Fertilizer-inducedpathogenicity of mycorrhizal fungi to sweetgum seedlings..soii Biology and Biochemistry (in press). KORMANIK, P. P. & MCGRAW. A.-C. (1982). Quantification of vesicular-arbuscular mycorrhizae in plant roots. In Methods and Principles of Mycorrhixal Research (Ed. by N. C. Schenck), pp American Phytopathological Society, St. Paul, Minnesota. MOSSE, B. & BOWEN, G. D. (1968). The distribution of Endogone spores in some Australian atid New Zealand soils, and in an experimental field soil at Rothamsted. Transactions of the British Mycoiogicat Society, SI, SCHENCK, N. C. & KINLOCH, R. A. (198). Incidence of mycorrhizal fungi on six field crops in monoculture on a newly cleared woodland site. Mycologia, 72, SCHENCK, N. C & SMITH, G. S. (1981). Distribution and occurrence of vesicular-arbuscular mycorrhizal fungi in Florida agricultural crops. and Crop Science Society of Florida Proceedings, SPURWAY, C. H. & LAWTON, K. (1949). testing-a practical system of soil diagnosis. Michigan Agricultural Experiment Station, East Lansing, Michigan. Technical Bulletin No 132. <4th revision).

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION

COMPONENTS OF VA MYCORRHIZAL INOCULUM AND THEIR EFFECTS ON GROWTH OF ONION New Phytol. (1981) 87, 3 5 5.161 355 OMPONENTS OF VA MYORRHIZAL INOULUM AND THEIR EFFETS ON GROWTH OF ONION BY A. MANJUNATH AND D. J. BAGYARAJ Depart?nent of Agricultural Microbiology, University of Agricultural