FEMS Microbiology Ecology 53 (1988) 285-290

Published by Elsevier

285

FEC 00175

Quantitative investigations on nematode-trapping hyphomycetes from woodland soils

Andrea Siinder and Gernot Lysek

Institut ftir Systematische Botanik und l’flanzengeographie der FU Berlin, Berlin, F. R.G.

Received 10 December 1987

Revision received 19 March 1988 Accepted 24 March 1988

Key words: Nematode-destroying fungi; Woodland soil

1. SUMMARY

From three different woodland types in Berlin (West) (poor sandy pine forest, mesotrophic beech forest and a sporadically inundated river bank), nematode-destroying hyphomycetes were isolated using the sprinkled plates method. 16.4% of the plates yielded isolates, one predacious fungus per 8 g of fresh soil. The fungi obtained belonged to ten species out of four different genera. The highest number of isolates was obtained from the beech forest, while the lowland forest yielded the highest number of species but the lowest amount of iso- lates. The differences between the various soil types are discussed. The results indicated that attempts to quantify the nematode-destroying hy- phomycetes may be successful.

2. INTRODUCTION

Nematode-destroying fungi are an interesting and ubiquitous group of soil fungi which have

Correspondence to: ti. Lysek, Inst. Systematische Botanik und Pflanzengeographie der Fu Berlin, Altensteinstr. 6, D-1000 Berlin 33, F.R.G.

been studied intensively with regard to their as- sumed ecological role and their potential use in biological control of plant parasitic nematodes [l-3]. While the physiology and the interactions with the nematode prey are known from recent intensive investigations [4-71 virtually nothing is known about the number, distribution and activity of these fungal organisms in the soil itself.

In our laboratory attempts to quantify the number of nematode-destroying fungi were started with the group of endoparasitic nematode-destroy- ing fungi, i.e., those fungi which complete their entire life cycle from germination to the end of the vegetative phase inside the corpses of infected eelworms, while only the finally produced conidio- phores break through the cuticle of the nematodes and form their spores outside. The quantification of these organisms by isolation of infected soil nematodes with the Baermann funnel technique was successful [8,9]. Serial isolations with this method allowed the calculation of the probable number of endoparasitic fungal species at a given site [9]. Similar results were obtained by Diirschner [lo] who isolated the spores by a centrifugation method. It was discovered that about 0.5% of a nematode population is infected and that the number of fungal species at a certain site is limited

0168-6496/88/$03.50 0 1988 Federation of European Microbiological Societies

286

to five to eight species. These results are in good agreement with attempts by other investigators [ll-151.

Predacious hyphomycetes which are partially saprophytic and partially predacious are more dif- ficult to assess. Attempts to quantify predacious fungi in soil and to study their ecology have been reported several times [12-14,16-191. Recently, Dackman et al. [ll] published a useful combina- tion of methods to quantify nematode capturing as well as endoparasitic fungi - unfortunately without identifying the occurring species. To ob- tain more detailed results and to quantify the nematode-trapping hyphomycetes, mass isolations using sprinkled plates were used.

3. MATERIALS AND METHODS

3.1. Sample sites

Samples were taken at three different sites in Berlin-West: Grunewald: Pine forest (Pinus syluestris L.) on dry sand. Boettcherberg: Beech forest (Fagus syluatica L.) on sandy moraines of the last glaciation. Pfauenisel: a small peninsula of an island in the river Havel, sporadically inundated and covered with poplar (Populus spp.) and willow (Salix spp.). Samples were taken at three different spots at each of these sites.

3.2. Sampling

The soil was taken monthly after removing the uppermost layer of plant debris from a depth up to 10 cm using autoclaved spatulas, was collected in sterile flasks and brought to the laboratory.

3.3. Isolation of nematode-trapping fungi Immediately after bringing it to the laboratory,

if frozen, after 3 days of thawing and incubation, the flasks were shaken and l-l.5 g of fresh soil was sprinkled on water-agar in petri dishes of 9 cm diameter [3]. Ten petri dishes per sample date and site were used for isolations.

After 5 days of growth at ambient temperature and in constant darkness, 30 to 70 nematodes from a culture of Turbatix aceti were added as

bait. The sprinkled plates were checked at regular intervals over a period of 3 months.

From those dishes which contained trapped and digested nematodes, the fungus was trans- ferred to another water-agar plate by inoculating dead nematodes, pieces of mycelium or, if availa- ble, conidia. The outgrowing colonies were subcul- tured to obtain pure cultures, and checked by addition of bait for their trapping capability. Per-

manent cultures were grown in agar slant tubes.

3.4. Identification

The strains obtained were identified using the key given by Cooke and Godfrey [20] and the original descriptions.

Permanent mounts were made using Karion F (Merck): the fungi were stained with lactophenol-

cotton-blue [21].

3.5. Chemical analysis of soil samples

The soil samples were analysed chemically by the Plant Protection Office Berlin (Pflanzen- schutzamt Berlin), according to the guidelines of the Society of Agricultural Investigation Offices (Verband landwirtschaftlicher Untersuchung- sanstalten) [22].

3.6. Herbarium specimens

Specimens of the microscopic mounts or dried- out petri dish cultures are used as herbarium specimens.

4. RESULTS

4.1. Species and isolates obtained

In the areas studied ten species of predacious fungi were found. Table 1 shows the species found, their frequency and their percentage of the total isolates. On the whole 107 fungal strains were obtained. As a total of 660 sprinkled plates (= 100%) were evaluated during the entire investiga- tion, 16.4% of the sprinkled plates yielded nema- tode-trapping fungi. This corresponds to one iso- late in about 8 g of fresh soil and hence shows a rather low density, while one or more propagules per fresh soil are reported from farmland soil [ll]. As seen in Table 2b, however, the soils investi-

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Table 1

Species, total number of isolates and frequency of isolations from the three sampling sites

Fungus Number Isolates

of in % of

isolates sprinkled

absolute plates

Arthrobotrys arthrobotryoides (Berl.) Lindau

Arthrobottys conoides Drechsler

Arthrobottys longispora Sopnmov Arthrobottys musiformis Drechsler

Arthrobottys oligospora Fresenius

Arthrobottys superba (Corda) Drechsler

Dactylaria eudermata Drechsler Dacrylaria thaumasia Drechsler

Dactyllela cionopaga Drechsler Unidentified zygomycete

Total

23 3.48

34 5.15 2 0.30

7 1.06 19 2.88

4

1 2

14

107

0.60

0.15

0.30 2.12

0.15

16.39

gated were very poor, and this may reduce the number of predacious fungi. The low density may be shown by preliminary experiments at one site (Grunewald), during which no predacious fungus was isolated at all.

Table 1 also shows Arthrobotrys conoides to be the most frequent species, followed by A. arthro- botryoides. The species Arthrobotvs oligospora often described to be the most frequent nema- tophagous species (e.g. [23-251) comes third in this study. In a study from Iowa [26]. A. conoides

was also isolated as the most frequent species. Other species may dominate as well [27-291. It is assumed that these differences are correlated to the respective soil parameters.

The temporal distribution is characterized by two peaks in December (22 isolations) and May (23 isolations). This may indicate that, like in endoparasitic nematode-destroying fungi [8], soil moisture has a dominant role. While the Arthro- botrys species, mentioned above as those most frequently isolated, were also found throughout the study, the other species isolated did not ex- hibit a distinct pattern.

4.2. Differences between the areas of investigation Between the three sites investigated marked

differences could be observed: as is shown in

Table 2a, the Boettcherberg yielded 52% (57 iso- lates) of all fungi found. Representatives of six different species thus form a nematophagous flora relatively rich in species. In the Grunewald 35 predators could be isolated, that is 33% of the total finds. Here the fungi could be attributed to five different taxa. Thus, the Grunewald, in com- parison, has a forest soil with the smallest number of species. On the Pfaueninsel only 15 fungal strains, that is 4.1% could be isolated. These few representatives, however, consist of seven different

species. A comparison of the individual sites in the three areas shows differences, which are, however, not as significant as those of the total areas.

Qualitatively, there are also distinctive dif- ferences: only three species occur at all sites (A.

arthrobotryoides, A. conoides and A. oligospora).

Arthrobottys longispora and Arthrobotrys musi-

formis are restricted to the beech wood; Dactylaria

thaumasia could only be isolated in the pine forest, while Dactylaria eudermata and the undetermined zygomycete are restricted to the Pfaueninsel. As far as conclusions can be drawn from the above, it is obvious that Dactylella cionopaga, D. thaumasia

and D. eudermata occur on poorer soils and that they are replaced by the Arthrobotrys species in the beech wood soil which is rich in nutrients. It is

striking that Duddingtonia jlagrans, usually iso- lated from garden soils, did not occur in these studies at all.

4.3. Correlation between soil types and occurring species

The results of the soil analyses are given in Table 2a. It is seen that the soil parameters differ widely at the investigated sites. Thus, the Grune- wald is characterized by low pH, high amount of humus, high nitrogen concentration and very low magnesium content. These characteristics can be attributed to the poor pine forest with its low pH and slow degradation of organic material. The dogs running free in this area may be responsible for the comparatively high nitrogen content.

The Boettcherberg is less poor, obviously the degradation of the organic material is more effi- cient, resulting in a low humus and nitrogen con- tent while, on the other hand, potassium and phosphate concentrations are comparatively high.

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Table 2a

Distribution of the isolated nematode capturing hyphomycetes between the three investigated sites

Fungus Number of isolates

Grunewald Boettcherberg Pfaueninsel Total

N % N 5% N % N

Arthrobotlys arthrobottyoides 4 11.5 14 24.6 Arthrobottys conoides 11 31.5 21 36.8 Arthrobotrys longispora 0 0 2 3.5 Arthrobotqv musiformis 0 0 7 12.3 Arthrobottys oligospora 5 14.3 12 21.0 Arthrobotrys superba 0 0 1 1.8 Dactylaria eudermata 0 0 0 0 Dactylaria thaumasia 2 5.7 0 0 Dactylaria cionopaga 13 37.1 0 0 Unknown species 0 0 0 0

Total 35 100 51 100

5 35.7 23 2 14.3 34

0 0 2 0 0 7

2 14.3 19

3 21.4 4 1 7.1 1

0 0 2 1 7.1 14

1 7.1 1

15 100 107

Table 2b

Chemical soil factors of the investigated sites. Each value gives the mean of three independent measurements.

Site

Grunewald Boettcherberg Pfaueninsel

Reaction (pH) 3.1 4.1 3.9 Humus (loss by ignition W)) 6.4 3.3 5.3 Nitrogen (mg N/l) 36.9 27.3 32.0 Magnesium (mg Mg2+/l) 0 10.7 35.0 Potassium (mg K,O/l) 30.7 39.0 25.0 Phosphorus (mg P202 /l) 71.1 213.7 86.0 Water soluble minerals (mg KCl/l) 371.0 539.0 232.0 Volume weight (g/l) 1019 1104 1056

The Pfaueninsel which is sporadically in- undated is characterized by high amounts of Mg2+ and soluble minerals. This is attributed to the material swept in by the inundations.

If these data are compared with the list of fungi given in Table 2b, the following correlations may be seen: The Pfaueninsel contains less nematode-trapping fungi than the other sites, with Arthrobotrys su- perba showing higher numbers and higher per- centage than in other sites. Dactylaria eudermata is found only here. The Boettcherberg is characterized by an overall high amount of nematode-trapping fungi; A. musiformis was found only here, while A. oligospora and A. conoides form a higher per- centage than at the other sites.

The Grunewald soils are characterized by Dacty- lella cionopaga, and less by Dactylaria thaumasia, which therefore may be more nitro- or xerophil.

5. DISCUSSION

These experiments demonstrate that estima- tions of ecological groups of fungi like the preda- cious hyphomycetes are possible and yield rea- sonable results. The number of species found is in the same order of magnitude as the endoparasitic nematode-destroying fungi [8,9]. In addition, the isolated fungi show a correlation to soil parame- ters, as it is seen especially from the values from the Boettcherberg.

There are several uncertain points, which should

not be neglected: The isolation method does not allow the conclusion that an isolated fungus is actively growing inside the soil or that it is only present as resting mycelium, conidium or

chlamydospore. In addition, the sprinkled plates favour those fungi which germinate or grow rapidly and reveal their predatory activity during the time of observation. As Cooke [16] pointed out, the activities in soil and in culture, i.e., on the water agar of the sprinkled plates, may differ widely and hence the fungi isolated may not be the only ones in the respective soil sample. To overcome these difficulties, other methods of isolation, which also allow a study on a large scale, must be developed.

A further uncertainty is due to the sampling method, since the sampling neglects the inhomo- geneities inside the soils. To overcome these, a project has now started to isolate the fungi from different microsites separately in order to see which differences exist between them.

In spite of these objections, it has been shown that this type of investigation may provide data to obtain ideas about the number of species, their abundance and the distribution of the nematode- trapping hyphomycetes in the soil.

ACKNOWLEDGEMENTS

The authors are indebted to the staff of the Institute, for technical and writing assistance, and to Dr. Birgit Nordbring-Hertz, Lund, for helpful discussions and critically reading the manuscript.

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