viability of soilborne spores of glomalean mycorrhizal ...

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Soil Biol. Biochem. Vol. 30, No. 8/9, pp. 1133±1136, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain S0038-0717(97)00194-6 0038-0717/98 $19.00 + 0.00

VIABILITY OF SOILBORNE SPORES OF GLOMALEAN MYCORRHIZAL FUNGI Z. Q. AN, B. Z. GUO and J. W. HENDRIX* Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0091, U.S.A. (Accepted 21 July 1997) SummaryÐSpores of glomalean (arbuscular) mycorrhizal fungi indigenous to soils in a central Kentucky cropping system were about 50% viable. The range was 35 to 60%, regardless of spore population density, time of year, or crop. Viability sometimes rose at the time new spores were produced in late summer and fall, but not appreciably. In the absence of extreme treatment of soil, such as fumigation or steaming, about half of the spores present in surface soil may be considered viable in the soil studied here. Elsewhere, a few viability tests should be conducted to con®rm this proportion. # 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Understanding the roles of glomalean (arbuscular) mycorrhizal fungi in the biology of their hosts must include understanding the roles of chlamydospores or azygospores in the biology of the fungi. Population dynamics of spores and propagules are often related (An et al., 1993b; Fischer et al., 1994) but sometimes di€er (An et al., 1993b), and a careful assessment of both is essential to understanding the host±fungal relationship. With drastic treatments, such as soil fumigation, dead spores that appear normal may persist in soil for extended periods (McGraw and Hendrix, 1984, 1986). Assessing viability of spores is necessary for determining their potential as propagules. Until recently, determining spore viability was dicult. Spore viability often is evaluated by germination tests (e.g. Tommerup and Kidby, 1979; Tommerup, 1983; Gemma and Koske, 1988). Because these fungi are obligate biotrophs, development of germination hyphae on agar media is limited. Spores from ®eld soils also are coated with soil materials, and the accompanying bacteria often interfere with germination tests. Dormant spores (Gemma and Koske, 1988) would not be detected as viable. Viability can also be assessed by inoculating host plants with single spores (Fang et al., 1983). With homogeneous spore populations, such as from a fresh pot culture, inoculation tests will detect a certain percentage (e.g. 70%) of viable spores consistently (An and Hendrix, 1988). However, spores from ®eld soils may seldom be homogeneous, particularly in experiments involving disparate treatments such as hosts, non-hosts such *Author for correspondence.

as cruciferous crops, and fallow (Black and Tinker, 1979). Germination tests and single-spore inoculations are labor-intensive, and days or weeks are required to obtain results. Others and ourselves developed a method using the vital stain MTT (An and Hendrix, 1988; Meier and Charvat, 1993; Walley and Germida, 1995) or INT (Walley and Germida, 1995). This method yields results in less than 2 d. Using this procedure with MTT, Diop et al. (1994) found the method useful in determining viability of spores in Senegal soils varying in a number of edaphic, climatic and host factors. Our objective in this study was to determine the viability of indigenous mycorrhizal fungal spores present in a central Kentucky cropping system over the course of a year. The main crops in the cropping system were dissimilar: tobacco (Nicotiana tabacum L.), a C4-broadleaf crop, and tall fescue (Festuca arundinacea Schreb), a C3-grass. The results of this experiment are indicative of the viability of mycorrhizal fungal spores in agricultural soils of this region.

MATERIALS AND METHODS

This study was conducted on two adjacent tracts of land with similar soil type (Elk silt loam, Finesilty, mixed, mesic, Ultic, Hapludalfs) and slope on a farm in Franklin County, central Kentucky. One tract had been in tall fescue pasture for at least 30 years, and the other had been in sorghumsudangrass hybrid (Sorghum bicolor L. Moench. X S. sudanense [Piper] Staph., SSGH) for 3 y. The fescue sod was plowed with a moldboard plow in the late fall of 1988. Early in 1989, the SSGH stubble was plowed under with a moldboard plow,

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Fig. 1. Populations densities of total and viable spores of glomalean mycorrhizal fungi in soils from a tobacco cropping system. First letter = long-term crop (S = sorghum-sudangrass hybrid, F = tall fescue). Second letter = short-term crop (F = tall fescue, T = burley tobacco). G. = Glomus. Gi. = Gigaspora.

and both tracts were strip-planted to either fescue (cv. Kentucky 31, sown on 1 March) or tobacco (cv. TN 86, transplanted on 1 May). Fescue plots were mowed occasionally during the summer and fall but were not disturbed otherwise during the

sampling period. Tobacco was harvested on 1 September. Wheat was broadcast-planted as a cover crop on tobacco plots in September, 3 weeks after tobacco was harvested, and plowed under on 11 April 1990.

Viability of spores of glomalean mycorrhizal fungi

Both tracts were very high in available P and K. Details of the mineral analyses, experimental design, and sampling procedures are given by An et al. (1993a,b) and Guo et al. (1994). Samples were taken from the upper 15 cm of soil. Population densities of spores of mycorrhizal fungi in soil were determined by wet-sieving four 100-g subsamples from each plot (An et al., 1990) and identifying according to Schenck and Perez (1987, 1988). Viable spores developing a red color with the tetrazolium bromide vital stain MTT [3-(4,5dimethylthiazol-yl)-2,5-diphenyl-2H-tetrazolium bromide] were determined by the procedure of An and Hendrix (1988). Spore suspensions were diluted 1:1 with a stock solution of 0.5 mg MTT mlÿ1 and incubated for 40 h. Before staining, species distinguished by color in the natural state were segregated into separate dishes with a micropipette. Of the 16 species in three Glomales genera identi®ed (An et al., 1993a), data for six species with very low and variable population densities are not presented here. In addition, data for Glomus constrictum Trappe were not obtained because the natural red color of its spores could not be distinguished from the red color associated with a positive reaction to the vital stain. Population densities of total and viable spores and percentages of viable spores were plotted as a function of time.

RESULTS

Regardless of time of year or treatment, the proportion of viable spores to total spores was between 35 and 60% for most species (Fig. 1). Occasionally, viabilities as high as 75% were recorded. As reported elsewhere for the tract with a long-term history of fescue (An et al., 1993b), population densities of viable spores decreased considerably in late spring, remained low throughout the summer, and increased to approximate mid-spring (18 April) numbers by fall (27 September) (Fig. 1). This pattern was true for all combinations of long- and short-term cropping history and for nearly all species. Where population densities of spores of a species were relatively high going into summer, viabilities in early- to mid-summer were usually somewhat lower than when spores were newly formed in the fall (Fig. 1). The fescue and SSGH long-term cropping histories greatly a€ected population densities of total and viable spores of G. macrocarpum. Population densities of G. macrocarpum were higher in plots with a SSGH background than in plots with a fescue background; however, viability of this species did not di€er appreciably in the various plots.

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DISCUSSION

Our results with soil-borne spores are consistent with those of Diop et al. (1994). We found viability of spores, all taken from surface soil, to range from 35 to 60%, regardless of season or treatment. Diop et al. found spore viability in surface soil in the rhizospheres of acacia trees to be about 40%. They, and ourselves, found population density and viability to be independent. They also sampled at greater depths and found viability to increase up to 80% with depth at some sites. Cuenca and Lovera (1992) also found spores in reclaimed disturbed sites in Venezuela to be about 60% viable. With spores from pot cultures, however, we sometimes found higher viability, up to 80% with fresh cultures of G. macrocarpum, while viability of spores stored for 5 years was 50% (An and Hendrix, 1988). Viability of cultures of Acaulospora spp. has been observed to be over 90% (Gazey et al., 1993). Meier and Charvat (1993) recorded viability of fresh and stored cultures of G. mosseae to be about 50%. We conclude that viability of soilborne spores of Glomus spp. will be considerably less than 100%, usually about the 35±60% range, in surface soils. The age of the spores appears not to be in¯uential. Most spores in the ®eld apparently germinate in the spring [Fig. 1 of An et al. (1993b)], so few found in the ®eld are more than a year old. However, the age of spores was not and could not be determined in this experiment, so the relationship of age of spores to their viability is speculative. It is possible that spores which, by whatever mechanism, do not germinate the ®rst spring following formation may persist in soil for several years without losing viability. With fresh pot cultures, viability may be higher but with time decreases to about 50% and can remain at that level for years if properly stored. Therefore, it appears that viability of soilborne spores may be assumed to be about 50% unless drastic soil treatments, such as fumigation, have been applied. This generalization may not be applicable to spores of genera other than Glomus. In our study, spores of species other than those of Glomus were not prevalent enough to allow generalization. AcknowledgementsÐWe thank Sam Tracey for competently growing the crops and Janet Finley, Chris Hoskins and Ningyan Zhang for assistance.

REFERENCES

An Z. Q., Guo B. Z. and Hendrix J. W. (1993a) Mycorrhizal pathogen of tobacco, cropping history and current crop e€ects on the mycorrhizal fungal community. Crop Protection 12, 527±531. An Z. Q., Guo B. Z. and Hendrix J. W. (1993b) Populations of spores and propagules of mycorrhizal fungi in relation to the life cycles of tall fescue and tobacco. Soil Biology & Biochemistry 25, 813±817.

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An Z. Q. and Hendrix J. W. (1988) Determining viability of endogonaceous spores with a vital stain. Mycologia 80, 259±261. An Z. Q., Hendrix J. W., Hershman D. E. and Henson G. T. (1990) Evaluation of the Most Probable Number (MPN) and wet-sieving methods for determining soilborne populations of endogonaceous mycorrhizal fungi. Mycologia 82, 576±581. Black R. and Tinker P. B. (1979) The development of endomycorrhizal root systems II. E€ect of agronomic factors and soil conditions on the development of vesicular±arbuscular mycorrhizal infection in barley and on the endophyte spore density. New Phytologist 83, 401± 413. Cuenca G. and Lovera M. (1992) Vesicular±arbuscular mycorrhizae in disturbed and revegetated sites from La Gran Sabana, Venezuela. Canadian Journal of Botany 70, 73±79. Diop T. A., Gueye M., Dreyfus B. L., Plenchette C. and Strullu D. G. (1994) Indigenous arbuscular mycorrhizal fungi associated with Acacia albida Del. in di€erent areas of Senegal. Applied and Environmental Microbiology 60, 3433±3436. Fang Y. C., McGraw A. C., Modjo H. and Hendrix J. W. (1983) A procedure for isolation of single spore cultures of certain endomycorrhizal fungi. New Phytologist 93, 107±114. Fischer C. R., Janos D. P., Perry D. A., Linderman R. G. and Sollins P. (1994) Mycorrhiza inoculum potentials in tropical secondary succession. Biotropica 26, 369±377. Gazey C., Abbott L. K. and Robson A. D. (1993) VA mycorrhizal spores from three species of Acaulospora: germination, longevity and hyphal growth. Mycological Research 97, 785±790. Gemma J. N. and Koske R. E. (1988) Seasonal variation in spore abundance and dormancy of Gigaspora gigan-

tea and in mycorrhizal inoculum potential of a dune soil. Mycologia 80, 211±216. Guo B. Z., An Z. Q. and Hendrix J. W. (1994) A mycorrhizal pathogen (Glomus macrocarpum Tul. and Tul.) of tobacco, e€ects of long- and short-term cropping on the mycorrhizal fungal community and stunt disease. Applied Soil Ecology 1, 269±276. McGraw A. C. and Hendrix J. W. (1984) Host and soil fumigation e€ects on spore population densities of species of endogonaceous mycorrhizal fungi. Mycologia 76, 122±131. McGraw A. C. and Hendrix J. W. (1986) In¯uence of soil fumigation and source of strawberry plants on population densities of spores and infective propagules of endogonaceous mycorrhizal fungi. Plant and Soil 94, 425±434. Meier R. and Charvat I. (1993) Reassessment of tetrazolium bromide as a viability stain for spores of vesicular± arbuscular mycorrhizal fungi. American Journal of Botany 80, 1007±1015. Schenck, N. C. and Perez, Y., Manual for the Identi®cation of VA Mycorrhizal Fungi, 1st ed. University of Florida, Gainesville, 1987. Schenck, N. C. and Perez, Y., Manual for the Identi®cation of VA Mycorrhizal Fungi, 2nd ed. University of Florida, Gainesville, 1988. Tommerup I. C. (1983) Spore dormancy in vesicular±arbuscular mycorrhizal fungi. Transactions of the British Mycological Society 81, 37±45. Tommerup I. C. and Kidby D. K. (1979) Preservation of spores of vesicular±arbuscular endophytes by L-drying. Applied and Environmental Microbiology 37, 831±835. Walley F. L. and Germida J. J. (1995) Estimating the viability of vesicular±arbuscular mycorrhizal fungal spores using tetrazolium salts as vital stains. Mycologia 87, 273±279.