Ecology of nematophagous fungi: Effect of soil moisture, organic matter, pH and nematode density on distribution

Soil Eiol. Biochem. Vol. 17, No. 4, pp. 499-507, Printed in Great Britain. All rights reserved

1985 Copyright

0038-0717/85 $3.00 + 0.00 ,Q 1985 Pergamon Press Ltd

ECOLOGY OF NEMATOPHAGOUS FUNGI: EFFECT OF SOIL MOISTURE, ORGANIC MATTER, pH AND NEMATODE DENSITY ON DISTRIBUTION N. F. GRAY Environmental

Sciences

Unit, Trinity (Accepted

College,

University

of Dublin,

Dublin

2, Eire

10 J~lanuffr~ 1985)

Summary-The effect of organic

matter, soil moisture, pH and the indigenous soil nematode density on the distribution of nematophagous fungi has been examined. Using 206 samples of soil and organic material, the presence and absence of groups and individual species of nematophagous fungi have been compared statistically to indicate which of these major factors are important in their dist~bution. The presence of predatory fungi was more influenced by pH and moisture than the other soil factors, while the presence of the conidia-forming endoparasites was influenced by organic matter. The presence of obligate parasites was associated with high soil nematode densities while facultative predators were independent of nematode density. Endoparasites with conidia that do not attract nematodes were found in soils with a higher nematode density than those species producing nematode-attracting conidia. This would increase the chance of htfection by the random ingestion of the conidia by a suitable host. Non-spontaneous trap forming predators were isolated from soils with low organic matter and low moisture. These species are able to compete well saprophytically under such conditions, and when nutrients or moisture temporarily improve, they are able to maintain their competitive advantage by utilizing the expanding nematode population. Spontaneous trap-forming predators were found in soils with relatively high organic matter and moisture. In such soils there is a rich microbial flora and fauna, and the ability to form traps spontaneousiy offers these predators a direct competitive advantage over saprophytes and non-spontaneous trap-forming predatory fungi. Attempts to model the data were generally unsuccessful due to the qualitative-quantitative nature of the data, the small data base and the resulting high number of distinct co-variate patterns formed by the independent variables. The methods of isolation used are compared with earlier investigations where isolations were made from extracts taken from large weights of sample material.

that a fundamental knowledge of the ecology of these fungi is essential before the value of such biological control methods can be assessed. The lack of success in using nematophagous fungi as biological control agents has been due, on the whole, to the choice of the control fungus (Gray, 1983a). The species used in such studies has rarely been selected on the basis of its suitability for the habitat where the potential target nematode is found, but rather on its ease of isolation and subsequent culture in the laboratory or its relative effectiveness in destroying nematodes as observed in the laboratory. A better understanding of the ecology of the group is a prerequisite to their successful exploitation in the biological control of parasitic nematodes. My aim was to ascertain whether nematophagous fungi are ubiquitous or whether distribution is restricted by biotic and abiotic factors in the soil. A survey of 206 samples of soil and plant material collected from sites throughout Ireland showed the group to exist in a wide variety of habitats (Gray 1983a, b). It was clear from the distribution and habitat data that many species were restricted to particular groups of habitats (Gray, 1983a). Using the same data base as before, I have attempted to identify the significant relationships between soil moisture, organic matter, pH and nematode density and individual species or groups of species with similar attack mechanisms (Table 1). The effect of maintains

INTRODUCTION numerous surveys of nematophagous fungi have been undertaken, none have examined the major environmental factors associated with the distribution of the major types of fungi or individual species. Laboratory studies on the group have been almost entirely non-ecological in nature (Barron, 1977) except for the pioneering work on the ecology of nematophagous hyphomycetes carried out by Cooke (1962, 1963a, b). A. M. Shepherd (unpublished Ph.D thesis, University of London, 195.5) found no relationship between the species of nematophagous fungi and pH, soil moisture, organic matter or nematode density of the soil in an earlier survey of Danish soils. However Nordbring-Hertz (1973) demonstrated that both nematode-induced and peptide-induced morphogenesis were markedly influenced by environmental factors such as nutrient level, pH, temperature, Iight and moisture. Attempts to use nematophagous fungi to control plantparasitic (Tribe, 1980) and animal-parasitic nematodes (Pandy, 1973) have not been encouraging, except for the recent work of Cayrol and Frankowski (1979) in France. They have successfully used strains of Arthrohotrys on a commercial basis to protect mushrooms from the mycophagous nematode Ditylenchus myceliophagous and tomatoes from infestations of Meloidogyne nematodes. Cooke (1962)

Although

499

500

N. F. GRAY Table

I. Species and groups of species used in data analysis. Groupings were made accordmg the mode of infection of endoparasites or the trapping mechanism of predators Endoparasites

Mode

M.vL-ocvtium

Encystment

Acrosralagmus goniodes Drechsler A. obouurus Drechsler Cephalosporium bulmoides Drechsler Merio coniospora Drechsler ~e~?at~~?u~it~~ leiospnrus Drechsler Spicuria cowospora Drechsler Verftcilliutn sphaerosporum Goodey

Adhesive

conidia

Conidia

ingested

Harposporium anguillulae Zopf H. helicoides Drechsier H. I~ili~~ra~~ Dixon

i

Trapping

Adhesive

Qstopage eladospora Drechsler C. loteralis Drechsler S@lqoge bodru Drechsler S. kioh.vpha Drechsler

I

Ductyklla cionopaga Drechsler D. lohara Duddington

>

Dactylaria haprotylo Drechslcr DartyMIa el~ip.~osporo Grove D. mam~~il/ata Dixon

1

Unmodified

Arrhrohotrys anchonia Drechsler Dnrtylariu gracilis Duddington Dncrylrlla ncrochwra Drechsler D. bembicodes Drechsler D. doe~veo~des Drechsler

these soil factors on the distribution of nematophagous fungi are discussed in relation to the earlier laboratory-based ecological studies of Cooke.

collection

AND METHODS

and isolation

zoospores

i

Predators

Sample fungi

of infectmn

of motile

I

Arlbrobotrys musiformis Drechsler A. oligospora Fresenius A. robusfa Duddington Dactylaria seap~oides Peach Dacrylella megnlospora Drechsler Trichothecium cysrosporium Duddington

MATERiALS

to

of nematophagous

Samples were collected for 2 yr, during the Spring of 1981 and the Summer of 1982. A total of 206 samples were collected from a wide variety of habitats from a number of sites throughout Ireland (Gray, 1983b). A 75 mm dia bulb planter was used to remove soil cores, 125-150mm in length, along with its associated plant or surface material. The cores were immediately placed in polythene bags and double-sealed to prevent evaporation and deterioration. Work by Gray and Bailey (1985) on the vertical distribution of nematophagous fungi has shown that the greatest diversity of species is isolated from the rich organic humus layers associated with the rhizosphere area of plants, with a reduction in species diversity and frequency of occurrence with depth, even though suitable prey may be available. Therefore subsamples were taken randomly from this distinct layer whenever possible for isolation of fungi. Where no distinct zones existed the material was gently mixed with a pestle a:ld mortar and subsamples taken randomly from the homogenous sample.

nets

adhesive

Adhesive

branches

Adhesive

knobs

Constricting

mechanism

hyphae

rings

Predatory nematophagous fungi were isolated using a modification of the soil sprinkling technique (Gray, 1984b) devised by Duddington (1955), and endoparasites by the Baermann funnel technique (Gray, 1984a). The methods used to collect, isolate and identify nematophagous fungi in this study are fully described by Gray (1984b). Measurement

of soil factors

Where distinct layers existed within cores, the subsamples removed for the measurement of the major soil factors were taken from the same layer from which subsamples for the isolation of the fungi were taken. If the soil sample was mixed before subsamples for isolation of fungi were removed, then the subsamples for the soil analysis were also taken randomly. Soil moisture was measured by drying I O-20 g of fresh soil in an ajr-circulated oven at IOYC until a constant weight. Soil moisture is expressed as a percentage of the fresh sample weight. The percentage of organic matter in soil samples was calculated by loss of ignition at 45O’C for 4 h (Allen, 1974), using triplicate samples (I g) of oven dried material. The organic content was expressed as the percentage loss in weight on ignition. The pH was measured using an Orion Research Microprocessor Ionaiyzer (Model 901) and pH electrode (Allen, 1974). The indigenous nematode density of the soil was estimated by separating the nematodes from fresh soil

Soil factors and nematophagous by the Baermann funnel method (Gray, 1984a). The nematodes were extracted by wrapping 30 g of fresh soil in a double layer of soft tissue. Placed in a standard Baermann funnel apparatus, the water level was adjusted until it came into contact with the soil. The nematodes wriggled through the tissue and sank down the funnel into a small collecting tube. After 24 h the collecting tube was removed and the nematode suspension concentrated up to 1 ml. Aliquots (10 ~1) of the nematode suspension were placed on slides and gently heated to immobilize the nematodes which were then counted at x 10 magnification. The nematode density was expressed as the number of nematodes gg’ of fresh soil. Statistical analysis Kent (1972) has demonstrated that it is likely that data of the type collected in the present study is not normally distributed. Therefore statistical tests were chosen which do not depend on the assumption of normality. The Mann-Whitney rank sum test is a non-parametric version of the two-sample t-test for independent samples, and is one of the most powerful of the non-parametric tests with a power efficiency approaching 95.5% as n increases (Siegel, 1956). The level of significance of the Mann-Whitney U statistic corresponds to a two-tail probability. Similar tests have been used successfully by Kent (1972), Dickinson and Kent (1972) and Dowding and Widden (1974) on soil fungal populations. Where there are several variables affecting the results being analysed, it is usual to partition their effect by using some form of multivariate analysis. The categorical nature of the dependent variables and the small sets of data with a large number of variables affecting them, failed to fulfill the assumptions necessary for the common multivariate techniques. However, stepwise logistic regression is specifically designed to model the probability of success of a categorical variable using both categorical and continous data. Stepwise logistic regression selects predictor (independent) variables in a stepwise manner and estimates the coefficients for a logistic regression. The dependent (absence or presence) variable is a binary variable coded as 0 or 1. The predicted portion of success (i.e. presence of species or groups of species) (s/n) follows the logistic model exp(u)/(l + exp(u)) where s is the sum of the binary (0, 1) dependent variable, n is the total sample size and u is a linear function of one or more of the independent variables. The hypothesis that the variable entered (or removed) at that step significantly improves prediction is tested using the chi-squared test. These variables entering the model do so in order of increasing P-value, the lowest P-value (i.e. the largest approximate F to enter) at step 0. No term is removed or entered from the model unless it has a p-value of > 0.15 or < 0.10 respectively, i.e it will not improve the prediction (Engelman, 1981). RESULTS

Significant differences (P < 0.05) between samples containing endoparasites and those without endoparasites were recorded for all four soil factors (Table 2). Endoparasites were present in samples with higher

fungi

501

moisture (P < 0.005) and organic matter (P < 0.001) contents, but with lower pH (P < 0.001). There was also a significant difference in nematode density, with endoparasites present in samples with significantly high densities of nematodes (P < 0.05). Predators as a single group were only significantly influenced by pH (P < 0.005), with the mean pH in samples containing predators being 5.5 compared to 6.2 for the samples without predatory nematophagous fungi. Endoparasites can be classified into three main groups according to their method of parasitizing host nematodes i.e. encystment, adhesive conidia and ingested conidia (Barron, 1977). All three groups were significantly associated with high nematode numbers (P < 0.05) while the adhesive and ingested conidia groups were both recorded in samples with higher organic matter contents (P < 0.05). Although the two groups which produce conidia were associated with soils with a relatively low pH, this was only significant for the group of species forming adhesive conidia (P < 0.05). Samples which contained endoparasites which reproduce by encystment had a lower moisture content than the rest of the samples, but this was not significantly different (P > 0.05). Like the endoparasites, the predatory nematophagous fungi, as a single group, were isolated from soils with a low pH. However by comparing the presence and absence of predators grouped by their attack mechanism (Table I), many significant associations became apparent (Table 2). Predators with adhesive hyphae, branches or knobs were all independent of the moisture and the organic matter content of soils, while predators with adhesive nets or constricting rings were significantly associated with these particular soil factors (P i 0.001). The net formers were associated with soils of much lower water and organic matter contents than the group of species forming constricting rings. The presence of net or adhesive knobs (P < 0.001) and constricting rings (P < 0.001) were isolated in soils with a low pH, although the adhesive hyphae (P < 0.05) were associated with a much higher pH range. Table 3 compares the presence and absence of the major component species for each soil factor examined. All species isolated from more than 5 soil samples are included, although caution is obviously necessary when comparing such small numbers of cases to a much larger data base. There were four commonly recorded endoparasitic species. All the encysting species were classified as Myzocytium spp. Harposporium anguillulae, which produces ingested conidia, was found to be significantly associated with soils with high nematode populations, relatively high water and organic matter contents (P < 0.05). M. coniospora was associated with similar soil conditions as H. anguillulae, that is a high nematode density (P < 0.05) and organic matter content (P < 0.05). Two net-forming predators, Arthrobotrys musiformis and A. robusta, were frequently isolated from Irish soils although only the former was significantly associated with any of the soil factors studied. A. musiformis was found in soils with low moisture (P < 0.001) and organic matter contents (P < 0.001) along with Dactylella cionopaga which captures nematodes on adhesive branches. The latter was significantly associated with nematode density

spore

Total fungi

Knobs

Branch

Hyphae

Ring

Net

Ingested spore

Adhesive

Encystment

Predator

Endoparasite

Prrcsent Absent

Present Absent

Present Absent

Present Absent

Present Absent

Pre%Xlt Absent

Present Absent

Present Absent

Present Absent

Absent

47.3

49 5 42.2

48.5 45.5

40.4 45.x

59.6 46.3

32.5 43.6

55.2 50.6

51.4 45.3

38.3 44.7

45.1 47.2

51.8 41.5

41.5

x

I 9x

4.65 2.83

5.74 1.77

6.23 I .73

3.45 1.71

1.90 1.78

6.11 1.96

4.35 1.69

4.39 1.74

2.14 1.77

0.3

13

0.461

0.609

0 734

0.000

0.000

0.177

0.160

0.079

0.491

0.003

128

26 41

16 143

7 153

26 162

42 143

14 I27

35 155

22 I34

99 147

75 70

94

2.03 2.60 2.62

n

Moisture (“/,) SEM P

33.0

36.9 ‘1.8

31.9 29.1

17.5 30.1

44.5 30.X

18.1 27.1

46.2 34.3

39.4 28.8

27.4 21.9

30.8 30.7

38.3 29.6

23.9

x

2.36

555 2.54

7.79 2.03

3.22 i .97

4.58 I .99

2.69 2.05

6.99 2.30

1.97

4.91

5.11 2.02

2.58 2.08

3.18 2.90

2.15

0.096

0.121

0.580

0.370

0.000

0.000

0.019

0.036

0.608

0.699

0.000

Organic matter (%) SEM P

n

128

26 41

16 143

I I53

26 162

42 143

14 127

35 155

22 134

99 147

75 70

94

X

5.6

5.1 6.5

59 6.0

6.X 5.8

5.0 5.x

6.0 6.0

5.5 5.8

5.3 5.9

6.1 5.9

5.5 5.8

5.4 6.2

6.1

0.12

135

47

150

17

8 165

29 174

42 153

16 I40

35 166

22 147

106 160

80 76

0.17

I

n IO2

32 0.00

0.000

0.718

0 047

0.001

0.228

0.279

0.040

0.378

0.003

0.001

P

027

0.22 0.1 I

0 23 0.11

0.24 O,li

0.16 0.11

0.40 0.12

0.25 0.11

0.26 0.11

0.13 0.11

0.15 0. I5

0.13

SEM

PM

z

61 2

53.3 84.5

85.8 69.4

13.5 64.6

so.7 69.0

52.3 69.5

135.4 70.9

94.6 60.4

124.8 59.6

51.9 58.5

78.2 86.9

57.4

8.62

14.03 46.43

15.70

21.35

4.06 14.25

13.70 13.73

12.88 15.36

39.82 16.68

22.70 13.81

33.46 15.40

7.70 14.16

13.41 29.42

21.06

Nematode

0. I x7

0.899

o.022

0.108

0.236

0.143

0.034

o.“‘4

0.020

0.769

0.029

P

density

135

32 4s

1: 149

164

28 173

42 153

15 139

36 166

22 145

10s 159

80 76

101

n

Table 2. Comparison of soil moisture, organic matter, pH and nematode density at sites where the main groups of nematophagous fungi were either present or absent. The mean (X), standard error af the mean (SEM), the level of significance of the Mann-Whitney statistic U using a normal two-tail approximation (P), and the number of samples (n) are reported. Variability of n is due to missing values or unidentified species not included in specific categories

spp

D. cionopaga

A. robusra

D. mommillata

D. bembicoides

M. coniospora

A. obovatus

Myzocytium

46.2

Present

21.1 46.6

Present Absent

33.9 46.1

45.8 48.5

Present Absent

Present Absent

58.2 46.1

45.4

60.7

45. t

63. I

61.3 45.1

45.5

44.7

46.2

38.3

41.2

Present Absent

Absent

Present Absent

Preset3 t Absent

Present Absent

Present Absent

Absent

P

5.96

6.15 I .73

0.41 I .69

9.55 1.77

6.61 I.68

1.70

4.73

7.3 I 1.71

9.84 I .67

6.78 1.67

4.39 1.71

1.77

0.958

0.137

0.000

0.814

0.073

0.006

0.049

0.083

0.609

0.079

Moisture (%) SEM P

14

I 155

I9 162

8 150

9 161

160

11

9 158

6 160

14 163

22 155

147

n

30.2

22.5 30.3

10.2 30.6

44.2 32.8

40.9 29.6

29.1

46.3

49.2 29.2

46.3 29.2

30.3 29.7

21.4 30.3

30.7

z?

8.97

6.39 I .97

0.42 I .99

12.46 2.08

6.99 1.92

1.99

7.36

8.78 1.97

8.14 I .95

6.64 1.96

5.11 2.02

2.08

0.477

0.746

0.000

0.176

0.050

0.007

0.016

0.040

0.914

0.608

Organic matter SX) SEM P

14

I 15.5

19 162

8 150

9 161

160

II

9 158

6 160

14 163

22 15s

147

n

5.9

6.3 5.8

6.2 5.8

4.4 5.8

4.5 5.9

5.9

4.6

5.9 5.9

6.6 5.8

5.1 5.8

6.1 5.9

5.8

X

0.24

0.22 0.11

0.14 0.11

0.42 0.11

0.29 0.10

0.10

0.34

0.53 0.10

0.77 0.10

0.32 0.10

0.26 0.11

0.11

SEM

0.001

0.002

0.638

0.082

0.068

0.378

P

0.609

0.376

0.234

0.001

PH

15

lb7

7

19 175

10 163

II 172

171

13

10 169

172

6

14 176

22 168

160

n

j?

93.7

64.1

38.3

59.7 67.1

25.4 61.4

30.52

13.83 14.08

17.55 13.67

14.37 14.56

9.33 13.88

13.98

68.1 34.5 69.0

21.99

44.28 14.00

88.11 13.63

27.82 13.17

33.46 14.07

14.16

46.1

122.1 68.0

205.2 63.7

89.3 61.8

124.8 64.7

58.5

Nematode SE(&)

0.020

0.824

0.020

0.147

1.000

0.168

0.046

0.023

0.131

0.020

P

density

15

7 166

19 174

10 162

I1 171

170

12

9 169

6 172

14 175

22 167

159

n

Table 3. Comparison of soil mois_ture, organic matter, pH and nematode density at sites where the major component species of nematophagous fungi were either present or absent. The mean (X), standard error of the mean @EM), the level of significance of the Mann-Whitney statistic U using a normal two-tail approximation (P), and the number of samples (n) are reported. Variability of n is due to missing values or unidentified species not included in specific categories

504

N. F. GRAY

(P < 0.05).

D. mammillata and D. ellipsospora, knobforming predatory fungi, were both isolated from soils with significantly lower pH values (P < O.OOl), although only D. mammillata was also associated with high organic matter contents (P cl 0.05). The constricting-ring forming species, D. bembicodes, was also isolated from soils with significantly higher moisture (P < 0.01) and organic matter contents (P < O.Ol), but from soils with a lower pH (P < 0.00s) than the rest of the soil samples. Soil moisture and organic matter were the only significantly correlated soil factors (P < 0.001). By comparing the correlations of soil factors of those sarnples containing specific groups or species of nematophagous fungi with the remaining soil samples, it was hoped that specific soil associations could be identified for particular categories of fungi. Although none of the predatory fungi displayed significantly different correlations of soil factors, the three endoparasitic groups did, with soil moisture and organic matter significantly correlated. In soils from which encysting endoparasites were isolated, nematode density was positively correlated with organic matter (P < 0.001) as was soil moisture and pH (P < 0.02). The pH of soils from which endoparasites with adhesive conidia were isolated, were positively correlated with the nematode density (P < 0.005). The results of the stepwise logistic regression analysis is given in Table 4 for groups of nematophagous fungi and in Table 5 for the commonest species isolated. The results confirm the relative importance of individual soil factors to particular fungal groups identified by the non-parametric analysis (Tables 2 and 3). The summary of the analysis in Table 4 indicates that the presence of predatory groups is more determined by pH and moisture, and the conidia-forming endoparasites by organic matter, than by the other factors tested. Apart from the encysting endoparasites, the indigenous nematode density of the soil appears to be the least important soil factor in determining the presence of nematophagous fungi.

DISCUSSION

The endoparasitic nematophagous fungi are obligate parasites, and unlike the predatory fungi they are unable to live saprophytically in the soil. This is confirmed by all the endoparasitic groups being isolated from soil with significantly higher densities of nematodes (x = 95-135 gg’) compared with predatory groups. Endoparasites forming adhesive conidia have been shown to chemically attract host nematodes (Jansson and Nordbring-Hertz, 1979) while ingested conidia are non-attractive. The latter rely on nematodes being attracted to the originally infected nematode by bacteria feeding on the corpse and also by the limited mycelium of the parasite which has been shown to attract nematodes (Jansson, 1982). Nematodes, attracted to the site, randomly ingest the conidia as they feed on the abundant bacteria. The mean density of nematodes in soil containing species forming non-attractant ingested-type conidia was much greater compared with the soil samples from which attracting adhesive-type conidia producing species were isolated (Table 2). This suggests that

Table 4. The importance of particular SOLI factors (independent variables) as selected by stepwise logistic regression on the presence of specific groups of “ematophagous fungi (dependent variable) Terms entered Into model at each step (P i 0.01) De”ende”t variable steo Indeoendent variable I 2

Endoparasitc

I

Predator Encystment Adhesive conidia Ingested conidia Net Ring

I 2 I

matter

PH PH Moisture Nematode densay organic matter

I

Oreanicmatter

I

M&w Moisture

I 2

I 0 I

Hyphae Branch Knobs Total

Organic

PH PH “H

I

PH

2

Organic

3

Moisture

matte!

Improvement in predictlo” (P-value) 0.090 0.02 I 0.004 0 (156 0.095 0.0 I6 0.014 0 000 0.000 0.002 0 037 0.000 0.001 0.05 I 0.07Y

such a non-specific method of attraction may rely on a greater density of soil nematodes to ensure infection compared with parasites which produce adhesive conidia. Microbial activity, and nematode density in particular, has often been associated with high levels of organic matter in soils. However in the present study no correlation was recorded between organic content and nematode density in the soil samples analysed, although there are obvious problems in correlating numbers of nematodes gg’ fresh weight of soil to organic content per oven-dried weight of soil. Those endoparasites producing conidia, both ingested and adhesive types, were however significantly associated with soils with high organic matter. Generally the conidia-forming endoparasites were isolated from samples with comparatively high soil moisture contents and low pH. A major limitation of this study is the use of moisture content of soil rather than water potential, especially as the type of soils used ranged from sandy loams to clay soils. While water content may be important in terms of mobility of zoospores, it is of much less value when assessing the effect of soil water on the distribution of soil fungi (Griffin, 1972).

Table 5. The importance of parttcular sol1 factors (Independent variables) as selected by stepwise logistic regression on the presence of selected species of nematophagous fungi (dependent variable) Terms entered into model at each step (P -C 0.01) Independent variable Dependent variable step Mvzocvtium

spp

1 2

A. ohonms

M. contosporu H. fquillulrre A. mu.rifivmis A. rohustu D. hemhrroirles

I

I I I 2

D.

eilipsospora

D. mcrmm,llrrla

0 056

Moisture Nematode density PH Predators Organic matter Moisture Organic matter

0.095 0.08 I 0.028 0.018 0.000 0 000

PH Moisture

0.001 0 044

PH PH

0.000 0 000

0

I 2

U cionopagu

in prediction (P-value)

0

1 I

Soil factors and nematophagous However the constraints on the study resulted in soil moisture being the only available measure of soil water. Although the predatory nematophagous fungi are all facultative predators, Cooke (1963a) demonstrated that they had different growth rates in soils and responded differently to the presence of nematodes. He also classified predators by their ability to spontaneously produce traps. Cooke concluded that the development of predaceous efficiency had been accompanied by a tendency to lose those characters associated with an efficient saprophytic existence in the soil; namely rapid growth rate and good competitive saprophytic ability. This is supported by the results of the present study (Table 2). Those species producing adhesive networks are isolated significantly more frequently (P < 0.001) from soils with low moisture and organic matter contents. In contrast ring-forming species are isolated significantly more frequently (P < 0.001) from soil with relatively high moisture and organic matter contents, although none of the groups of species, with the exception of species forming adhesive branches, were associated with nematode density. The latter conditions favour a more active microbial community with a greater biomass of organisms including nematodes. Under conditions of active microbial activity the property of spontaneous trap formation may confer an ecological advantage on the organism, especially if the fungus is sensitive to competition from other microorganisms. The poorer conditions of the soil which favour the net-forming species are the conditions where non-spontaneous trap formers would flourish. Their good saprophytic ability allows them to compete favourably with other species for the limited nutrients, however when conditions temporarily improve they are able to maintain a competitive advantage by utilizing the subsequent increase in the nematode population (Cooke, 1963b). Little is known and few records exist of the distribution of nematophagous fungi with unmodified adhesive hyphae. Although they are capable of very high levels of predaceous activity (Gray, 1984~) they are often observed without having caught any nematodes even though potential prey may be abundant. It may be more appropriate to include this group into the non-spontaneous trap forming category. The results, although not significant, support this view with species forming unmodified adhesive hyphae being isolated from similar soil conditions as netforming species. The present data shows that although the ring- and knob-forming species are associated with soils with a low pH (P < O.OOl), those species with unmodified adhesive hyphae were isolated significantly more frequently from soils with a higher pH (P < 0.005). Whether individual species have adapted to specific pH ranges thus reducing interspecific competition remains unclear, although the presence of many of the predatory groups of fungi is determined more by pH than any of the other factors tested (Table 4). However more soil data are required for individual species before and variability within groups in relation to specific soil factors can be established. The logistic regressions at step 1 are essentially performing the same function as the Mann-Whitney

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tests, that is identifying the variables most associated with the presence or absence of particular species or groups of species. It is not surprising that the results of the stepwise logistic regression analysis (Tables 4 and 5) confirm the more general trends identified by the non-parametric statistics. The data base is rather small for multivariate analysis as the relatively large number of continuous variables resulted in a large number of distinct co-variate patterns within the model, which were occasionally equal to the total number of cases in the original data. By transforming each continuous variable set into several categorical groups the number of distinct co-variate patterns was only slightly reduced and was never less than 70% of the total number of cases. Although Tables 4 and 5 do indicate those variables which have a significant effect on the probability of a species or groups of species being present in a particular soil, without a much greater data base a reliable predictive model cannot be constructed. The major problem of field-based studies in microbial ecology is to ensure that the sampling method employed provides representative and reproducible results. In this study extreme care was taken to ensure that the maximum number of species of nematophagous fungi were isolated from each site by sampling specific areas of soil cores which have been shown to contain the greatest diversity of species (Gray and Bailey, 1985; Mankau and McKenry, 1976; Peterson and Katznelson, 1965). Samples of soil, not extracts, were used for isolations to ensure that those species which had not produced conidia or infected nematodes would also be recovered. This was supported by frequent and comprehensive microscopic examination of plates (Gray, 1984b). This approach is different to that recommended by Mankau (1975) and McCulloch (1977), who suggest that the diversity of fungal species isolated will be increased by using larger samples of soil, and that variability between replicates will be reduced. Both of these methods use a water separation technique similar to that of Barron (1969). However Barron (1978) demonstrated the limitation of his technique in a comparative study of isolation methods, and showed that the Baermann funnel technique was equally, if not more, efficient for the isolation of endoparasites. A major problem of such separation techniques is that while some infected nematodes and conidia are recovered, the living mycelia of fungi remains in the soil to a great extent. Such techniques are most effective for fine mineral soils, but neither the method of Mankau or MZulloch was found to be suitable for the organically rich soils used in the present study, which often contained fine root material and extensive fungal mycelia. Mankau (1975) used a 5-25 g soil sample which was suspended in water, washed through a series of small sieves and the residue collected on a filter paper disc which was plated onto diluted corn meal agar (CMA). While this isolation method has advantages over others using CMA media, especially as it allows some quantitative assessment to be made, the method encourages unwanted contamination from bacteria and myxomycetes which is suppressed by using minimal medium. Further contamination of the CMA occurs whenever the plates are examined under high

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magnifications, which requires the cover to be removed from the plate allowing airborne contaminants to colonize. This method also relies on the natural development of indigenous prey organisms which results in a longer development time for most species, and requires more frequent and longer microscopic examinations to ensure identification of all the species present. It is clear from previous studies (Gray, 1984b) that many nematophagous species will not form traps or conidia under such high nutrient conditions, but will grow saprophytically even in the presence of abundant prey. McCulloch (1977) also used a separation technique to remove nematodes and other microfauna from soil samples, which were subsequently plated onto minimal medium. No details on the efficiency of this technique in removing conidia, chlamydospores or living mycelial fragments, as well as infected nematodes, is given. She concluded that the low species diversity encountered during other surveys was primarily due to the small subsamples used for isolating infrequent species, the main reason for the low frequency of isolation in previous surveys has been shown to be the frequency of examination of the plates (Barron, 1978; Gray, 1984b). McCulloch isolated a total of 57 species from 1722 samples, which she compares to the species diversity obtained in relation to the number of samples collected in similar surveys. However, 31 of these species were isolated from less than 1o/0of the samples taken and included a number of species which have been shown in other surveys to be extremely abundant elsewhere (Fowler, 1970; Gray, 1984a; Gray and Lewis-Smith, 1984; Shepherd, 1955). Whether these species would have been isolated using a smaller bulk of soil is not known. However it appears that the high incidence of certain species isolated by McCulloch may be influenced by the size of the conidia and the frequency conidia are produced in the soil, rather than their true distribution. Clearly it is not possible to compare the effectiveness of this method with others used in similar surveys (Duddington, 1951; Fowler, 1970; Gray, 1983a; Gray et al., 1982) on the basis of species diversity per unit number of samples. What is urgently required is a comparative study of all the major isolation techniques to establish the most effective method and to determine whether sample size is significant. However in the context of the present study, neither of the methods discussed appeared as appropriate as the techniques used. REFERENCES Allen S. E. (1974) Chemicul Analysis of Erological Materials. Blackwell, Oxford. Barron G. L. (1969) Isolation and maintenance of endoparasitic nematophagous Hyphomycetes. Canadian Journal of Botany 47, 1899-1902. Barron G. L. (1977) The Nematode-Destroying Fungi (Topics in Mycobiology: I). Canadian Biological Publications, Guelph, Ontario. Barron G. L. (1978) Nematophagous fungi: endoparasites of Rhadbitis terricola. Microbial Ecology 4, 157-163. Cayrol J.-C. and Frankowski J.-P. (1979) Une methode de lutte biologique contre les nematodes a galles des racines appartenant au genre Meloidogyne. Revue Horticole 184, 23-30. Cooke R. C. (1962) The ecology of nematode-trapping fungi in soil. Annals of Applied Biology 50, 507-513.

Cooke R. C. (1963a) Ecological characteristics of nematodetrapping Hyphomycetes. I. Preliminary studies. Annals of Applied Biology 52, 43 1-437. Cooke R. C. (1963b) Succession of nematophagous fungi during the decomposition of organic matter in soil. Nature 197, 205. Cooke R. C. (1964) Ecological characteristics of nematodetrapping Hyphomycetes. II. Germination of conidia in soil. Annals of Applied Bio1og.v 54, 375-379. Dickinson C. H. and Kent J. W. (1972) Critical analysis of fungi in two sand dune soils. Transactions of the Briti.rh MyFological Society 58, 269-280. Dowdine P. and Widden P. (1974). Some relationshius between fungi and their environment in tundra regions. In Soil Organisms and Decomposition in Tundra (A. J. Holding et al.. Eds), pp. 123-150. Tundra Biome Steering Committee (Stockholm). Duddington C. L. (1951) The ecology of predaceous fungi. I: Preliminary survey. Transactions of the British Mycological Society 34, 322-33 1. Duddington C. L. (1955)Notes on the technique of handling predaceous fungi. Transactions of the British Mycological Society 38, 97-103. Engelman L. (1981) Stepwise logistic regression. In BMDP Statistical Software (1981) (W. J. Dixon, Ed.), pp. 330-344. University of California Press, Los Angeles. Fowler M. (1970) New Zealand predaceous fungi. Nen Zealand Journal of Botany 8, 2833302. Gray N. F. (1983a) Ecology of nematophagous fungi: distribution and habitat. Annals of Applied Biology 102, 501-509. Gray N. F. (1983b) The distribution of nematophagous fungi in Ireland. Bulletin of the Irish Biogeographical Society 7, 19-37. Gray N. F. (1984a) Ecology of nematophagous fungi: comparison of the soil sprinkling method with the Baermann funnel technique in the isolation of endoparasites. Soil Biology & Biochemistry 16, 81-83. Gray N. F. (1984b) Ecology of nematophagous fungi: collection, isolation and maintenance of predatory and endoparasitic fungi. Mycopathologia 86, 143-153. Grav N. F. (1984c) Ecologv of nematophaeous fungi: predatory and endbparasityc species new to Ireland. Irish Naturalists’ Journal 21, 337-341. Gray N. F. and Bailey F. (1985) Ecology of nematophagous fungi: vertical distribution in a deciduous woodland. Plant and Soil In press. Gray N. F. and Lewis-Smith R. I. (1984) The distribution of nematophagous fungi in the maritime Antarctic. Mycopathologia 85, 81-92. Gray N. F., Wyborn C. H. E. and Smith R. I. L. (1982) An ecological study of nematophagous fungi from the maritime Antartic. Oikos 38, 194201. Griffin D. M. (1972) Ecology of Soil Fungi. Chapman & Hall. London. Jansson H.-B. (1982) Attraction of nematodes to endoparasitic nematophagous fungi. Transactions of the British M.ycological Society 79, 25-29. Jansson H.-B. and Nordbring-Hertz B. (1979) Attraction of nematodes to living mycelium of nematophagous fungi. Journal of General Microbiology 112, 89-93. Kent J. W. (1972) Application of statistical techniques to the analysis of fungal populations. Transactions of the British Mycological Society 58, 253-268. Mankau R. (1975) A semiquantitative method enumerating and observing parasites and predators of soil nematodes. Journal of Nematology ‘7, 119-I22. Mankau R. and McKenry M. V. (1976) Spatial distribution of nematophagous fungi associated with Meloidogyne incognita on peach. Journal of Nemutology 8, 294-295. McCulloch J. S. (1977) A survey of nematophagous fungi in Queensland. Queensland Journal of Agricultural and Animal Sciences 34, 25-34.

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Nordbring-Hertz B. (1973) Nematode-induced morphogensis in the predacious fungus Arthrobotrys oligosoora. NematoloPia 23. 443-45 1. Pandy V. S. (1973)-Predarory activity of nematode trapping fungi against the larvae of Trichostrongylus axei and Ostertagia ostertagi. Journal of Helminthiiogy 47, 35-48. Peterson E. A. and Katznelson H. (1965) Studies on the

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relationship between nematode-trapping fungi in the vicinity of plant roots. Canadian Journal of Microbiology 11. -1 491495. ~- ~~ Siegel S. (1956) Non-Parametric Stutistics for the Behavioural Sciences. McGraw-Hill. Tokyo. Tribe H. T. (1980) Prospects for the-biological control of plant-parasitic nematodes. Parasitology 81, 619-639.