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Microfungal communities in soil, litter and casts of Lumbricus terrestris L. (Lumbricidae): a laboratory experiment
Applied Soil Ecology 14 (2000) 17–26
Microfungal communities in soil, litter and casts of Lumbricus terrestris L. (Lumbricidae): a laboratory experiment Alexei V. Tiunov a,∗ , Stefan Scheu b a b
Laboratory of Soil Zoology, Institute of Ecology and Evolution, Leninsky Prospect 33, 117071, Moscow, Russia Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 3, D-64287, Darmstadt, Germany Received 8 July 1999; received in revised form 29 November 1999; accepted 1 December 1999
Abstract The anecic earthworm Lumbricus terrestris L. was kept in laboratory microcosms containing beech forest soil without litter, with beech leaf litter or with lime leaf litter. The structure of microfungal communities in soil, litter and fresh and aged (100 days) earthworm faeces was analysed using the washing and plating technique. The passage of mineral soil through the gut of L. terrestris affected the structure of the fungal community only little. In contrast, in the litter treatments the structure of the fungal community in fresh earthworm casts significantly differed from that in soil and litter. The majority of soil and litter inhabiting fungi survived passage through the gut of L. terrestris and the fungal community in casts consisted of a mixture of soil and litter inhabiting fungi. However, the frequency of Cladosporium spp., Alternaria spp., Absidia spp., and other taxa was strongly reduced in fresh casts. The degree of colonization of litter particles (number of isolates per number of plated particles) also decreased, but some fungi (mainly Trichoderma spp.) benefited from gut passage and flourished in fresh casts. During ageing of cast material the dominance structure of the fungal community changed. Both the degree of colonization of organic particles and the species diversity increased and approached that in soil. However, the structure of the fungal community in casts remained cast specific even after 100 days of incubation. It is concluded that the feeding and burrowing activity of L. terrestris accelerates the colonization of litter by the edaphic mycoflora but also extends the range of occurrence of litter-associated fungi into mineral soil layers. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Soil fungi; Fungal community; Anecic earthworms; Lumbricus terrestris; Litter decomposition
1. Introduction Fungi and earthworms are important members of soil communities and their interactions are assumed to significantly affect microbial-mediated processes in soil. Although the importance of these interactions is widely accepted, they are little studied and the information available is in part contradictory (Brown, 1995). Earthworms may influence soil fungi by a ∗ Corresponding author. Tel.:+7-095-958-1449; fax: +7-095-954-5534/952-2592. E-mail address: [email protected] (A.V. Tiunov).
variety of mechanisms, including the alteration of the physical and chemical state of the environment (‘engineering’ sensu Jones et al., 1997), comminution and translocation of litter, dispersal of fungal propagules and grazing on fungal tissue (Visser, 1985). Various authors suggested that fungi are a major component of the diet of earthworms (Dash et al., 1984, 1986; Edwards and Fletcher, 1988; Tiwari et al., 1990; Doube and Brown, 1998). Earthworms have been shown to prefer food substrates colonized by certain fungal species and fungi have been found to be damaged by gut passage (Cooke, 1983; Moody et al., 1995, 1996; Marfenina and Ishchenko, 1997).
0929-1393/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 5 0 - 5
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Even low levels of selective grazing by invertebrates has been shown to significantly alter the distribution and succession of fungi on litter (Parkinson et al., 1979; Newell, 1984). Therefore, it is likely that earthworms alter the composition of fungal communities in soil and the fungal succession in decomposing litter. However, there is only fragmentary information on effects of earthworms on the structure of fungal communities in soil. Domsch and Banse (1972) reported a strong alteration of the fungal dominance structure in casts of Lumbricus terrestris in comparison to the food substrates. In contrast, Tiwari and Mishra (1993) did not find significant differences in the structure of fungal communities in soil and field-collected earthworm casts. In micro- and mesocosm experiments, the presence of the epigeic earthworm species Dendrobaena octaedra (McLean and Parkinson, 1998) and the endogeic species Octolasion tyrtaeum (Scheu and Parkinson, 1994) affected the structure of fungal communities only little, although the frequency of some species changed significantly. Detailed studies aiming at separating different mechanisms which may contribute to earthworm-mediated changes in the structure of fungal communities in soil are lacking. We studied microfungal communities in fresh and aged faeces of the widespread anecic earthworm L. terrestris L. This large deep-burrowing species builds permanent vertical burrows, but feeds mainly on organic materials on the soil surface. Due to the feeding and burrowing activity of L. terrestris, large amounts of mineral soil are translocated upward to the soil surface and mixed with litter, whereas litter material is transferred deep into the mineral soil. We addressed the following questions: how does passage through the gut of L. terrestris affect the structure of the fungal community as compared to soil and litter? Which of the soil and litter inhabiting fungal species survive, and which are damaged by gut passage? How does the fungal community in faeces change during ageing of cast material?
2. Materials and methods 2.1. Experimental design Mineral soil, beech leaf litter and specimens of L. terrestris were obtained from a 115–120 year old
beech (Fagus sylvatica) stand, the ‘Göttinger Wald’ (Lower Saxony, Germany). Soil material was taken in June 1996 at a depth of 3–10 cm from the mineral soil surface and sieved (<4 mm). Overwintered F. sylvatica leaf litter was collected from the soil surface at the same time. Lime (Tilia cordata) leaf litter was collected in May 1996 in a lime grove 30 km south of Moscow (Russia) and kept until the start of the experiment in polyethylene bags at 5◦ C. The experiment was carried out in narrow containers (inner dimensions: 650 mm high, 310 mm long, 10 mm wide). Three combinations of beech forest soil and litter were studied: soil without litter (B0 treatment), soil+beech litter (BB treatment), and soil+lime litter (BL treatment). Containers were filled with soil to a level of 500 mm. The soil was compacted to a bulk density of 0.65 kg dry weight 1000 cm−3 which is typical for the upper soil layers at the Göttinger Wald. The moisture content of the soil was kept at the field level (62% of dry wt.). About 25 g of Tilia or Fagus litter was added to each BL and BB container and replenished periodically as it was consumed by earthworms. In each microcosm two adult L. terrestris (average individual weight 6.0±0.2 g) were placed. Containers were kept at 15◦ C in permanent darkness. Five replicates were set up per treatment. Further details are given in Tiunov and Scheu (2000). After a preliminary period of 2 weeks, when a burrow system was established by the earthworms, containers were opened every second day and freshly deposited faeces were collected (average age of 1 day). For the analysis of the microfungal community, fresh casts and control litter samples were collected 26–30 days after the start of the experiment from four containers of each treatment. Simultaneously, control soil samples were taken from B0 (two replicates), BL and BB treatments (one replicate each). Soil and litter samples were taken from the zones not affected by L. terrestris (at least 50 mm away from the nearest burrow). Faecal material of age 100 days was obtained by incubating casts between two layers of soil separated by 0.5 mm gauze. The container with soil and casts was incubated in a polyethylene bag at 15◦ C. A plastic tube (i.d. 3 mm) was inserted into the bag to ensure free gas exchange.
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2.2. Isolation of fungi The composition of the fungal communities in the soil, litter and casts was analysed by a modified washing and plating method (Parkinson and Williams, 1961). One gram (fresh weight) of soil, cast or litter material was blended for 20 s in 100 ml sterile water. About 0.5 g of the blended material was placed in the washing apparatus (vol. ca. 100 ml) between 0.5 and 0.2 mm meshes. The material was washed by applying a constant flow (0.5 l min−1 ) of sterile water for 10 min. During the washing procedure the apparatus was gently shaken. Washed organic particles of size 0.2–0.5 mm were collected on sterile filter paper. Fifty particles per sample were plated on malt extract agar containing streptomycin and tetracycline (0.1 and 0.05 g l−1 , respectively) to suppress growth of bacteria. One particle was placed per Petri-dish. The Petri-dishes were incubated at room temperature. When two or more fungal species were growing from the single particle, pure cultures were prepared by transferring hyphal inocula to separate plates. The frequency of occurrence of fungal taxa (as percentages) was calculated as the number of particles with fungal growth per total number of isolates ×100. On average, 44.5 isolates were obtained per 50 plated particles.
2.3. Statistical analysis The main goal of the analysis was to compare the structure of whole fungal communities in the studied substrata, rather than to follow changes in abundance of single fungal species or genera. For this purpose, ordinations and discriminant analysis were used. Rare fungi (fewer than two isolates), yeast-like forms and loosely-defined groups (i.e., ‘other sterile dark’, ‘other Trichoderma’; see further) were not included in the analysis. The remaining 46 taxa represented ca. 70% of the isolates. The logic of the mathematical analysis follows the scheme proposed by Puzachenko and Kuznetsov (1998). Based on the relative frequency of fungal taxa, the square matrix of nonparametric Gamma correlation (analogous to Kendall τ ) between the studied samples was calculated. The distance matrix was obtained by subtracting correlation coefficients
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(range 1 to −1) from 1. Thus, distances varied from 0 (perfect similarity between two samples) to 2 (no similarity). The distance matrix was analysed further by nonmetric multidimensional scaling. This ordination technique ‘rearranges’ objects in n-dimensional space, so as to arrive at a configuration that best approximates the observed distances. The number of meaningful dimensions was evaluated by comparing actual stress values with the theoretical exponential function of stress. The coordinates of the samples in n-dimensional space were used for discriminant function analysis (DFA), with ‘substrate’ (soil, litter or casts) as a grouping variable. Squared Mahalanobis distances between group centroids and the reliability of sample classification were determined. Typically, only two significant discriminant functions (canonical roots) were derived. Thus, the results of DFA are graphically presented in 2-dimensional space. For the interpretation of the discriminant axes in respect to the frequency of fungal taxa, linear correlations were calculated between the discriminant function scores for each sample and the relative dominance of fungal taxa. A similar procedure was used to analyse the similarity matrix between fungal taxa. Prior to the analysis, fungi were grouped into soil- and litter-associated species using cluster analysis and Ward’s minimum variance method. All calculations were made using the STATISTICA software package. Differences between means were tested using Tukey’s honestly significant difference (HSD) at the 0.05 probability level.
3. Results 3.1. ‘Soil’ and ‘litter’ fungi The fungal community in beech litter was dominated by Mortierella gamsii, T. pseudokoningii and Mucor hiemalis. The most frequent fungi in lime litter were T. koningii, T. hamatum, M. hiemalis and two Alternaria spp. Most of these fast growing fungi were not isolated from the soil (Table 1). The fungal community in soil was more diverse. The most frequent fungi were two species of the genus Acremonium, Humicola sp. 1, Absidia cylindrospora, Cylindrocarpon destructans, Mortierella minutissima, several species of Penicillium and Volutella sp.
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Table 1 Frequency (percentage of the total number of isolates) of dominant fungi in soil, litter and fresh casts of L. terrestrisa Soil
B0 casts
Beech litter
BB casts
Lime litter
BL casts
1.6 ab 0a 0a 0 0
0a 0a 0a 0 0
14.8 b 6.9 b 3.3 b 2.6 1.1
6.7 b 13.2 b 0.7 ab 0 1.4
0.5 a 0a 0a 0 0
0a 0a 0a 0 0
‘Lime litter species’ Trichoderma koningii Oudem. Trichoderma hamatum Bain. Mucor hiemalis Wehmer Alternaria sp. 2 Alternaria alternata (Fr.) Keissler Tolypocladium sp.
0a 0.9 a 0 0a 0 0
0a 1.4 a 0 0.6 a 0 0
0a 4.4 a 5.8 0.5 a 0 0
0a 2.2 a 3.0 0a 0 0
29.3 b 9.5 ab 5.9 5.1 b 2.4 1.6
39.4 b 14.5 b 3.5 0.6 a 0 1.8
‘Soil species’ Acremonium spp. Humicola sp. 1 Absidia cylindrospora Hagem Sterile dark sp. 2 Cylindrocarpon destructans (Zinssm.) Scholten Mortierella minutissima van Tiegh. Penicillium spp. Volutella sp. Sterile dark sp. 3 Sterile dark sp. 4 Chrysosporium sp. 1 Phialophora sp. 1 Sterile hyaline sp. 2 Sterile hyaline sp. 3 Paecilomyces spp. Sterile hyaline sp. 4 Sporothrix sp. Chloridium sp. Trichoderma polysporum (Link ex Pers.) Rifai Sterile hyaline sp. 5 Chrysosporium sp. 2 Trichocladium sp.
11.2 a 5.7 a 5.5 a 3.9 ab 4.4 3.8 a 3.6 3.1 2.8 a 2.6 ab 2.6 a 2.1 a 2.0 a 1.9 1.8 1.5 1.5 1.5 0.9 0 0 0
9.0 ab 5.7 a 2.1 ab 6.4 a 4.3 2.6 a 4.6 4.4 0b 3.4 b 0b 0b 0b 2.0 1.4 2.0 1.3 0.6 0.6 0.6 0 1.4
1.7 ab 0b 0.5 b 0.7 b 0 0b 1.2 1.6 0.6 ab 0c 0b 0b 0b 0 1.1 1.8 0 0 1.2 0 0 0
5.6 ab 2.0 ab 1.4 ab 4.3 ab 0.5 3.4 a 2.9 5.7 1.2 ab 0.7 ac 0b 0b 0.7 ab 1.2 0 1.2 0.8 0.7 0 0 1.5 0
0.5 b 0b 0b 0b 0 0b 0.5 0 0b 0c 0b 0b 0b 0 0 0 0 0 0 0 0 0
3.9 ab 1.8 ab 0b 1.7 ab 0.6 0b 0.6 0.6 0b 0c 0b 0b 0b 0 0 0.6 0.6 0 0 1.2 0 0
Total number of taxa isolated Average number of taxa per sample
37 22.5 a
29 19.0 a
26 14.0 bc
32 16.8 ab
18 11.5 c
24 11.5 c
‘Beech litter species’ Mortierella gamsii Milko Trichoderma pseudokoningii Sterile hyaline sp. 1 Cladosporium spp. Sterile dark sp. 1
Rifai
a The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil +lime litter) treatments. Values within rows followed by different letters are significantly different (Tukey’s HSD test based on arcsine-transformed data, n=4, p<0.05).
Cluster analysis of the similarity matrix of 38 dominant fungal taxa singled out three main clusters, which were interpreted as ‘beech litter species’, ‘lime litter species’ and ‘soil species’. The reliability of the separation was further confirmed by the discriminant analysis (p<0.001). Fungal taxa in Table 1 are grouped according to this clustering. However, the arbitrary character
of the grouping should be noted, as some ‘soil fungi’ (e.g. T. polysporum or Sterile hyaline sp. 4) were equally abundant in the beech litter, while M. hiemalis had similar occurrence in both types of litter. Although we applied several clustering procedures, no specific cluster of ‘cast-associated’ fungi could be singled out. Trichocladium sp., Sterile hyaline sp. 5
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and Chrysosporium sp. 2 were isolated only from the casts, but the frequency of these fungi was low and they did not reliably separate from the ‘soil fungi’. 3.2. Fresh casts The structure of the fungal community in food substrates and egested materials can only be compared directly in the B0 treatment, where earthworms exclusively consumed mineral soil. Only a few fungal taxa were significantly affected by gut passage in this treatment (Table 1). However, Mucorales (Absidia spp. and Mortierella spp.) tended to be less frequent and other fungal species, e.g. Chrysosporium sp. 1 and Phialophora sp., were not isolated from casts. The total number of fungal taxa was lower in casts (29) than in soil (37). Comparing food substrate and casts is more difficult in BB and BL treatments because L. terrestris consumed both soil and litter. Alterations in the structure of fungal communities in casts of these treatments might be due to gut passage or to mixing of soil and litter materials. Generally, ‘soil fungi’ in casts accounted for 35 and 13% of the total number of isolates in BB and BL treatments, respectively (Fig. 1). Of
Fig. 1. Number of organic particles colonized by litter-associated fungi (‘litter fungi’), soil-associated fungi (‘soil fungi’) and uncolonized particles in soil, litter and fresh casts of Lumbricus terrestris (means+SD). The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. 50 particles were plated per replicate, n=4.
Fig. 2. Discriminant analysis plane of soil, litter and fresh casts of Lumbricus terrestris according to the fungal dominance structure. The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Ellipses represent 95% confidence limits.
the ‘litter fungi’, Mucorales (Mortierella gamsii and M. hiemalis), Cladosporium spp., and two species of Alternaria tended to be less abundant in casts than in the litter offered as food substrate. In contrast, the frequency of three Trichoderma spp. was greater in casts (Table 1). However, most of these tendencies were not statistically significant. In each of the three treatments the number of organic particles from which no fungi developed was significantly greater in L. terrestris casts compared to food materials. The mean number of uncolonized particles was 10% in soil, 5% in beech litter and 0.5% in lime litter, but reached 25, 26 and 14% in B0, BB and BL casts, respectively (Fig. 1). The dominance structure of fungal communities in casts from BB and BL treatments differed considerably from that in food materials (Fig. 2). The materials studied were reliably discriminated in 2-dimensional space. The first axis, which accounted for 60.8% of the discriminatory power, separated litter (negative values) and soil (positive values). Although the control soil samples were taken from three different treatments (B0, BB and BL), they did not differ significantly from each other and formed a compact group. The second axis (38.5% of the discriminatory power) separated Fagus and Tilia litter. Casts from BB and BL treatments were intermediate in position between soil and litter, but were more similar to the corresponding litter than to the soil. However, they could be reliably
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Table 2 Squared Mahalanobis distances between group centroids and reliability of discrimination based on data on fungal dominance structurea
Soil Beech litter Lime litter B0 casts BB casts
B0 casts
BB casts
BL casts
5.3 nsb 180.9∗∗∗ 186.9∗∗∗ 0 89.4∗∗∗
73.8∗∗∗ 19.2∗ 114.7∗∗∗ 89.4∗∗∗ 0
102.5∗∗∗ 130.3∗∗∗ 26.4∗∗ 86.7∗∗∗ 100.8∗∗∗
a
See text for details. ns=not significant. ∗ p<0.01. ∗∗ p<0.005. ∗∗∗ p<0.0001. b
distinguished from each other and from both soil and litter (p<0.01). In contrast, casts from the B0 treatment could not be discriminated from the soil (Table 2). The two discriminant axes can be interpreted easily considering the dominance structure of the fungal communities. The occurrence of dominant ‘soil fungi’ positively correlated with the first axis, but not with the second axis. ‘Beech litter fungi’ were negatively correlated with both axes. ‘Lime litter fungi’ were negatively correlated with the first axis and positively with the second (Table 3).
Table 3 Linear correlation (r-values) between the frequency of fungal taxa and discriminant axesa Axis 1
Axis 2
‘Lime litter species’ Trichoderma hamatum Mucor hiemalis Alternaria sp. 2 Trichoderma koningii Tolypocladium sp.
−0.517∗∗ −0.515∗∗ −0.482∗ −0.472∗ −0.221 ns
‘Beech litter species’ Mortierella gamsii Trichoderma pseudokoningii Sterile hyaline sp. 1 Sterile dark sp. 1
−0.323 −0.171 −0.313 −0.180
‘Soil species’ Humicola sp. 1 Sterile dark sp. 2 Mortierella minutissima Sterile dark sp. 4 Acremonium spp. Cylindrocarpon destructans Absidia cylindrospora Penicillium spp. Sterile hyaline sp. 3 Sporothrix sp. Sterile hyaline sp. 2 Chloridium sp. Phialophora sp. 1 Chrysosporium sp. 1 Paecilomyces spp.
ns ns ns ns
0.757∗∗∗ 0.677∗∗∗ 0.657∗∗∗ 0.652∗∗ 0.636∗∗ 0.626∗∗ 0.613∗∗ 0.585∗∗ 0.517∗∗ 0.500∗ 0.480∗ 0.480∗ 0.480∗ 0.448∗ 0.410∗
0.599∗∗ 0.010 nsb 0.435∗ 0.727∗∗∗ 0.507∗ −0.646∗∗ −0.630∗∗ −0.511∗ −0.434∗ 0.077 −0.125 −0.264 −0.014 −0.074 0.086 −0.126 −0.183 −0.094 0.074 −0.134 −0.098 −0.019 −0.008 −0.138
ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
a
See text for details. ns=not significant. ∗ p<0.05. ∗∗ p<0.01. ∗∗∗ p<0.001. b
3.3. Aged casts The frequency of certain fungal taxa significantly changed during the 100-day incubation of soil and cast material (Table 4). However, in most cases the changes were not uniform in the different substrates. The frequency of T. koningii, Volutella sp. and Humicola sp. 1 tended to decline during ageing of soil and casts. In contrast to the fresh samples, Sterile hyaline sp. 1, Sterile dark sp. 1, Tolypocladium sp. and Trichocladium sp. were not isolated from the aged samples (Tables 1 and 4). Conversely, the frequency of A. cylindrospora, T. pseudokoningii, T. polysporum and Penicillium spp. increased in most aged substrates. T. viride, Fusarium sp., Wardomyces sp. and other fungi were isolated only from aged soil and casts. The total number of isolated taxa also increased, and the number of uncolonized particles in aged casts did not exceed 10%.
The changes in the fungal community structure in aged materials were reflected by reliable discrimination between ‘fresh’ and ‘aged’ substrates. The discrimination was strongest between ‘fresh’ and ‘aged’ soil (p=0.003) and between ‘fresh’ and ‘aged’ casts from BL treatment (p=0.007) whereas it was less reliable between ‘fresh’ and ‘aged’ casts in B0 and BB treatments (p=0.037 and 0.054, respectively). During incubation, cast materials of different treatments were kept in the same soil container, separated only by 2–5 cm. However, after 100 days of ageing, fungal communities in L. terrestris casts retained specific features (Fig. 3). The three types of cast material were separated from each other in 2-dimensional discriminant space (p<0.01). Fungal communities were
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Table 4 Frequency (percentage of the total number of isolates) of dominant fungi in aged soil and casts of Lumbricus terrestrisa Holding soil
B0 casts
BB casts
BL casts
‘Beech litter species’ Mortierella gamsii Trichoderma pseudokoningii
0a 3.4 a
0.5 a 0.5 a
13.1 ⇑b 13.4 b
0.5 a 5.5 ⇑ab
‘Lime litter species’ Trichoderma koningii Trichoderma hamatum Mucor hiemalis
0a 4.8 ⇑ 0a
0a 2.7 0a
0a 4.6 4.2 b
23.8 ⇓b 2.1 ⇓ 5.5 b
‘Soil species’ Acremonium spp. Humicola sp. 1 Absidia cylindrospora Sterile dark sp. 2 Cylindrocarpon destructans Mortierella minutissima Penicillium spp. Volutella sp. Sterile dark sp. 3 Sterile dark sp. 4 Chrysosporium sp. 1 Phialophora sp. 1 Sterile hyaline sp. 2 Sterile hyaline sp. 3 Paecilomyces spp. Sterile hyaline sp. 4 Sporothrix sp. Chloridium sp. Trichoderma polysporum Sterile hyaline sp. 5 Chrysosporium sp. 2 Chaetomium sp.
5.8 1.2 ⇓ 8.1 3.0 4.0 1.8 7.9 0⇓ 1.2 0.6 1.6 0.6 0⇓ 1.2 2.3 2.3 1.2 1.2 ab 4.6 1.2 0 1.1
9.4 6.4 4.3 3.4 3.2 1.7 6.3 2.8 2.7 ⇑ 2.0 3.3 ⇑ 0 0 1.1 0.5 1.4 1.7 2.2 ⇑a 1.2 0 1.2 0
3.6 1.6 3.1 3.1 2.6 ⇑ 1.0 6.2 1.6 0.5 0 0.5 1.5 0.5 2.1 1.1 0 0 0b 0.5 0.5 1.1 0.5
4.8 1.1 2.8 2.7 1.7 1.1 3.8 1.6 0.5 0 0.5 0 0 0.5 0.5 0 1.0 0.6 0 0.5 0.5 1.6
‘Isolated only from aged materials’ Trichoderma viride Pers. ex Gray Petriellidium sp. Fusarium sp. Wardomyces sp.
2.9 a 0.5 0.6 0
1.2 ab 1.1 1.2 1.1
2.5 ab 0 0 0
0b 0 0 0.5
Total number of taxa isolated Average number of taxa per sample
39 22.5 ab
37 24.0 ⇑b
34 19.5 ⇑ac
38 18.8 ⇑c
⇑
⇑
ab
⇑
a The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Bold⇑ values indicate significant increase in dominance during 100 days of incubation, underlined⇓ values indicate significant decrease (t-test, p<0.05, n=4). Values within rows followed by different letters are significantly different (Tukey’s HSD test based on arcsine- transformed data, n=4, p<0.05).
significantly different in BB and BL casts and in soil (p<0.05). In contrast, aged casts from the B0 treatment could not be reliably separated from the soil (p=0.27). 4. Discussion Two very different situations were modelled in our experiment. (i) L. terrestris consumed only mineral
soil (B0 treatment), therefore most changes of the soil fungal community in fresh casts can be attributed to gut passage. However, as L. terrestris is not a geophagous species, this strongly simplified system poorly reflects natural processes. (ii) Earthworms consumed soil and litter simultaneously (BB and BL treatments) and, therefore, the microfungal community in the casts was formed by fungi originating
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Fig. 3. Discriminant analysis plane of aged soil and casts of Lumbricus terrestris according to the fungal dominance structure. The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Ellipses represent 95% confidence limits.
from both substrates. Beech leaf litter appears to be low quality food for L. terrestris because in the BB treatment earthworm body mass decreased by 12.1% over 45 days. In contrast, Tilia litter was readily consumed and earthworm body mass increased by 9.1% in the BL treatment. The average quantity of litter materials in fresh casts was about 20% (w/w) in the BL treatment but less than 10% in the BB treatment (Tiunov and Scheu, 2000). The soil washing technique, which, by removing fungal spores, isolates predominantly active mycelial fungi, was used to assess the composition of the fungal community in food materials and casts of L. terrestris. The results can be summarised as follows: 1. The passage of mineral soil through the gut of L. terrestris only weakly affected the dominance structure of the soil microfungal community. 2. The fungal community in casts deposited after feeding on litter and soil significantly differed from that in soil and litter. The community is formed by mixing of soil and litter colonizing species and by selectively changing the occurrence of particular species due to gut passage. The composition of the fungal community in casts of L. terrestris strongly depended on the type of litter consumed. 3. Certain soil and litter inhabiting fungi (e.g. A. cylindrospora, Cladosporium spp., Alternaria spp.) were detrimentally affected by gut passage. The colonization rate (number of isolates per number of plated particles) also strongly decreased in
casts. However, some fungi (mainly Trichoderma spp.) flourished in the fresh casts. 4. The dominance structure of fungi changed considerably during ageing of casts. Both the colonization rate and the diversity of species increased. However, the fungal community in casts continued to differ from that in soil for a long time. L. terrestris is a large earthworm species and ingests large organic particles. Therefore, it does not conform to typical fungal feeders, defined as animals that selectively graze fungal material (Shaw, 1992). However, the composition of fungi in food material may affect its palatability to earthworms, particularly to litter feeding species like L. terrestris (Cooke, 1983; Moody et al., 1995). It is also likely that fungi are important agents for the conditioning of litter in middens of L. terrestris. The decrease in the colonization of organic particles by fungi and the decline in frequency of certain species indicate that at least some fungi were damaged by gut passage or by specific environmental conditions in fresh casts. These ‘sensitive’ species comprised about 5–10, 20–25 and 10–15% of the total number of isolates in lime litter, beech litter and soil, respectively. The feeding activity of L. terrestris appears to suppress early litter colonizers (Cladosporium, Alternaria) and secondary sugar fungi (Mucorales) in favour of Trichoderma spp. Knowledge on digestion of fungal hyphae during gut passage through earthworms is very limited; however, by using fluorescence microscopy it has been concluded that fungal hyphae are digested in part by L. terrestris (Wolter and Scheu, 1999). The preference of invertebrate fungivores for darkly pigmented fungi has been stressed frequently (Shaw, 1992; Maraun et al., 1998). Digestion of Alternaria alternata and Cladosporium cladosporioides by the epigeic earthworm Eisenia fetida has been reported by Marfenina and Ishchenko (1997). Therefore, it is likely that the decrease in frequency of Alternaria, Cladosporium and Phialophora species in casts of L. terrestris observed in the present study was caused by digestion of these species. Trichoderma spp. are rejected as food substrate by many fungivores (Shaw, 1992) but this pattern is not consistent in earthworms. Cooke (1983) and Striganova et al. (1988) found Mucorales to be preferred to Trichoderma spp. In contrast, Moody et al. (1995)
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reported M. hiemalis and Trichoderma sp. to be of similar palatability to L. terrestris and two other earthworm species. Also, the viability of spores of Trichoderma sp. and M. hiemalis has been found to be reduced strongly by gut passage (by 99 and 90%, respectively), presumably due to the action of intestinal fluids (Moody et al., 1996). T. koningii and T. viride, and also Mortierella ramanniana were found to be digested by the tropical earthworm species Drawida assamensis (Tiwari et al., 1990). In contrast, Domsch and Banse (1972) reported a strong increase in the frequency of T. hamatumand Mortierella zonata in casts of L. terrestris. In accordance with this finding, the frequency of the three dominant Trichoderma spp. (T. pseudokoningii, T. koningii and T. hamatum) was strongly increased in casts of L. terrestris in our experiment. Three mechanisms might have been responsible for these changes: (i) L. terrestris may have selectively consumed litter particles colonized by Trichoderma spp.; (ii) in the gut of earthworms and in fresh casts, Trichoderma spp. might have been competitively superior to other fungal species, which might be due to the ability of Trichoderma spp. to colonize new substrates quickly but also due to damage to competing fungal species during gut passage; (iii) species of the genus Trichoderma were little damaged by gut passage or balanced the damage by compensatory growth (Hedlund and Augustsson, 1995). We assume the last mechanism to be most important since the number of particles colonized by Trichoderma spp. was consistently higher in casts than in ingested materials. Fungal species which occupy resources earlier may have a competitive advantage over later incomers (Stahl and Christensen, 1992). Thus, ‘tolerant’ species like Trichoderma may benefit from the feeding activity of L. terrestris. This may be of profound importance for the succession of fungi on decomposing litter, since species of the genus Trichoderma are known to be effective competitors and produce a wide range of antifungal substances (Domsch et al., 1980). Domsch and Banse (1972) reported that the percentage of T. hamatum in casts of L. terrestris increased strongly during 30 days of incubation. In our experiment the frequency of Trichoderma spp. was not affected by cast incubation in B0 and BB treatments, but it was reduced in the BL treatment (from 28.5% in fresh to 16.3% in aged casts). Simultaneously, the fre-
25
quency of Absidia, Penicillium and other ‘soil fungi’ increased. The role of earthworms in the dispersal of beneficial or harmful soil fungi is controversial (Edwards and Fletcher, 1988; Brown, 1995; Pattinson et al., 1997). In our study the majority of soil and litter inhabiting fungi were able to survive gut passage through L. terrestris. Therefore, we assume that the feeding and burrowing activity of L. terrestris accelerates the colonization of litter by the edaphic mycoflora but also extends the range of litter-associated fungi to mineral soil layers. The vertical movement of L. terrestris ranges from the soil surface to a depth of up to 2 m, which corresponds with the range of cast deposition (Joergensen et al., 1998). It is likely that in temperate ecosystems the role of L. terrestris and other anecic earthworm species in vertically transporting fungal propagules through the soil profile can hardly be effectively substituted by any other mechanism.
Acknowledgements Financial support by the Volkswagen-Stiftung, Federal Republic of Germany, is gratefully acknowledged. We thank Dr. Yu.G. Puzachenko for valuable advice on statistical analysis.
References Brown, G.G., 1995. How do earthworms affect microfloral and faunal community diversity? Plant and Soil 170, 209–231. Cooke, A., 1983. The effects of fungi on food selection by Lumbricus terrestris L. In: Satchell, J.E. (Ed.), Earthworm Ecology. Chapman and Hall, London, pp. 365–373. Dash, H.K., Beura, B.N., Dash, M.C., 1986. Gut load, transit time, gut microflora and turnover of soil, plant and fungal material by some tropical earthworms, plant and fungal material by some tropical earthworms. Pedobiologia 29, 13–20. Dash, M.C., Satpathy, B., Behera, N., Dei, C., 1984. Gut load and turnover of soil, plant and fungal material by Drawida calebi, a tropical earthworm. Rev. Ecol. Biol. Soil 21, 387–393. Domsch, K.H., Banse, H.J., 1972. Mykologische Untersuchungen an Regenwurmexkrementen. Soil Biol. Biochem. 4, 31–38. Domsch, K.H., Gams, W., Anderson, T.H., 1980. Compendium of Soil Fungi. Academic Press, London, 859 pp. Doube, B.M., Brown, G.G., 1998. Life in a complex community: functional interactions between earthworms, organic matter, microorganisms, and plant growth. In: Edwards C.A. (Ed.), Earthworm Ecology. St. Lucie Press, Boca Raton, pp. 179–211.
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Edwards, C.A., Fletcher, K.E., 1988. Interactions between earthworms and microorganisms in organic-matter breakdown. Agric. Ecosyst. Environ. 24, 235–247. Hedlund, K., Augustsson, A., 1995. Effects of enchytraeid grazing on fungal growth and respiration. Soil Biol. Biochem. 27, 905– 909. Joergensen, R.G., Küntzel, H., Scheu, S., Seitz, D., 1998. Movement of faecal indicator organisms in earthworm channels under a loamy arable and grassland soil. Appl. Soil Ecol. 8, 1–10. Jones, C.G., Lawton, J.H., Shachak, M., 1997. Positive and negative effects of organisms as physical ecosystem engineers. Ecology 78, 1946–1957. Maraun, M., Migge, S., Schaefer, M., Scheu, S., 1998. Selection of microfungal food by six oribatid mite species (Oribatida, Acari) from two different beech forests. Pedobiologia 42, 232–240. Marfenina, O.E., Ishchenko, I.A., 1997. Earthworms preference for soil microscopic fungi. Izvestiya Akademii Nauk Seriya Biologicheskaya 4, 504–506 (in Russian). McLean, M.A., Parkinson, D., 1998. Impacts of the epigeic earthworm Dendrobaena octaedra on microfungal community structure in pine forest floor: a mesocosm study. Appl. Soil Ecol. 8, 61–75. Moody, S.A., Briones, M.J.I., Piearce, T.G., Dighton, J., 1995. Selective consumption of decomposing wheat straw by earthworms. Soil Biol. Biochem. 27, 1209–1213. Moody, S.A., Piearce, T.G., Dighton, J., 1996. Fate of some fungal spores associated with wheat straw decomposition on passage through the guts of Lumbricus terrestris and Aporrectodea longa. Soil Biol. Biochem. 28, 533–537. Newell, K., 1984. Interactions between two decomposer basidiomycetes and a collembolan under Sitka spruce: distribution, abundance and selective grazing, abundance and selective grazing. Soil Biol. Biochem. 16, 227–233. Parkinson, D., Visser, S., Whittaker, J.B., 1979. Effects of collembolan grazing on fungal colonization of leaf litter. Soil Biol. Biochem. 11, 529–535. Parkinson, D., Williams, S.T., 1961. A method for isolating fungi from soil microhabitats, Plant . Plant and Soil 13, 347–355.
Pattinson, G.S., Smith, S.E., Doube, B., 1997. Earthworm Aporrectodea trapezoides had no effect on the dispersal of a vesicular-arbuscular mycorrhizal fungi, Glomus intraradices. Soil Biol. Biochem. 29, 1079–1088. Puzachenko, Yu.G., Kuznetsov, G.V., 1998. Ecological differentiation of rodents in tropical semi-evergreen broad-leaved forests of North Vietnam. Zoologicheskij Zhurnal 77, 117–132 (in Russian). Scheu, S., Parkinson, D., 1994. Effects of earthworms on nutrient dynamics, carbon turnover and microorganisms in soils from cool temperate forests of the Canadian Rocky Mountains — laboratory studies. Appl. Soil Ecol. 1, 113–125. Shaw, P.J.A., 1992. Fungi, fungivores, and fungal food webs. In: Carroll, G.C., Wicklow, D.T. (Eds.), The Fungal Community. Marcel Dekker, New York, pp. 295–310. Stahl, P.D., Christensen, M., 1992. In vitro mycelial interactions among members of a soil microfungal community. Soil Biol. Biochem. 24, 309–316. Striganova, B.R., Marfenina, O.E., Ponomarenko, V.A., 1988. Some aspects of the effect of earthworms on soil fungi. Izvestiya Akademii Nauk Seriya Biologicheskaya 5, 715–719 (in Russian). Tiunov, A.V., Scheu, S., 2000. Microbial biomass, biovolume . biovolume and respiration in Lumbricus terrestris cast material of different age. Soil Biol. Biochem.32, 265–275. Tiwari, S.C., Mishra, R.R., 1993. Fungal abundance and diversity in earthworm casts and in uningested soil. Biol. Fertil. Soils 16, 131–134. Tiwari, S.C., Tiwari, B.K., Mishra, R.R., 1990. Microfungal species associated with the gut content and casts of Drawida assamensis Gates. Proc. Indian Acad. Sci. (Plant Sci.) 100, 379–382. Visser, S., 1985. Role of soil invertebrates in determining the composition of soil microbial communities. In: Fitter, A.H., Atkinson, D., Read, D.J., Usher, M.B. (Eds.), Ecological Interactions in Soil. Blackwell, Oxford, pp. 297–317. Wolter, C., Scheu, S., 1999. Changes in bacterial numbers and hyphal lengths during the gut passage through Lumbricus terrestris (Lumbricidae, Oligochaeta). Pedobiologia 43, 891– 900.
Microfungal communities in soil, litter and casts of Lumbricus terrestris L. (Lumbricidae): a laboratory experiment Alexei V. Tiunov a,∗ , Stefan Scheu b a b
Laboratory of Soil Zoology, Institute of Ecology and Evolution, Leninsky Prospect 33, 117071, Moscow, Russia Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 3, D-64287, Darmstadt, Germany Received 8 July 1999; received in revised form 29 November 1999; accepted 1 December 1999
Abstract The anecic earthworm Lumbricus terrestris L. was kept in laboratory microcosms containing beech forest soil without litter, with beech leaf litter or with lime leaf litter. The structure of microfungal communities in soil, litter and fresh and aged (100 days) earthworm faeces was analysed using the washing and plating technique. The passage of mineral soil through the gut of L. terrestris affected the structure of the fungal community only little. In contrast, in the litter treatments the structure of the fungal community in fresh earthworm casts significantly differed from that in soil and litter. The majority of soil and litter inhabiting fungi survived passage through the gut of L. terrestris and the fungal community in casts consisted of a mixture of soil and litter inhabiting fungi. However, the frequency of Cladosporium spp., Alternaria spp., Absidia spp., and other taxa was strongly reduced in fresh casts. The degree of colonization of litter particles (number of isolates per number of plated particles) also decreased, but some fungi (mainly Trichoderma spp.) benefited from gut passage and flourished in fresh casts. During ageing of cast material the dominance structure of the fungal community changed. Both the degree of colonization of organic particles and the species diversity increased and approached that in soil. However, the structure of the fungal community in casts remained cast specific even after 100 days of incubation. It is concluded that the feeding and burrowing activity of L. terrestris accelerates the colonization of litter by the edaphic mycoflora but also extends the range of occurrence of litter-associated fungi into mineral soil layers. ©2000 Elsevier Science B.V. All rights reserved. Keywords: Soil fungi; Fungal community; Anecic earthworms; Lumbricus terrestris; Litter decomposition
1. Introduction Fungi and earthworms are important members of soil communities and their interactions are assumed to significantly affect microbial-mediated processes in soil. Although the importance of these interactions is widely accepted, they are little studied and the information available is in part contradictory (Brown, 1995). Earthworms may influence soil fungi by a ∗ Corresponding author. Tel.:+7-095-958-1449; fax: +7-095-954-5534/952-2592. E-mail address: [email protected] (A.V. Tiunov).
variety of mechanisms, including the alteration of the physical and chemical state of the environment (‘engineering’ sensu Jones et al., 1997), comminution and translocation of litter, dispersal of fungal propagules and grazing on fungal tissue (Visser, 1985). Various authors suggested that fungi are a major component of the diet of earthworms (Dash et al., 1984, 1986; Edwards and Fletcher, 1988; Tiwari et al., 1990; Doube and Brown, 1998). Earthworms have been shown to prefer food substrates colonized by certain fungal species and fungi have been found to be damaged by gut passage (Cooke, 1983; Moody et al., 1995, 1996; Marfenina and Ishchenko, 1997).
0929-1393/00/$ – see front matter ©2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 9 - 1 3 9 3 ( 9 9 ) 0 0 0 5 0 - 5
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Even low levels of selective grazing by invertebrates has been shown to significantly alter the distribution and succession of fungi on litter (Parkinson et al., 1979; Newell, 1984). Therefore, it is likely that earthworms alter the composition of fungal communities in soil and the fungal succession in decomposing litter. However, there is only fragmentary information on effects of earthworms on the structure of fungal communities in soil. Domsch and Banse (1972) reported a strong alteration of the fungal dominance structure in casts of Lumbricus terrestris in comparison to the food substrates. In contrast, Tiwari and Mishra (1993) did not find significant differences in the structure of fungal communities in soil and field-collected earthworm casts. In micro- and mesocosm experiments, the presence of the epigeic earthworm species Dendrobaena octaedra (McLean and Parkinson, 1998) and the endogeic species Octolasion tyrtaeum (Scheu and Parkinson, 1994) affected the structure of fungal communities only little, although the frequency of some species changed significantly. Detailed studies aiming at separating different mechanisms which may contribute to earthworm-mediated changes in the structure of fungal communities in soil are lacking. We studied microfungal communities in fresh and aged faeces of the widespread anecic earthworm L. terrestris L. This large deep-burrowing species builds permanent vertical burrows, but feeds mainly on organic materials on the soil surface. Due to the feeding and burrowing activity of L. terrestris, large amounts of mineral soil are translocated upward to the soil surface and mixed with litter, whereas litter material is transferred deep into the mineral soil. We addressed the following questions: how does passage through the gut of L. terrestris affect the structure of the fungal community as compared to soil and litter? Which of the soil and litter inhabiting fungal species survive, and which are damaged by gut passage? How does the fungal community in faeces change during ageing of cast material?
2. Materials and methods 2.1. Experimental design Mineral soil, beech leaf litter and specimens of L. terrestris were obtained from a 115–120 year old
beech (Fagus sylvatica) stand, the ‘Göttinger Wald’ (Lower Saxony, Germany). Soil material was taken in June 1996 at a depth of 3–10 cm from the mineral soil surface and sieved (<4 mm). Overwintered F. sylvatica leaf litter was collected from the soil surface at the same time. Lime (Tilia cordata) leaf litter was collected in May 1996 in a lime grove 30 km south of Moscow (Russia) and kept until the start of the experiment in polyethylene bags at 5◦ C. The experiment was carried out in narrow containers (inner dimensions: 650 mm high, 310 mm long, 10 mm wide). Three combinations of beech forest soil and litter were studied: soil without litter (B0 treatment), soil+beech litter (BB treatment), and soil+lime litter (BL treatment). Containers were filled with soil to a level of 500 mm. The soil was compacted to a bulk density of 0.65 kg dry weight 1000 cm−3 which is typical for the upper soil layers at the Göttinger Wald. The moisture content of the soil was kept at the field level (62% of dry wt.). About 25 g of Tilia or Fagus litter was added to each BL and BB container and replenished periodically as it was consumed by earthworms. In each microcosm two adult L. terrestris (average individual weight 6.0±0.2 g) were placed. Containers were kept at 15◦ C in permanent darkness. Five replicates were set up per treatment. Further details are given in Tiunov and Scheu (2000). After a preliminary period of 2 weeks, when a burrow system was established by the earthworms, containers were opened every second day and freshly deposited faeces were collected (average age of 1 day). For the analysis of the microfungal community, fresh casts and control litter samples were collected 26–30 days after the start of the experiment from four containers of each treatment. Simultaneously, control soil samples were taken from B0 (two replicates), BL and BB treatments (one replicate each). Soil and litter samples were taken from the zones not affected by L. terrestris (at least 50 mm away from the nearest burrow). Faecal material of age 100 days was obtained by incubating casts between two layers of soil separated by 0.5 mm gauze. The container with soil and casts was incubated in a polyethylene bag at 15◦ C. A plastic tube (i.d. 3 mm) was inserted into the bag to ensure free gas exchange.
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2.2. Isolation of fungi The composition of the fungal communities in the soil, litter and casts was analysed by a modified washing and plating method (Parkinson and Williams, 1961). One gram (fresh weight) of soil, cast or litter material was blended for 20 s in 100 ml sterile water. About 0.5 g of the blended material was placed in the washing apparatus (vol. ca. 100 ml) between 0.5 and 0.2 mm meshes. The material was washed by applying a constant flow (0.5 l min−1 ) of sterile water for 10 min. During the washing procedure the apparatus was gently shaken. Washed organic particles of size 0.2–0.5 mm were collected on sterile filter paper. Fifty particles per sample were plated on malt extract agar containing streptomycin and tetracycline (0.1 and 0.05 g l−1 , respectively) to suppress growth of bacteria. One particle was placed per Petri-dish. The Petri-dishes were incubated at room temperature. When two or more fungal species were growing from the single particle, pure cultures were prepared by transferring hyphal inocula to separate plates. The frequency of occurrence of fungal taxa (as percentages) was calculated as the number of particles with fungal growth per total number of isolates ×100. On average, 44.5 isolates were obtained per 50 plated particles.
2.3. Statistical analysis The main goal of the analysis was to compare the structure of whole fungal communities in the studied substrata, rather than to follow changes in abundance of single fungal species or genera. For this purpose, ordinations and discriminant analysis were used. Rare fungi (fewer than two isolates), yeast-like forms and loosely-defined groups (i.e., ‘other sterile dark’, ‘other Trichoderma’; see further) were not included in the analysis. The remaining 46 taxa represented ca. 70% of the isolates. The logic of the mathematical analysis follows the scheme proposed by Puzachenko and Kuznetsov (1998). Based on the relative frequency of fungal taxa, the square matrix of nonparametric Gamma correlation (analogous to Kendall τ ) between the studied samples was calculated. The distance matrix was obtained by subtracting correlation coefficients
19
(range 1 to −1) from 1. Thus, distances varied from 0 (perfect similarity between two samples) to 2 (no similarity). The distance matrix was analysed further by nonmetric multidimensional scaling. This ordination technique ‘rearranges’ objects in n-dimensional space, so as to arrive at a configuration that best approximates the observed distances. The number of meaningful dimensions was evaluated by comparing actual stress values with the theoretical exponential function of stress. The coordinates of the samples in n-dimensional space were used for discriminant function analysis (DFA), with ‘substrate’ (soil, litter or casts) as a grouping variable. Squared Mahalanobis distances between group centroids and the reliability of sample classification were determined. Typically, only two significant discriminant functions (canonical roots) were derived. Thus, the results of DFA are graphically presented in 2-dimensional space. For the interpretation of the discriminant axes in respect to the frequency of fungal taxa, linear correlations were calculated between the discriminant function scores for each sample and the relative dominance of fungal taxa. A similar procedure was used to analyse the similarity matrix between fungal taxa. Prior to the analysis, fungi were grouped into soil- and litter-associated species using cluster analysis and Ward’s minimum variance method. All calculations were made using the STATISTICA software package. Differences between means were tested using Tukey’s honestly significant difference (HSD) at the 0.05 probability level.
3. Results 3.1. ‘Soil’ and ‘litter’ fungi The fungal community in beech litter was dominated by Mortierella gamsii, T. pseudokoningii and Mucor hiemalis. The most frequent fungi in lime litter were T. koningii, T. hamatum, M. hiemalis and two Alternaria spp. Most of these fast growing fungi were not isolated from the soil (Table 1). The fungal community in soil was more diverse. The most frequent fungi were two species of the genus Acremonium, Humicola sp. 1, Absidia cylindrospora, Cylindrocarpon destructans, Mortierella minutissima, several species of Penicillium and Volutella sp.
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Table 1 Frequency (percentage of the total number of isolates) of dominant fungi in soil, litter and fresh casts of L. terrestrisa Soil
B0 casts
Beech litter
BB casts
Lime litter
BL casts
1.6 ab 0a 0a 0 0
0a 0a 0a 0 0
14.8 b 6.9 b 3.3 b 2.6 1.1
6.7 b 13.2 b 0.7 ab 0 1.4
0.5 a 0a 0a 0 0
0a 0a 0a 0 0
‘Lime litter species’ Trichoderma koningii Oudem. Trichoderma hamatum Bain. Mucor hiemalis Wehmer Alternaria sp. 2 Alternaria alternata (Fr.) Keissler Tolypocladium sp.
0a 0.9 a 0 0a 0 0
0a 1.4 a 0 0.6 a 0 0
0a 4.4 a 5.8 0.5 a 0 0
0a 2.2 a 3.0 0a 0 0
29.3 b 9.5 ab 5.9 5.1 b 2.4 1.6
39.4 b 14.5 b 3.5 0.6 a 0 1.8
‘Soil species’ Acremonium spp. Humicola sp. 1 Absidia cylindrospora Hagem Sterile dark sp. 2 Cylindrocarpon destructans (Zinssm.) Scholten Mortierella minutissima van Tiegh. Penicillium spp. Volutella sp. Sterile dark sp. 3 Sterile dark sp. 4 Chrysosporium sp. 1 Phialophora sp. 1 Sterile hyaline sp. 2 Sterile hyaline sp. 3 Paecilomyces spp. Sterile hyaline sp. 4 Sporothrix sp. Chloridium sp. Trichoderma polysporum (Link ex Pers.) Rifai Sterile hyaline sp. 5 Chrysosporium sp. 2 Trichocladium sp.
11.2 a 5.7 a 5.5 a 3.9 ab 4.4 3.8 a 3.6 3.1 2.8 a 2.6 ab 2.6 a 2.1 a 2.0 a 1.9 1.8 1.5 1.5 1.5 0.9 0 0 0
9.0 ab 5.7 a 2.1 ab 6.4 a 4.3 2.6 a 4.6 4.4 0b 3.4 b 0b 0b 0b 2.0 1.4 2.0 1.3 0.6 0.6 0.6 0 1.4
1.7 ab 0b 0.5 b 0.7 b 0 0b 1.2 1.6 0.6 ab 0c 0b 0b 0b 0 1.1 1.8 0 0 1.2 0 0 0
5.6 ab 2.0 ab 1.4 ab 4.3 ab 0.5 3.4 a 2.9 5.7 1.2 ab 0.7 ac 0b 0b 0.7 ab 1.2 0 1.2 0.8 0.7 0 0 1.5 0
0.5 b 0b 0b 0b 0 0b 0.5 0 0b 0c 0b 0b 0b 0 0 0 0 0 0 0 0 0
3.9 ab 1.8 ab 0b 1.7 ab 0.6 0b 0.6 0.6 0b 0c 0b 0b 0b 0 0 0.6 0.6 0 0 1.2 0 0
Total number of taxa isolated Average number of taxa per sample
37 22.5 a
29 19.0 a
26 14.0 bc
32 16.8 ab
18 11.5 c
24 11.5 c
‘Beech litter species’ Mortierella gamsii Milko Trichoderma pseudokoningii Sterile hyaline sp. 1 Cladosporium spp. Sterile dark sp. 1
Rifai
a The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil +lime litter) treatments. Values within rows followed by different letters are significantly different (Tukey’s HSD test based on arcsine-transformed data, n=4, p<0.05).
Cluster analysis of the similarity matrix of 38 dominant fungal taxa singled out three main clusters, which were interpreted as ‘beech litter species’, ‘lime litter species’ and ‘soil species’. The reliability of the separation was further confirmed by the discriminant analysis (p<0.001). Fungal taxa in Table 1 are grouped according to this clustering. However, the arbitrary character
of the grouping should be noted, as some ‘soil fungi’ (e.g. T. polysporum or Sterile hyaline sp. 4) were equally abundant in the beech litter, while M. hiemalis had similar occurrence in both types of litter. Although we applied several clustering procedures, no specific cluster of ‘cast-associated’ fungi could be singled out. Trichocladium sp., Sterile hyaline sp. 5
A.V. Tiunov, S. Scheu / Applied Soil Ecology 14 (2000) 17–26
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and Chrysosporium sp. 2 were isolated only from the casts, but the frequency of these fungi was low and they did not reliably separate from the ‘soil fungi’. 3.2. Fresh casts The structure of the fungal community in food substrates and egested materials can only be compared directly in the B0 treatment, where earthworms exclusively consumed mineral soil. Only a few fungal taxa were significantly affected by gut passage in this treatment (Table 1). However, Mucorales (Absidia spp. and Mortierella spp.) tended to be less frequent and other fungal species, e.g. Chrysosporium sp. 1 and Phialophora sp., were not isolated from casts. The total number of fungal taxa was lower in casts (29) than in soil (37). Comparing food substrate and casts is more difficult in BB and BL treatments because L. terrestris consumed both soil and litter. Alterations in the structure of fungal communities in casts of these treatments might be due to gut passage or to mixing of soil and litter materials. Generally, ‘soil fungi’ in casts accounted for 35 and 13% of the total number of isolates in BB and BL treatments, respectively (Fig. 1). Of
Fig. 1. Number of organic particles colonized by litter-associated fungi (‘litter fungi’), soil-associated fungi (‘soil fungi’) and uncolonized particles in soil, litter and fresh casts of Lumbricus terrestris (means+SD). The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. 50 particles were plated per replicate, n=4.
Fig. 2. Discriminant analysis plane of soil, litter and fresh casts of Lumbricus terrestris according to the fungal dominance structure. The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Ellipses represent 95% confidence limits.
the ‘litter fungi’, Mucorales (Mortierella gamsii and M. hiemalis), Cladosporium spp., and two species of Alternaria tended to be less abundant in casts than in the litter offered as food substrate. In contrast, the frequency of three Trichoderma spp. was greater in casts (Table 1). However, most of these tendencies were not statistically significant. In each of the three treatments the number of organic particles from which no fungi developed was significantly greater in L. terrestris casts compared to food materials. The mean number of uncolonized particles was 10% in soil, 5% in beech litter and 0.5% in lime litter, but reached 25, 26 and 14% in B0, BB and BL casts, respectively (Fig. 1). The dominance structure of fungal communities in casts from BB and BL treatments differed considerably from that in food materials (Fig. 2). The materials studied were reliably discriminated in 2-dimensional space. The first axis, which accounted for 60.8% of the discriminatory power, separated litter (negative values) and soil (positive values). Although the control soil samples were taken from three different treatments (B0, BB and BL), they did not differ significantly from each other and formed a compact group. The second axis (38.5% of the discriminatory power) separated Fagus and Tilia litter. Casts from BB and BL treatments were intermediate in position between soil and litter, but were more similar to the corresponding litter than to the soil. However, they could be reliably
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Table 2 Squared Mahalanobis distances between group centroids and reliability of discrimination based on data on fungal dominance structurea
Soil Beech litter Lime litter B0 casts BB casts
B0 casts
BB casts
BL casts
5.3 nsb 180.9∗∗∗ 186.9∗∗∗ 0 89.4∗∗∗
73.8∗∗∗ 19.2∗ 114.7∗∗∗ 89.4∗∗∗ 0
102.5∗∗∗ 130.3∗∗∗ 26.4∗∗ 86.7∗∗∗ 100.8∗∗∗
a
See text for details. ns=not significant. ∗ p<0.01. ∗∗ p<0.005. ∗∗∗ p<0.0001. b
distinguished from each other and from both soil and litter (p<0.01). In contrast, casts from the B0 treatment could not be discriminated from the soil (Table 2). The two discriminant axes can be interpreted easily considering the dominance structure of the fungal communities. The occurrence of dominant ‘soil fungi’ positively correlated with the first axis, but not with the second axis. ‘Beech litter fungi’ were negatively correlated with both axes. ‘Lime litter fungi’ were negatively correlated with the first axis and positively with the second (Table 3).
Table 3 Linear correlation (r-values) between the frequency of fungal taxa and discriminant axesa Axis 1
Axis 2
‘Lime litter species’ Trichoderma hamatum Mucor hiemalis Alternaria sp. 2 Trichoderma koningii Tolypocladium sp.
−0.517∗∗ −0.515∗∗ −0.482∗ −0.472∗ −0.221 ns
‘Beech litter species’ Mortierella gamsii Trichoderma pseudokoningii Sterile hyaline sp. 1 Sterile dark sp. 1
−0.323 −0.171 −0.313 −0.180
‘Soil species’ Humicola sp. 1 Sterile dark sp. 2 Mortierella minutissima Sterile dark sp. 4 Acremonium spp. Cylindrocarpon destructans Absidia cylindrospora Penicillium spp. Sterile hyaline sp. 3 Sporothrix sp. Sterile hyaline sp. 2 Chloridium sp. Phialophora sp. 1 Chrysosporium sp. 1 Paecilomyces spp.
ns ns ns ns
0.757∗∗∗ 0.677∗∗∗ 0.657∗∗∗ 0.652∗∗ 0.636∗∗ 0.626∗∗ 0.613∗∗ 0.585∗∗ 0.517∗∗ 0.500∗ 0.480∗ 0.480∗ 0.480∗ 0.448∗ 0.410∗
0.599∗∗ 0.010 nsb 0.435∗ 0.727∗∗∗ 0.507∗ −0.646∗∗ −0.630∗∗ −0.511∗ −0.434∗ 0.077 −0.125 −0.264 −0.014 −0.074 0.086 −0.126 −0.183 −0.094 0.074 −0.134 −0.098 −0.019 −0.008 −0.138
ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns
a
See text for details. ns=not significant. ∗ p<0.05. ∗∗ p<0.01. ∗∗∗ p<0.001. b
3.3. Aged casts The frequency of certain fungal taxa significantly changed during the 100-day incubation of soil and cast material (Table 4). However, in most cases the changes were not uniform in the different substrates. The frequency of T. koningii, Volutella sp. and Humicola sp. 1 tended to decline during ageing of soil and casts. In contrast to the fresh samples, Sterile hyaline sp. 1, Sterile dark sp. 1, Tolypocladium sp. and Trichocladium sp. were not isolated from the aged samples (Tables 1 and 4). Conversely, the frequency of A. cylindrospora, T. pseudokoningii, T. polysporum and Penicillium spp. increased in most aged substrates. T. viride, Fusarium sp., Wardomyces sp. and other fungi were isolated only from aged soil and casts. The total number of isolated taxa also increased, and the number of uncolonized particles in aged casts did not exceed 10%.
The changes in the fungal community structure in aged materials were reflected by reliable discrimination between ‘fresh’ and ‘aged’ substrates. The discrimination was strongest between ‘fresh’ and ‘aged’ soil (p=0.003) and between ‘fresh’ and ‘aged’ casts from BL treatment (p=0.007) whereas it was less reliable between ‘fresh’ and ‘aged’ casts in B0 and BB treatments (p=0.037 and 0.054, respectively). During incubation, cast materials of different treatments were kept in the same soil container, separated only by 2–5 cm. However, after 100 days of ageing, fungal communities in L. terrestris casts retained specific features (Fig. 3). The three types of cast material were separated from each other in 2-dimensional discriminant space (p<0.01). Fungal communities were
A.V. Tiunov, S. Scheu / Applied Soil Ecology 14 (2000) 17–26
23
Table 4 Frequency (percentage of the total number of isolates) of dominant fungi in aged soil and casts of Lumbricus terrestrisa Holding soil
B0 casts
BB casts
BL casts
‘Beech litter species’ Mortierella gamsii Trichoderma pseudokoningii
0a 3.4 a
0.5 a 0.5 a
13.1 ⇑b 13.4 b
0.5 a 5.5 ⇑ab
‘Lime litter species’ Trichoderma koningii Trichoderma hamatum Mucor hiemalis
0a 4.8 ⇑ 0a
0a 2.7 0a
0a 4.6 4.2 b
23.8 ⇓b 2.1 ⇓ 5.5 b
‘Soil species’ Acremonium spp. Humicola sp. 1 Absidia cylindrospora Sterile dark sp. 2 Cylindrocarpon destructans Mortierella minutissima Penicillium spp. Volutella sp. Sterile dark sp. 3 Sterile dark sp. 4 Chrysosporium sp. 1 Phialophora sp. 1 Sterile hyaline sp. 2 Sterile hyaline sp. 3 Paecilomyces spp. Sterile hyaline sp. 4 Sporothrix sp. Chloridium sp. Trichoderma polysporum Sterile hyaline sp. 5 Chrysosporium sp. 2 Chaetomium sp.
5.8 1.2 ⇓ 8.1 3.0 4.0 1.8 7.9 0⇓ 1.2 0.6 1.6 0.6 0⇓ 1.2 2.3 2.3 1.2 1.2 ab 4.6 1.2 0 1.1
9.4 6.4 4.3 3.4 3.2 1.7 6.3 2.8 2.7 ⇑ 2.0 3.3 ⇑ 0 0 1.1 0.5 1.4 1.7 2.2 ⇑a 1.2 0 1.2 0
3.6 1.6 3.1 3.1 2.6 ⇑ 1.0 6.2 1.6 0.5 0 0.5 1.5 0.5 2.1 1.1 0 0 0b 0.5 0.5 1.1 0.5
4.8 1.1 2.8 2.7 1.7 1.1 3.8 1.6 0.5 0 0.5 0 0 0.5 0.5 0 1.0 0.6 0 0.5 0.5 1.6
‘Isolated only from aged materials’ Trichoderma viride Pers. ex Gray Petriellidium sp. Fusarium sp. Wardomyces sp.
2.9 a 0.5 0.6 0
1.2 ab 1.1 1.2 1.1
2.5 ab 0 0 0
0b 0 0 0.5
Total number of taxa isolated Average number of taxa per sample
39 22.5 ab
37 24.0 ⇑b
34 19.5 ⇑ac
38 18.8 ⇑c
⇑
⇑
ab
⇑
a The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Bold⇑ values indicate significant increase in dominance during 100 days of incubation, underlined⇓ values indicate significant decrease (t-test, p<0.05, n=4). Values within rows followed by different letters are significantly different (Tukey’s HSD test based on arcsine- transformed data, n=4, p<0.05).
significantly different in BB and BL casts and in soil (p<0.05). In contrast, aged casts from the B0 treatment could not be reliably separated from the soil (p=0.27). 4. Discussion Two very different situations were modelled in our experiment. (i) L. terrestris consumed only mineral
soil (B0 treatment), therefore most changes of the soil fungal community in fresh casts can be attributed to gut passage. However, as L. terrestris is not a geophagous species, this strongly simplified system poorly reflects natural processes. (ii) Earthworms consumed soil and litter simultaneously (BB and BL treatments) and, therefore, the microfungal community in the casts was formed by fungi originating
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Fig. 3. Discriminant analysis plane of aged soil and casts of Lumbricus terrestris according to the fungal dominance structure. The casts originated from B0 (soil only), BB (soil+beech litter) and BL (soil+lime litter) treatments. Ellipses represent 95% confidence limits.
from both substrates. Beech leaf litter appears to be low quality food for L. terrestris because in the BB treatment earthworm body mass decreased by 12.1% over 45 days. In contrast, Tilia litter was readily consumed and earthworm body mass increased by 9.1% in the BL treatment. The average quantity of litter materials in fresh casts was about 20% (w/w) in the BL treatment but less than 10% in the BB treatment (Tiunov and Scheu, 2000). The soil washing technique, which, by removing fungal spores, isolates predominantly active mycelial fungi, was used to assess the composition of the fungal community in food materials and casts of L. terrestris. The results can be summarised as follows: 1. The passage of mineral soil through the gut of L. terrestris only weakly affected the dominance structure of the soil microfungal community. 2. The fungal community in casts deposited after feeding on litter and soil significantly differed from that in soil and litter. The community is formed by mixing of soil and litter colonizing species and by selectively changing the occurrence of particular species due to gut passage. The composition of the fungal community in casts of L. terrestris strongly depended on the type of litter consumed. 3. Certain soil and litter inhabiting fungi (e.g. A. cylindrospora, Cladosporium spp., Alternaria spp.) were detrimentally affected by gut passage. The colonization rate (number of isolates per number of plated particles) also strongly decreased in
casts. However, some fungi (mainly Trichoderma spp.) flourished in the fresh casts. 4. The dominance structure of fungi changed considerably during ageing of casts. Both the colonization rate and the diversity of species increased. However, the fungal community in casts continued to differ from that in soil for a long time. L. terrestris is a large earthworm species and ingests large organic particles. Therefore, it does not conform to typical fungal feeders, defined as animals that selectively graze fungal material (Shaw, 1992). However, the composition of fungi in food material may affect its palatability to earthworms, particularly to litter feeding species like L. terrestris (Cooke, 1983; Moody et al., 1995). It is also likely that fungi are important agents for the conditioning of litter in middens of L. terrestris. The decrease in the colonization of organic particles by fungi and the decline in frequency of certain species indicate that at least some fungi were damaged by gut passage or by specific environmental conditions in fresh casts. These ‘sensitive’ species comprised about 5–10, 20–25 and 10–15% of the total number of isolates in lime litter, beech litter and soil, respectively. The feeding activity of L. terrestris appears to suppress early litter colonizers (Cladosporium, Alternaria) and secondary sugar fungi (Mucorales) in favour of Trichoderma spp. Knowledge on digestion of fungal hyphae during gut passage through earthworms is very limited; however, by using fluorescence microscopy it has been concluded that fungal hyphae are digested in part by L. terrestris (Wolter and Scheu, 1999). The preference of invertebrate fungivores for darkly pigmented fungi has been stressed frequently (Shaw, 1992; Maraun et al., 1998). Digestion of Alternaria alternata and Cladosporium cladosporioides by the epigeic earthworm Eisenia fetida has been reported by Marfenina and Ishchenko (1997). Therefore, it is likely that the decrease in frequency of Alternaria, Cladosporium and Phialophora species in casts of L. terrestris observed in the present study was caused by digestion of these species. Trichoderma spp. are rejected as food substrate by many fungivores (Shaw, 1992) but this pattern is not consistent in earthworms. Cooke (1983) and Striganova et al. (1988) found Mucorales to be preferred to Trichoderma spp. In contrast, Moody et al. (1995)
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reported M. hiemalis and Trichoderma sp. to be of similar palatability to L. terrestris and two other earthworm species. Also, the viability of spores of Trichoderma sp. and M. hiemalis has been found to be reduced strongly by gut passage (by 99 and 90%, respectively), presumably due to the action of intestinal fluids (Moody et al., 1996). T. koningii and T. viride, and also Mortierella ramanniana were found to be digested by the tropical earthworm species Drawida assamensis (Tiwari et al., 1990). In contrast, Domsch and Banse (1972) reported a strong increase in the frequency of T. hamatumand Mortierella zonata in casts of L. terrestris. In accordance with this finding, the frequency of the three dominant Trichoderma spp. (T. pseudokoningii, T. koningii and T. hamatum) was strongly increased in casts of L. terrestris in our experiment. Three mechanisms might have been responsible for these changes: (i) L. terrestris may have selectively consumed litter particles colonized by Trichoderma spp.; (ii) in the gut of earthworms and in fresh casts, Trichoderma spp. might have been competitively superior to other fungal species, which might be due to the ability of Trichoderma spp. to colonize new substrates quickly but also due to damage to competing fungal species during gut passage; (iii) species of the genus Trichoderma were little damaged by gut passage or balanced the damage by compensatory growth (Hedlund and Augustsson, 1995). We assume the last mechanism to be most important since the number of particles colonized by Trichoderma spp. was consistently higher in casts than in ingested materials. Fungal species which occupy resources earlier may have a competitive advantage over later incomers (Stahl and Christensen, 1992). Thus, ‘tolerant’ species like Trichoderma may benefit from the feeding activity of L. terrestris. This may be of profound importance for the succession of fungi on decomposing litter, since species of the genus Trichoderma are known to be effective competitors and produce a wide range of antifungal substances (Domsch et al., 1980). Domsch and Banse (1972) reported that the percentage of T. hamatum in casts of L. terrestris increased strongly during 30 days of incubation. In our experiment the frequency of Trichoderma spp. was not affected by cast incubation in B0 and BB treatments, but it was reduced in the BL treatment (from 28.5% in fresh to 16.3% in aged casts). Simultaneously, the fre-
25
quency of Absidia, Penicillium and other ‘soil fungi’ increased. The role of earthworms in the dispersal of beneficial or harmful soil fungi is controversial (Edwards and Fletcher, 1988; Brown, 1995; Pattinson et al., 1997). In our study the majority of soil and litter inhabiting fungi were able to survive gut passage through L. terrestris. Therefore, we assume that the feeding and burrowing activity of L. terrestris accelerates the colonization of litter by the edaphic mycoflora but also extends the range of litter-associated fungi to mineral soil layers. The vertical movement of L. terrestris ranges from the soil surface to a depth of up to 2 m, which corresponds with the range of cast deposition (Joergensen et al., 1998). It is likely that in temperate ecosystems the role of L. terrestris and other anecic earthworm species in vertically transporting fungal propagules through the soil profile can hardly be effectively substituted by any other mechanism.
Acknowledgements Financial support by the Volkswagen-Stiftung, Federal Republic of Germany, is gratefully acknowledged. We thank Dr. Yu.G. Puzachenko for valuable advice on statistical analysis.
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