Independent spread of vesicular-arbuscular mycorrhizal fungi in soil

Independent spread of vesicular-arbuscular mycorrhizal fungi in soil

Trans. Br, mycol. Soc. 74 (2) 407-446 (1980) Printed in Great Britain NOTES AND BRIEF ARTICLES INDEPENDENT SPREAD OF VESICULAR-ARBUSCULAR MYCORRHIZA...

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Trans. Br, mycol. Soc. 74 (2) 407-446 (1980)

Printed in Great Britain


Soil Microbiology Department, Rothamsted Experimental Station, Harpenden, Herts., AL5 2JQ

Vesicular-arbuscular mycorrhizal fungi cannot be grown in axenic culture, although limited growth from germinating spores or infected root pieces often occurs. Such growth ceases when the emerging hyphae are separated from the parent spore or when the root piece dies. It is therefore widely accepted that the fungus does not grow saprophytically. The limited colonization of dead and senescent roots of some Chenopodiaceae and Cruciferae reported by Hirrel, Mehravaran & Gerdemann (1978) occurred only if a living onion or citrus plant was present in the same pot and is not evidence of independent saprophytic growth by the fungus. This note reports results of two experiments showing limited spread of infectivity from inoculum placed in the soil without a living host plant. In experiment 1, two y-irradiated (1 Mrad) soils were used, an agricultural soil from Woburn Experimental Farm and a soil from Geesecroft wilderness, a mixed deciduous woodland. Irradiation at this relatively low dosage eliminates the endophytes but does not fully sterilize the soil. The spread of four endophyte species was tested. They were E., similar to that described by Gilmore (1968), a form of Glomus jasciculatus (Gerdemann & Trappe, 1974); YV, a form of Glomus mosseae (Gerdemann & Trappe, 1974) multiplied in pot cultures at Rothamsted; Acaulospora laevis (Gerdemann & Trappe, 1974) and Gigaspora margarita (Becker & Hall, 1976) originating from Florida and multiplied in pot cultures at Rothamsted. Inoculum of each endophyte, consisting of roots, mycelium and spores was placed in pouches buried in irradiated soil in pots with the open end tied well above soil level (Fig. 1 a). The pouches were made from two thicknesses of 12 x 12 ern nylon fabric. There were two replicates of each endophyte in each soil and controls had pouches containing irradiated soil. All pots were covered and kept on moist sand in constant level watering trays. After 6 months the pouches were removed and the holes filled with fresh irradiated soil

which was sown with clover to act as an indicator of infectivity. Two months later the roots were washed from the soil, stained in trypan blue and lactophenol (phillips & Hayman, 1970) and examined microscopically. In both soils indicator plants in all the inoculated treatments had become mycorrhizal, with many infected roots. Thus all four endophytes had spread from the pouches into the surrounding soil, had survived there for 6 months and become sufficiently well established to act as new foci of infection. The anatomical characteristics of infection in the indicator plants were those of the particular endophyte used as inoculum (Warner & Mosse, 1978). Soil in control pots remained uninfective. The significance of these results depends on the reliability of the pouches as a barrier to the mechanical transfer of inoculum, particularly resting spores, into the surrounding soil. Such transfer could conceivably be in drainage water, by soil fauna or movement of soil particles on the surface. Although such eventualities were guarded against, VA infection can develop from only a few spores (Daft & Nicolson, 1969), even from inoculation with single spores (Kruckelmann, 1973). Resting spores of G. margarita, A. laevis and Glomus mosseae range between 100-500 usn, but those of E, are only about 50 pm and could conceivably have passed through the nylon fabric. The regularity of infection with all four endophytes argues against chance infection by single escaped spores, but in a second experiment further precautions were taken against the possibility of mechanical transfer of spores. In addition to the pouches, tubes were made from a polypropylene fabric with a basket weave (K458, supplied by Fothergill & Harvey). Under the microscope this material appeared to have almost no continuous spaces between the superimposed fibres so that it was impossible for spores to pass through it. The tubes were folded over at either end, sealed and placed in the soil (Fig. 1 b). In order to establish how far infectivity would spread from the inoculum, some tubes were placed inside

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Notes and brief articles






Arrangement of nylon pouches (a) and polypropylene tubes (b, c) as described in text. Table


Percentage infection in indicator plants (mean offour replicates)

Inoculum Site of soil tested Outside nylon pouch (Fig. 1 a) Outside polypropylene tube (Fig. 1b) Between polypropylene tubes (Fig. 1 c) Outside outer polypropylenetubes (Fig. 1 c)

larger tubes of the same fabric with a 7'5 rom layer of irradiated soil between them (Fig. 1 c). Two endophytes, E, and A. laevis were used as inocula. E, inoculum consisted of heavily infested soil containing mycelium and spores, or infected roots washed under a spray of water to remove any externally adhering resting spores. A. laevis inoculum consisted of infected roots, mycelium and spores. To prevent chance contamination of the outer surface of the containers during filling, they were temporarily covered with masking tape while the inoculum was being placed inside them. Woburn soil mixed with 50 % sterilized sand was used and there were four replicates of each treatment. Containers were buried in the soil for 4 months, removed and replaced with fresh soil which was sown with clover seed. Infectivity of the soil layer between the double tubes was also tested by removing the soil and sowing seed in it. The clover indicator plants were harvested after 60 days and the infection assessed quantitatively; 250 root pieces selected from a random sample by a line intersect method (Marsh, 1971) were examined for presence or absence of infection and results expressed as % infection. From all inocula infectivity had spread into the Trans. Br. mycol. Soc. 74 (2), (1980).


mycelium+ spores E, roots

Ai laeois

15 16 18

60 56 59

33 60 27




Control o o o o

surrounding soil but not beyond the outer polypropylene tube (Table 1). When the inoculum consisted of infected root pieces without externally attached resting spores the clover roots were most heavily infected, showing that spread of infectivity was independent of such spores and that root inoculum was apparently more infective than sievings, This confirms observations by Hall (1976) and Powell (1976) who also found that infection occurred more quickly from root pieces than from spores. Control soils again remained non-infective. The nature of the infective material was not established, but we believe that only hyphae could pass through the containers. Such hyphae might have arisen from infected root pieces or from germinating resting spores. Limited hyphal growth can be obtained from infected root pieces in agar or hanging drop cultures or from germinating resting spores. But hyphae from infected root pieces usually cease growth and die when the root piece becomes moribund, while germ tubes from resting spores generally retract their cytoplasm and the resting spore returns to dormancy if no suitable host root is encountered. When separated from the parent spore the germ tube ceases growth. Hyphae growing through the containers might

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Notes and briefarticles

Fig. 2. Small vegetative spores (VS) formed on mycelium arising from large resting spores (RS). form a resting structure, either a vegetative or a resting spore, in the surrounding soil. But new resting spores have never developed either on germ tubes growing from resting spores or on hyphae grow ing from root pieces. Even in the presence of a live host they very rarely develop in axenic culture in agar. Unless the fungus behaved very differently in soil and agar cultures it seems improbable that the inoculum in the containers would be able to form new resting spores in the surrounding soil when it was not attached to a living host plant. Small vegetative spores, up to 30 /lm, are often formed on the soil mycelium and on germ tubes arising from resting spores (Fig. 2). They form at the tip of short branch hyphae and sometimes continue to grow sympodially and can then give rise to another vegetative spore (Mosse & Hepper, 1975). These spores are not shed unless they are detached mechanically. They have never been germinated experimentally and it is not known whether root infection can originate from them, but they are incapable of independent growth in culture when separated from the parent material, either living plant root or resting spore. Trans. Br. mycol. Soc. 74 (2), (1980).

In the present experiments the hyphae growing out of the container into the surrounding soil would have to retain their viability for 6 months and retain sufficient vigour to infect, after separation from the parent material in the container. We therefore believe that the results presented here indicate some saprophytic ability of these fungi in soil that would enable them to establish a base (possibly in particles of organic material) from which they can infect a host plant. The conclusion is supported by the results of Ocampo & Hayman (in prep.) which show an increase of infectivity in fallow soil kept in the greenhouse for 12 weeks after inoculation.


W. N. & HALL, 1. R. (1976). Gigaspora margarita, a new speciesin the Endogonaceae. Mycotaxon 4, 155-160 . DAFT, M. J. & NICOLSON, T . H. (1969). Effect of Endogone mycorrhiza on plant growth. III. Influence


of inoculum concentration on growth and infection in tomato. N ew Phytologist 68, 953--963.

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Notes and briefarticles


GERDEMANN, J. W. & TRAPPE, J. M. (1974). The Endogonaceae in the Pacific Northwest. Mycologia Memoir 5, 1.J]6. GILMORE, A. E. (1968). Phycomycetous mycorrhizal organisms coIlected by open pot culture methods. Hi/gardia 39, 87-105. HALL, 1. R. (1976). Response of Coprosma robusta to different forms of endomycorrhizal inoculum. Tran sactions of the British Mycological Society 67, 409-411. HIRREL, M. C., MEHRAVARAN, H. & GERDEMANN, J. W. (1978). Vesicu1ar-arbuscular mycorrhizae in the Chenopodiaceae and Cruciferae: do they occur? Canadian Journal of Botany 56,2813-2817 . KRUCKELMANN, H. W. (1973). Die vesikular-arbuscuHire Mykorrhiza und ihre Beeinflussung in landwirtschaftlichen Kulturen. Dissertation. Universitat Braunschweig. Pp. 54.

MARsH, B. A'B. (1971). Measurement of length in random arrangements of lines. Journal of Applied Ecology 8, 265-267. MOSSE, B. & HEPPER, C. (1975). Vesicular-arbuscular mycorrhizal infections in root organ cultures. Phy siological Plant Pathology 5, 215-223. PHILLIPS, J. M. & HAYMAN, D. S. (1970). Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycologi cal Society 55, 158-161. POWELL, C. LL. (1976). Development of mycorrhizal infections from Endogone spores and infected root segments. Transaction s of the British M y cological Society 66, 439-445. WARNER, A. & MOSSE, B. (1978). Establishment and spread of introduced endophytes. Rathamsted Report for 1977, part 1, p. 239.


School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ

The ability of Gaeumannomyces graminis (Sacc.)

An.. & Olivier var. tritici Walker to utilize an inorganic source of nitrogen was demonstrated by White (1941) to be dependent on the presence of biotin, and the extent of such growth could be more than doubled by addition of thiamine. Ward & Henry (1961), however, did not find that biotin alone supported growth. Ward (1961) suggested that vitamin carry-over in the agar inoculum could explain the apparent variability in vitamin requirement, and was able to demonstrate an absolute requirement for both vitamins for mycelial development in liquid culture using a vitaminstarved, homogenised mycelial inoculum (Ward & Colotello, 1960). Balis (1970), using pieces from a culture on water agar as inoculum, showed that Phialophora radicicola Cain var. graminicola Deacon (recently described as the possible conidial state of Gaeumannomyces cylindrosporus Hornby et al., 1977) had a partial requirement for both biotin and thiamine for maximum growth. Deacon (1974b) compared the growth of 5 isolates of P. radicicola var, graminicola in a medium which contained

'* Present address: Bundesforschunsanstalt fur Landwirtschaft, Institut fur Biochemie des Bodens, BundesaIlee 50, D-3300 Braunschweig-Volkenrode, West Germany. Trans. Br. mycol. Soc. 74 (2), (1980).

thiamine or thiamine and biotin, and demonstrated that growth was enhanced by addition of biotin. The interaction between G. graminis var , tritici and P. radicicola var. graminicola, first described by Scott (1970), has been found to decrease the severity of the disease in small scale experiments (Balis, 1970; Deacon, 1973, 1974a). Speakman & Lewis (1978) reponed that when G. graminis var . tritici and P. radicicola var, gramin icola were separately inoculated on the same wheat root, growth over the surface ceased when the colonies met. Different patterns of lignification and suberization of endodermis and stele in the colonized root sections were recorded; however, no attempt was made to explain the cessation of external growth. We report here that both G. graminis var . tritici and P. radicicola var . graminicola have a partial requirement for biotin and thiamine, and that different isolates of the two species vary in their requirements for the vitamins for maximum growth. The possible role of the competition for biotin and thiamine in the rhizosphere in the interaction between these fungi on the wheat root is also discussed. Isolates of G. graminis var, tritici (code numbers: G1 and G2) and P. radicicola var. graminicola

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