Translocation of mycorrhizal fungi by earthworms during early succession

Soil Biol. Biochem. Vol. 25, No. 8, pp. 1021-1026, Printed in Great Britain. All rights reserved

TRANSLOCATION EARTHWORMS Imperial

1993 Copyright

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0038-0717/93 56.00 + 0.00 1993 Pergamon Press Ltd

OF MYCORRHIZAL FUNGI BY DURING EARLY SUCCESSION

ALAN C. GANGE* College at Silwood Park, Ascot, Berks SL5 7PY, U.K. (Accepted

20 February

1993)

Summary-The distribution of propagules of vesicular-arbuscular mycorrhizal (VAM) fungi by earthworms was investigated in a number of natural plant communities, differing in successional age. Propagule numbers were quantified using direct spore counts and the Most Probable Number (MPN) dilution method. Propagule number was low in early successional sites and was progressively higher in sites of 3 yr and over. Results of spore counts and MPN analysis showed similar patterns. In all sites, earthworm cast material contained higher numbers of spores and infective propagules than nearby field soil. These differences were significant in sites of 5 yr and over. There was a positive relationship between MPN of propagules and spore counts in cast material, but spore counts underestimated total propagule numbers. It is suggested that, due to feeding on senescing roots, earthworms concentrate mycorrhizal propagules in their cast soil. Due to the fact that surface worm casts were more abundant in early succession, the deposition of propagules was comparable to that in older communities. Earthworms may therefore be important agents in the distribution of mycorrhizal fungi, and thereby influence plant establishment in early succession.

INTRODUCHON

Vesicular-arbuscular mycorrhizal (VAM) fungi occur in a wide variety of habitats and in many

experiments have been shown to enhance plant nutrition and growth, through the formation of a mutualistic association with plant roots (Allen, 1991). With few exceptions, these fungi sporulate in the soil and consequently depend on external agencies for longrange dispersal (Gerdemann and Trappe, 1974). The dispersal process is clearly efficient, as fungal populations can recover quickly following elimination, or severe reduction, by soil cultivation or fungicide application (An et al., 1990a). It is generally considered that activities by the soil fauna bring mycorrhizal propagules to the surface, where they are subsequently dispersed by wind and rain. Fossorial mammals may be important (e.g. Allen, 1987), and a wide range of invertebrates (but most commonly earthworms) have been suggested as vectors (Thaxter, 1922; McIlveen and Cole, 1976; Rabatin and Stinner, 1989; Reddell and Spain, 1991). The latter three papers demonstrated that mycorrhizal spores can pass through earthworm guts without loss of viability. This is clearly important if worms are to be an effective dispersal agent, in view of the fact that spores of ferns and bryophytes suffer high mortality in the digestive tract (van Tooren and During, 1988).

*Present address: Department of Biology, Royal Holloway University of London, Egham Hill, Egham, Surrey TW20 OEX, U.K. SBB 251&D

Reddell and Spain (1991) have suggested that due to the feeding activities of worms in the rhizosphere, spore numbers may be higher in earthworm cast material, compared with surrounding soil. Although these authors briefly considered mycorrhizal propagules other than spores, in general the possibility of worms dispersing fragments of hyphae or fungal-infected root appears to have been neglected. Worms generally feed on decaying organic matter, but feeding in the rhizosphere or on newlysenesced roots may increase the likelihood of distributing these other propagules. Propagules other than spores are important in initiating infection, as Moorman and Reeves (1979) found no relationship between spore numbers and the infectivity of soils. Direct counting of spores from soil (to measure mycorrhizal infectivity), employed in all previous experiments with earthworms, is open to a number of criticisms. The wet sieving technique used may be too coarse to retain spores of certain species, some species only sporulate within roots, and any non-spore propagules will be missed (Porter, 1979). A method which can enumerate the infective propagules of VAM fungi in a soil is the dilution series technique, involving calculation of the ‘Most Probable Number’ (MPN) (Alexander, 1982). Although also having drawbacks, this method has been effectively employed in a variety of soils (Porter, 1979; An et al., 1990b) and was used here. My aim was to present the MPN of VAM propagules in worm cast material and surrounding field soil, in a number of differently-aged plant communities, at one time. The estimates are compared with direct spore counts, and the role of

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earthworms as distributors of VAM fungi during early plant succession is discussed.

MATERIALS

AND METHODS

At Silwood Park, Berkshire, there are a number of plant communities which have been allowed to develop naturally following a disturbance caused by ploughing. Details of site preparation and establishment are given in Brown and Gange (1989). The soil is sandy and acidic (pH 5.4, using a standard pH meter). The communities chosen in this study were three early successional sites (1, 3 and 5 yr old) and two mid successional sites (8 and 11 yr old). Sites were divided into 3 x 3 m plots and are part of a much larger experiment involving application of insecticide and fungicide to the soil (Brown and Gange, 1989; Gange et al., 1990). Five control plots (with no pesticide application) were selected in each site. In February 1991, the number of visible earthworm casts in each plot was counted. As the major experiments involve non-destructive sampling of the plant communities, it was not possible to remove dead vegetation in order to count all worm casts. Cast material was carefully removed and each cast taken to the laboratory. Five soil cores, 3 cm dia and 5 cm deep, were taken from random coordinates in each plot. In the laboratory, soil was weighed wet and after air drying, to determine the water content. After weighing, the cast and field soil material from each site was pooled and 20 1 g samples of each soil type taken for spore counts. Spores were extracted with the wet sieving method (Gerdemann and Nicolson, 1963). Cast number per plot and spore number g-’ of dry soil were analysed by ANOVA.

The Most Probable Number of infective particles was estimated by preparing a IO-fold dilution series for each type of soil from every site. Test soils were mixed with sterilized soil from their site of origin. A dilution down to 10e4 was used, with five replicates at each dilution. 70 g of mixed soil was placed in a 5 cm dia flower pot and one germinating seed of Plantago lanceolata was placed in each pot. This species is known to be highly mycorrhizal in the experimental sites. Pots were watered with 100ml water per week and maintained at 15°C for I6 weeks. After this time, the roots were washed free of soil, stained in cotton blue in acid glycerol (Koske and Gemma, 1989) and examined under 200 x magnification for the presence of VAM fungal infection. A control series of plants was grown in sterilized soil only from each site. No mycorrhizal fungi were detected in these. Using the number of plants infected at each dilution, the MPN and 95% confidence limits were calculated from the tables given in Alexander (1982).

RESULTS

The number of earthworm casts me2 is shown in Fig. 1. The greatest number of casts was found on the 1 yr site, this being significantly different from all other sites (F4,,9= 35.38, P
2.4

a

+l 0

--

b

b

8

11

b I

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3

I I 5 Site age (years)

Fig. 1. Mean number of worm casts me2 in sites of different successional age. Bars with the same letter are not significantly different at P = 0.05 (Tukey test).

Mycorrhizal distribution by earthworms

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ma $ i! ii a4

0

1

3

5

a

Site age (years) 0 fieldsoll, n wmlcast Fig. 2. Mean number of VAM spores g-’ dry soil from worm cast and field soil in sites of different successional age. Bars with the same letter are not significantly different at P = 0.05(Tukey test).

between the 3, 5, 8 and 11 yr sites, but all were 5 yr. There was also a difference between worm cast different from the 1 yr site. Propagule numbers in material and field soil (F,,190= 52.98, P < O.OOl),with casts having significantly higher spore counts in the field soil increased slightly with site age, but the only significant difference (P < 0.05) was between the 1 5, 8 and 11 yr sites. There was a dramatic increase in propagule numand 11 yr old sites. Of more interest were the differences between worm cast soil and field soil. In all ber g-’ of dry worm cast material with successional cases, worm casts contained higher numbers of age of site (Fig. 3). The pattern mirrored that of spore number (Fig. 2) with the greatest increase during propagules and these differences were significant at P = 0.05 in the 5, 8 and 11 yr old sites. Indeed, early succession. This was shown by the fact that there were no significant differences (at P = 0.05) propagule numbers in cast material were 20 times

a0

1

3 0

5 Site age (years) fbldsoil

n

a

11

womlcast

Fig. 3. Most Probable Number of infective VAM propagules g-l dry soil from worm cast and field soil in sites of different successional age. Bars with the same letter are not significantly different at P = 0.05, as determined by 95% confidence limits.

ALANC. GANGE

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Od 0

______*--______----_______----4

0

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Spores / g of soil Fig.4. Relation between the MPN of infective propagules and spore number g-’ dry soil from worm cast material. Each Point is a site of different suceessiona1age. Fitted line: y = 4.38~ + 6.34. Dotted line: y = X. higher in the 8 yr site than in field soil, and 10 times higher in the 11 yr site. These differences were therefore similar to, but more dramatic, than those found with spore number. There was a si~~~nt relationship between propagule number and spore number g-’ of dry worm cast (r = 0.946, P < 0.05) (Fig. 4). However, only when mycorrhizal abundance was low were these numbers similar, and at high density, spore counts seriously underestimated the soil infectivity,

with the slope of the fitted line being significantly greater than one (t = 3.91, P -c 0.01). Using the figures for worm cast weight, multiplied by frequency and MPN infectivity, the deposition intensity of infective propagules per unit area was calculated for the different sites (Fig. 5). In early succession, this represented about 40mm2 rising to 100 mm2 in the 11 yr site. Due to the high frequency of worm casts in the 1 yr site, there was no significant difference in propagule deposition between this and

b b

;n a

1

3

5

8

-

11

Site age (years)

Fig. 5. Number of deposited infective propagules in worm cast material in sites of different successional age. Bars with the same letter are not significantly different at P = 0.05, as determined by 95% confidence limits.

Mycorrhizal distribution by earthworms the 3 and 5 yr sites. Deposition was greater in the two older sites due to the high MPN values (Fig. 3), as there was little difference in cast weight between the sites. DISCUSSION

These results endorse those of McIlveen and Cole (1976) and Reddell and Spain (199 1) in demonstrating that earthworms are important distribution agents of VAM fungi. Furthermore, the fact that the dominant casting worm in the sites, Lumbricus terrestris, is an effective dispersal agent of fungi confirms the findings of McIlveen and Cole (1976) and Rabatin and Stinner (1989). However, previous work has usually concentrated on spore dispersal, although Reddell and Spain (199 1) presented some evidence to suggest that other mycorrhizal propagules could be involved. My study has found that in mid-successional plant communities, when fungal density is quite high, counts of spores by wet sieving will seriously underestimate fungal infectivity. The fact that the fitted regression line in Fig. 4 is significantly different from y =x suggests that earthworm cast material contains a high proportion of non-spore fungal material. Examination of sieved soil material, soaked in cotton blue, under the microscope indicated large amounts of hyphae and infected root fragments. Such material is known to initiate infections and explains why spore counts may not accurately represent the infectivity of a soil (Moorman and Reeves, 1979). My results also demonstrate that earthworm cast material may contain considerably higher concentrations of fungal propagules, compared with nearby field soil. This may be a result of worms feeding in the rhizosphere or in organic matter-rich areas, where mycorrhizal fungi sporulate (Allen, 1991). However, as earthworms consume large amounts of decomposing fine roots (Spain et al., 1990), this is a likely cause of the concentration of propagules in cast material. Furthermore, since infections were initiated in P. Zanceolata, it appears that mycorrhizal propagules are not harmed by passage through the earthworm gut. If mortality was very high, then we might expect fewer propagules in cast material compared with field soil, a situation reported for bryophyte spores (van Tooren and During, 1988). The data also suggest that propagules were not seriously affected by the soil dilution procedure, a criticism which has been levelled against this method in the past (Mosse et al., 1981). It is well known that severe soil disturbance, such as cultivation, reduces the abundance of mycorrhizal fungi (Jasper et al., 1989) and that subsequently fungal density increases with the successional age of a community (Johnson et al., 1991). My results reflect these changes, with propagule numbers being low in early succession and increasing dramatically after about 5 yr. Furthermore, the data also indicate that

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differences in propagule number between cast material and field soil are dependent on the age of a site. When mycorrhizal fungi reach relatively high densities, cast material contains substantially higher concentrations of propagules than field soil. This is to be expected if the arguments concerning earthworm feeding (above) hold, as more plant species are mycorrhizal in the older communities and infection levels are higher (Allen, 1991). My study has only examined worm cast material at one point in time. February was chosen for the observations, since casting is common at this time (Edwards and Lofty, 1977). It is appreciated that both casting and spore production show seasonal changes, but the aim of my study was to examine successional, rather than temporal differences. In addition, the work sought to record cast deposition of fungal propagules, irrespective of worm species. However, future research should distinguish between worm species and their ability to transmit VAM propagules. In this study, it is likely that most cast material was of L. terrestris, as casts were morphologically-indistinguishable between sites and this species dominated the worm fauna (Gange, unpublished). At present, the spatial scale at which earthworms are important distributors of VAM fungi is unknown. L. terrestris forms mainly vertical burrows and may cast above and below ground (B. Doube, personal communication). It is therefore possible that this species is important at a local scale, although nocturnal worm movement on the surface may be important in more distant dispersal. Other longer range dispersal agents may include wind, water, vertebrates or even horizontally-burrowing earthworms. Earthworms are known to be important distribution agents of seeds in the soil (McRill and Sagar, 1973; Grant, 1983). It has been suggested that as casts contain greater amounts of nutrients than field soil (Lee, 1985) seeds germinating in casts may have a higher probability of establishment, due to increased availability of light and nutrients and reduced competition (Piearce et al., 1993). Indeed, these authors have demonstrated that there are significant positive relationships between Plantago, Trifolium and Ranunculus species and wormcast frequency in grassland. The fact that mycorrhizal inoculum may be abundant in wormcast material may also aid seedling establishment, and this may be important in early succession when perennial forbs are becoming established. Reducing mycorrhizal abundance with fungicides leads to a lowering of plant species richness, an effect resulting from decreased establishment of the perennial forbs (Gange et al., 1993). Due to the density of worm casts being higher in early succession, the deposition of infective propagules by these animals is likely to be important for those mycorrhizal plant species establishing at this time. Therefore, earthworms may contribute to the heterogeneous nature of mycorrhizal distribution in natural

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communities, through their feeding and casting activities. In this way, they may also lead to patchy plant establishment in these communities, in a manner similar to that proposed in agroecosystems by Rabatin and Stinner (1989). Acknowledgemenrs--I am grateful to the Natural Environment Research Council for financial support while working with Dr V.K. Brown at Silwood Park. Rosemary Setchfield and John Hollier kindly commented on the manuscript and Ian Clarke helped with the soil dilutions.

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Allen M. F. (1991) The Ecology of Mycorrhizae. Cambridge University Press, Cambridge. An Z. Q., Grove J. H., Hendrix J. W., Hershman D. E. and Henson G. T. (199Oa) Vertical distribution of endogonaceous mycorrdizal fungi associated with soybean, as affected by soil fumigation. Soil Biology & Biochemistry 22,715-719.

An Z. Q., Hendrix J. W., Hershman D. E. and Henson G. T. (199Ob) Evaluation of the “Most Probable Number” (MPN) and wet-sieving methods for determining soilborne populations of endogonaceous mycorrhizal fungi. Mycologia 82, 576-58 1. Brown V. K. and Gange A. C. (1989) Herbivory by soil-dwelling insects depresses plant species richness. Functional Ecology 3, 66767 1.

Edwards C. A. and Lofty J. R. (1977) Biology of Earthworms. Chapman & Hall, London. Gange A. C., Brown V. K. and Farmer L. M. (1990) A test of mycorrhizal benefit in an early successional plant community. New Phytologist 115, 85-91. Gange A. C., Brown V. K. and Sinclair G. S. (1993) VA mycorrhizal fungi: a determinant of plant community structure in early succession. Functional Ecology. In press. Gerdemann J. W. and Nicolson T. H. (1963) Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Transactions of the British Mycological Society 46, 235-244.

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Johnson N. C., Zak D. R., Tilman D. and PtIeger F. L. (1991) Dynamics of vesicular-arbuscular mycorrhizae during old field succession. Oecologia 86, 349-358. Koske R. E. and Gemma J. N. (1989) A modified procedure for staining roots to detect VA mycorrhizas. Mycological Research 92, 486488. Lee K. E. (1985) Earthworms: Their Ecology and Relationships with Soils and Land Use. Academic Press, Sydney.

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Thaxter R. cl922) A revision-of the Endogoneae. Proceedings of the American Academy of Arts and Science 57, 291-334. van Tooren B. F. and During H. J. (1988) Viable plant diaspores in the guts of earthworms. Acta Botanica Neerlandica 37, 181-185.