FEMS Microbiology Ecology Xh ( 1002) 255-26s 0 1992 Federation of Europcan Microhiological Societies 016X-h49h/Y2/$05.00 Published by Elscvicr

FEMSEC 0037 I

The physiology and ecology of a novel, obligate mycophagous flagellate

Wilma E. Hekman, Paul J.H.F. Van den Boogert * and Kor B. Zwart

DLO-lnsrirurr for Soil Frrriliry Rrsrarch, Huren, The Nerherlunds

Received 8 July 1991 Revision received and accepted 1 1 October 1991

Key words: Protozoa; Biological control; Nematophagous fungus; Drechrneriu coniosporu; Conidial survival

1 . SUMMARY

To determine if conidia of the nematophagous fungus Drechmeria coniosporu are subject to pre- dation by soil protozoa, several sandy soils were enriched with 10' conidia of this fungus per g dry soil. After incubation of the samples at 20°C for three weeks, a flagellate was detected as the most dominant mycophagous protozoan. Conidia of several fungi, with minimum diameters between 2 and 16 pm, supported growth of this flagellate, irrespective of Pigmentation. Bacteria however could not be used for growth, although bacteria and also latex beads of the same size were in- gested. This is, to our knowledge, the first report of an obligate mycophagous soil-borne flagellate. The flagellate was able to grow at the expense of the conidia of D. coniosporu in liquid culture,

Correspondence ro: K.B. Zwart, DLO-Institute for Soil Fertil- ity Research, P.O. Box 30003, 9750 RA Haren, The Nether- lands. * Present address: DLO Research Institute for Plant Protec-

tion, P.O. Box 9060, NL-6700-GW, Wageningen, The Netherlands.

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with a specific growth rate of about 0.1 hK'; the optimum temperature was 20-24°C. Approxi- mately 10 D. coniosporu conidia were required for one flagellate division. In sterilized soil, en- riched with 10' D. coniospora conidia per g dry soil, the specific growth rate was 0.014 h- ' , when the soil was at 50 or 65% of its water-holding capacity (WHC). In drier soil, i.e. 25% WHC, no growth took place. During growth of the flagel- late in soil, the number of D. coniosporu was reduced by about 20%, which was in the same order of magnitude as expected on the basis of the requirement of 10 D. coniosporu conidia for one flagellate division. Since many conidia re- mained in the soil after growth of the flagellate, we conluded that although the flagellate is an interesting organism, it does not play a very im- portant role in the survival of D. coniosporu coni- dia in the soil.

2. INTRODUCTION

Several soil-inhabiting mycophagous protozoa have been described, most of which are faculta-

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tively mycophagous since they use bacteria be- sides fungi for growth [l]. Some, however, are obligate mycophagous organisms like the ciliate Grossglockneriu acuta [2] and the amoebae The- carnoeba granifera minor [3] and Cashia rny- cophuga [4]. To our knowledge, mycophagous flagellates have not yet been described. In a study on the ecology of the nematophagous fungus Drechmeria coniospora we discovered a flagellate which was able to grow on the conidia of this fungus in rather high concentrations in several soils. This might be the first observation of a mycophagous flagellate.

D. coniospora is an obligate parasite of nema- todes [5] and produces numerous conidia on these nematodes after killing them [6,7]. The protozoan predation of D. coniospora might to a great ex- tent determine its pcpulation dynamics and thus its potential effectiv Zness as biological control agent of plant parasitic nematodes [8,91. In this paper we describe the isolation procedure and some morphological properties and growth char- acteristics of this flagellate. In addition, we will discuss the possible role of the flagellate in the population dynamics of D. coniospora conidia in the soil.

3. MATERIALS AND METHODS

3.1. Organisms and cultirlation Drechmeria coniospora isolate Lund was gener-

ously provided by Dr. H-B. Jansson. The fungi, yeasts and bacteria used as alternative food sources were from our own collection, or were kindly provided by J. de Haan (H.L. Hilbrands Laboratorium, Assen), W. Cams (Centraalbureau voor Schimmelcultures, Baarn) and G. Bollen (Agricultural University, Wageningen) (all in The Netherlands), respectively.

The fungi were grown on malt peptone agar (MPA) plates [lo]. Fungal conidia were isolated two weeks after inoculation by streaking the plates with a metal rod in the presence of a few ml mineral medium (PJ) used for protozoan isolation [ 111 and filtering the resulting suspension through nylon cloth (pore size 40 pm).

Hansenula polymorpha de Morais et Maya CBS 4732 and Saccharomyces cereuisia were provided

by M. Veenhuis, University of Groningen (The Netherlands). Yeasts and the bacteria Pseu- domonas fluorescens and Bacillus suhtilis were grown in the mineral medium of Van Dijken et al. [12] supplemented with 0.5% glucose and 0.5% yeast extract (MMGY medium) for 24 h.

A mixture of soil bacteria was obtained by suspending 10 g of soil in 100 ml sterile deminer- alized water. The suspension was filtered through a 0.8-pm filter to remove fungal material and protozoa and 1 ml of the filtrate was added to 100 ml MMGY medium in a 250-ml Erlenmeyer flask. After incubation on a shaking incubator (200 rpm) at 25°C for 48 h the bacterial mixture was washed three times with PJ prior to use. All organisms were washed three times with an equal volume of PJ before use.

3.2. isolation of the mycophagous flagellate To enrich and isolate mycophagous protozoa,

soil was collected from three different locations in The Netherlands. A reclaimed peat soil with a pH-KCI of 4.9 and an organic matter content of 4.5% from Rolde, a loamy sand of Pleistocene origin, with a pH-KCI of 4.6 and an organic-matter content of 3.4% from Haren, and a light sandy soil of Pleistocene origin with a pH-KCI of 5.2 and an organic-matter content of 2.6% from Wa- geningen .

Soils were sieved through a mesh with 3-mm pores. Soil samples of 80 g were amended with lo9 D. coniospora conidia in PJ per g dry soil; the equivalent amount of PJ was added to control samples. Soil moisture was adjusted to 50% of the water holding capacity (WHC) and the sam- ples were incubated for three weeks at 20°C. To determine the amount and the type of my- cophagous protozoa, a ten-fold difuted soil sus- pension (v/w) was made in PJ. The suspension was further diluted serially (1 : 4) according to Darbyshire et al. [131 in a conidial suspension of D. coniospora (10’ per ml) in a microtiter plate (MTP, Greiner, Alphen a/d Rijn, The Nether- lands). After incubation at 20°C for 7 days the types of protozoa in each well were determined using an inverted microscope (Leitz Labovert, 250x, Leitz, Wetzlar, F.R.G.). The number of protozoa per g soil were estimated using the Most

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Probable Numbcr (MPN) method [14]. The flag- ellate used for further experiments was isolated from the highest dilutions of a microtiter plate. To obtain a monoculture of the flagellate a single cell was picked up manually, using a capillary pipette. Rifampicin (100 p g ml-’ water) was used to suppress growth of bacteria in the stock cultures of the flagellate.

3.3. Food specificity o f the flagellate To study the food specificity of the flagellate,

several conidia-producing fungi, yeast cells, sev- eral bacteria and also inert food particles of dif- ferent sizes were tested for their ability to sustain growth of the flagellate. Suspensions in a concen- tration of approximately 10‘ cells per ml of these potential food sources in PJ were added to MTP wells in triplicate. Flagellate cells from a culture which was pre-grown on D. coniospora were added at lo3 cells per ml. The presence of food particles in the flagellate cells and the occurrence of subsequent growth of the flagellate were stud- ied using an inverted microscope.

3.4. Growth of the flagellate in liquid To characterize growth of the flagellate at the

expense of D. coniospora conidia, two types of experiments were performed in which the organ- ism was cultivated in a conidia suspension of this fungus. For shake cultures, 100-ml Erlenmeyer flasks were used with 40 ml PJ and 10’ D. co- niospora conidia per ml. After inoculation with lo3 flagellate cells per ml, growth was measured as an increase in cell number, determined with a haemocytometer. For standing cultures, macro- plates (Greiner) with 35-mm diameter wells con- taining 3 ml liquid Per well were used. After inoculation with conidia and flagellates, cell num- bers of both Organisms were determined at timed intervals with an inverted microscope. The spe- cific growth rate was determined as described by Stanier et al. [15]. The effect of external ion concentration on the growth rate of the flagellate was determined by suspending the conidia in 0-, 0.1-, 1.0-, 10.0- and 100.0-fold concentrated PJ and subsequently following the growth in stand- ing cultures as described above. The effect of temperature on the growth rate of the flagellate

was measured by incubating standing cultures in incubators at 0, 5, 10, 15, 20, 25 and 25°C.

3.5. Growth of the flagellate in soil l o study the development of the number of

flagellates and D. coniospora conidia in soil un- der controlled conditions at different moisture contents, Rolde soil was air-dried overnight and y-sterilized (6 Mrad). The soil was thoroughly mixed with D. coniospora conidia (10‘ per g dry soil) and extra PJ where necessary, to bring the moisture content to just below the required level (25, 50 or 60% WHC). PVC containers (inner diameter 6.0 cm; height 2.4 cm), made aseptic with 70% ethanol, were filled with the equivalent of 75 g dry soil each (ten containers per moisture treatment) to a bulk density of 1.1 g cm-3. Since mixing of the flagellates with the soil would result in serious cell damage, flagellates were added to the soil as follows: in the surface of the soil, ten wells were made ( 1 cm deep and 2 mm diameter) and to each well 100 pI of a flagellatc suspension was added to a final concentration of 2 x lo4 cells per g dry soil. With the addition of the flagellate suspension, the final moisture content was 25, 50 or 65% WHC respectively. Control containers (50% WHC) were prepared containing either only D. coniospora conidia, or only flagel- lates. The containers were covered with parafilm to prevent water loss; they were incubated at 20°C for 43 days.

The development of flagellate and conidia numbers with time was followed by sampling con- tainers at intervals and suspending all the soil from a container in a ten-fold amount of PJ (v/dry weight). The number of flagellates per g dry soil was determined using the MPN method [ 141.

The number of D. coniospora conidia was de- termined after adjusting the soil suspension men- tioned above to 0.1% sodium pyrophosphate by mixing with a 7.5% stock solution, followed by blending for 30 s in a Waring blender (Cole Parmer Instrument Company, Chicago, IL; set- ting “lo”). After centrifugation (400 X g , 5 mid , 10 pl of the supernatant was spread over 7.5-mm diameter wells of a microscope slide (Flow Labo- ratories Irvine, Scotland). After drying, fixation

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and staining with europium chelate, the slides were prepared for microscopical counting of the conidia according to Bloem et al. [16]. Data on flagellate and conidia numbers were subjected to univariate analysis of variance (ANOVA) after log transformation using Genstat programs (Gen- stat release 5.1, Rothamsted Experimental Sta- tion, Harpenden, U.K.). In case the ANOVA was significant, LSDs were used to indicate significant differences between means.

3.6. Electron microscopy For SEM cells were prefixed in 4% formalde-

hyde for 15 min and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 30 min. For TEM cells were fixed in ice-cold 2.5% glutaraldehyde (5% solution in sodium cacodylate buffer, mixed 1 : 1 with the culture medium) for 30 min, post-fixed in a mixture of 1% OsO, and 5% K,Cr,O, for 60 min and stained in 1% uranylacetate overnight. After dehydration, em- bedding in Epon 812 and ultrasectioning the cells were observed in a Philips EM 300.

4. RESULTS

4.1. Enrichment and isolation of the flagellate The standing crop of mycophagous protozoa,

able to grow on D. coniospora conidia in PJ medium, was approximately lo3 protozoa per g dry soil, and varied depending on the soil type (Table 1). After incubation with lo9 D. co- niospora conidia per g dry soil for three weeks, the number of mycophagous protozoa increased 30-fold (Haren and Wageningen) and 426-fold (Rolde) to a maximum of 4.26 X lo7 protozoa per g.

Flagellates and amoebae were among the pro- tozoa detected in the MTP test wells. In the higher dilutions naked and testaceous amoebae and one type of flagellate were found. The flagel- lates, however, predominated.

4.2. Morphology of the flagellate Cells of the flagellate were oval shaped, ap-

proximately 10 p m wide and 15 p m long, but cell size varied with nutritional state. They had an

Table 1 The number of mycophagous protozoa, expressed as log MPN, in three different soils, before and after amendment with 10’ Drechmeriu coniosporu conidia per g dry soil

Soil log MPN

Without conidia With conidia -

Haren 3.00 (0.25) 4.49 (0.20) Rolde 2.85 (0.32) 5.48 (0.21) Wageningen 3.26 (0.22) 4.75 (0.20)

Numbers in parentheses indicate the confidence interval ( p =

0.05).

irregular surface as observed in SEM prepara- tions (Fig. 1) and became temporarily amoeboid during the ingestion of particles. Cysts, usually formed during the stationary phase, were thin- walled and isodiametric in shape. Each cell con- tained two flagella, a short one and a long one, approximately 5 p m and 25 p m long, respec- tively, protruding from a small anterior cell de- pression (Figs. 1 and 2). Each cell contained one nucleus. In addition, the cytoplasm contained mi- tochondria, ER, a Golgi apparatus, lipid droplets and many vesicles (Fig. 2). Conidia, where pre- sent, were found in food vacuoles (Fig. 3). No contractile vacuoles or kinetoplasts were ob- served.

The flagellate cells moved in a shivering way, with the posterior part directed upwards. The shorter flagellum rotated during and in synchrony with this movement. The longer flagellum on the other hand was trailing behind the cell at a slight angle always to the left of the cell axis during movement in liquid.

4.3. Food specificity of the flagellate Of the conidia from 30 fungal species, tested

as alternative food source besides D. coniospora, 19 were digested, resulting in growth of the flag- ellate, 8 were ingested, but did not support growth and 4 were not ingested at all. The yeast cells tested also resulted in multiplication of the flagel- late. Bacteria on the other hand did not support growth (Table 2), although bacteria and also latex beads of similar sizes were ingested. Hyphae of D. coniospora, incidentally formed in the MTp wells, were attacked by the flagellate too.

Fig. 1. SEM o f the mycophagous l l i ig~l l i i t~ after growth on the yeast Hun.scnit1u pdynwrp/iu. The hactcria prescnl iirc ;I

contamination from the stock culture of the flagellates. These bacteria grow on lysed yeast cells hut do not support growth 01 thc flagellate. The bar indicates 5 pm.

Size probably was the determining factor for ingestion since all particles with a minimum length between 2 and 16 p m , the latter being the maxi-

mum size of the organism itself, wcrc taken up, whereas larger particles, with one exception, i.c. Fusuriiiriz cu/Inorum, were not ingcstcd. Thesc

Fig. 2. TEM of the mycophagous flagellate after growth on conidia of t h e nematophagous fungus Drechmeria ~ o ~ z ~ o . $ ~ c J ~ u .

The bar indicates 5 p m .

Fig. 3. Drechmcriu coniosporu conidia in several stages of hreakdown in food vacuoles of the mycophagous tlagellate.

The har indicates I pm.

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results indicate that the organism was rather non-specific in its choice of potential food parti- cles.

In contrast to uptake, growth was obviously not determined by the size of the food particles, since there was a large variation in the size of the ingested particles which did not result in growth.

The presence of pigmentation in the conidial wall did not determine the occurrence of growth ei- ther, since 3 of the 10 pigmented conidia caused flagellate growth. The factors that control the digestion of food particles and subsequent growth in this flagellate are not yet known. The electron micrograph with ingested D. coniospora conidia

Table 2

Food selectivity of the mycophagous flagellate

Potential food source Type a Size (pm) Reaction

Fusarium acuminatum M - - h 53-90 - Cochliobolus satious MTP 53-90 - Fusarium culmorum MTP 36-54 0 Arthrobotrys oligospora U - - 18-27 - Alternaria porrri MTP 17-19 - Latex beads - - - 15-17 - Hormiactis fimrcola B - - 14-36 + Cylindrocarpon destructans UM- - 13-36 0 C. destructans UM- P 11 -45 0 Fusarium oxysporum UM- - 9-54 + F. oxysporum UM- - 9-18 + Verticillium biguttatum U - - 9-13 + Cladosporium cladosporioides u-P 9-13 + F. oxysporum f.sp. melongenae U - - 7-27 + plectosphaerella cucumerina B - P 7-13 + Botrytris aclada U - - 7-13 + Drechrneria coniospora (Rolde) U - - 1-9 + D. coniospora (Lund) U - - 7-9 + F. oxysporum f.sp. asparagi U - - 5-20 + C. herbarum UB- P 5-18 0 V. albo atrum U - - 5-11 + Microdochium bolleyi U - - 5-11 + Gliocladiurn roseum U - - 5-7 + C. sphaerospermum u - P 5-7 0

5-6 0 Latex beads Trichoderma hamatum U - - 4-5 + V. lecanii U - - 4-9 + P. simplicissimum U - - 4-5 0 P. melanii u - P 4-5 + G. nigrosirem u - P 4-5 0 K chlamydosporium U - - 3-5 + V. balanoides U - - 3-5 + Hansenula polymotpha yeast 3-5 + Saccharomyces cerevisiae yeast 3-5 +

1-2 0 Latex beads - - - Soil bacteria (mixture) bact. 0.5-1 0 Pseudomonas fluorescens bact. 0.5-1 0 Bacillus subtilis bact. 0.5-1 0

a U: unicellular; B: bicellular; M: multicellular: UM: uni- to multicellular; T thick-walled; P: pigmented; + : growth; 0 ingestion

- - -

only, no growth; -: no ingestion. according to Domsch et al. (1980).

' Indicates the minimum to maximum length of fungal conidia, cells or other particles.

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f 008 m m 2 om- 5

R c

c

2 4 6 0

-

time (days) Fig. 4. Increase of flagellate number (A - A) with con- current decrease in the number of Drechrneriu coniospora

conidia ( - M ) in liquid standing culture.

(Fig. 3) shows intracellular digestion of both cyto- plasm and cell walls of the conidia by the flagel- late.

4.4. Growth of the flagellate in liquid 4.4.1. General characteristics. The flagellate was

grown in both shake and standing cultures. In shake cultures the specific growth rate on D. coniospora conidia was approximately 0.1 h- ' at 19°C. Growth was not affected by the rotation rate of the shaker between 150 and 300 rpm. Below 150 rpm clumping of the flagellate cells occurred. In standing cultures a similar growth rate was found, although there was more cell clumping than in shake cultures. With standing cultures, the determination of the cell density was less time-consuming than with shake cultures and therefore this type of cultivation was preferred.

The increase in flagellate number in standing cultures is shown in Fig. 4; this coincides with a decrease in D. coniospora conidia. At the final density of approximately 3 x lo3 flagellates x mm-' less than one conidium was left. It was estimated that approximateIy ten conidia were needed for the reproduction of one flagellate.

4.4.2. Temperature. The variation in specific growth rate of the flagellate growing on D. CO-

niospora conidia at different temperatures is shown in Fig. 5 . The optimum temperature for growth was between 19 and 24°C. The specific growth rate was sharply reduced at 30°C. At this temperature the cultures formed cysts or died

I . erp1

I . ".""

0 5 10 15 20 25 30 35

temperature ("C)

Fig. 5. Specific growth rate of the mycophagous flagellate in liquid during growth on Drechrneriu coniospora conidia at

different temperatures.

rapidly following logarithmic growth. For further experiments a temperature of 19°C was used as a matter of routine.

4.4.3. Concentration of minerals. To test the effect of mineral concentration on the specific growth rate of the flagellate, the organism was grown in PJ ranging from 0 to 100 times the standard concentration. The results are shown in Fig. 6; they indicate that the standard concentra- tion of PJ was the optimum concentration for growth.

4.5. Growth of the flagellate in sail To study flagellate growth in the soil and the

effect of soil moisture on this process, sterilized Rolde soil was used at three different moisture contents. An increase occurred only in soil at 50

times Standard p J

Fig. 6. Specific growth rate of the mycophagous flagellate in liquid culture during growth on Drechmeria coniospora Conk

dia as dependent on different concentrations of PJ.

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and 65% WHC, not in soil at 25% WHC ( P < 0.001) (Fig. 7a). At the lowest moisture content the number of flagellates decreased in time. The specific growth rates at 50 and 65% WHC calcu- lated from day 0 until day 15 were 0.015 and 0.013 h - ' respectively. The maximum cell num- ber achieved after 15 days was approximately lo6 cells per g dry soil in both cases. This indicated that a minimum amount of soil moisture, between 25 and 50% WHC, was necessary to allow net flagellate growth.

The final conidia number found, calculated as the average of the values between day 19 and day 43, was lower ( P < 0.01) in soils which showed flagellate growth than in soils in which no flagel- late growth occurred, i.e. soils without flagellate additions or soils at 25% WHC, as shown in Fig. 7b. At the end of the experiment, the soils at 50% WHC containing flagellates and conidia contained 20% less conidia than the controls without flagellates (Fig 7b). Therefore it was esti- mated that 20% of the conidia were consumed during the experiment.

No flagellates were found in control micro- cosms with D. coniosporu conidia added only (data not shown). In controls with no conidia and flagellates only, the number of flagellates re- mained constant, probably as encysted cells (Fig. 7a). Surprisingly, the conidia number increased in time at 25% WHC and in the control with only

7 1

conidia and no flagellates added. Possible reasms for this will be discussed later.

A linear correlation (Y = 0.115X - 0.723; t = 17.9; X ranging from 5 X lo6 to 100 X lo6 coni- dia per g soil) between the number of flagellates produced and the inoculum density of conidia was found in an additional experiment (not shown). This indicates that approximately 9 coni- dia were needed for the reproduction of one flagellate in soil.

4.6. Dispersion of the flagellate As shown in Fig. 7, growth of the flagellate

and the concurrent decrease in the number of D. coniosporu conidia took place under suitable moisture conditions, but no net growth occurred after 15 days, even though the number of conidia detected in the soil samples was still relatively high. To test whether this discontinuation of growth was due to insufficient dispersion of the flagellate from the inoculation sites, microcosms were prepared as described in 3.5, with y-steri- lized Rolde soil at 50% WHC and lo8 D. co- niosporu conidia, except that one well was made in the centre of the microcosm to which the flagellate suspension was added. After three weeks of incubation at 20°C, samples of approxi- mately 1 g were taken with a 6-mm diameter borer at distances of 0, 6, 12 and 18 mm respec- tively, from the centre of the microcosm. The

1.8 I

1.6 I B n 0.8 ' ' ' ' ' ' ' ' ' '

0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50 time (days) time (days)

Fig. 7. Change in the log flagellate number (A) and conidia number (B) with time in a gnotobiotic microcosm under different soil moisture conditions. - D , 25% WHC; A - A , 50% WHC; 0- 0,65% WHC; A - A , 50% WHC; no conidia

(A), no flagellates (B).

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Table 3

The number of mycophagous flagellates expressed as log MPN at different locations in soil microcosms, immediately after inoculation and after 3 weeks of incubation at 20°C (The microcosms were inoculated with flagellates at the centre.)

Location d " r = O I = 3 weeks F h F + D F F + D

0 3.91 (0.39) 3.71 (0.30) 3.03 (0.28) 5.98 (0.29) 1 2 6 < 2 < 2 < 2 5.97(1.11) 3 12 < 2 < 2 < 2 4.15 (0.10) 4 18 < 2 < 2 < 2 < 2

a d , distance from the centre of the microcosm (mm). F, soil containing flagellates only. F+ D, soil containing flagellates and D. coniosporu.

The numbers in parentheses indicate the confidence interval at P = 0.05; < 2 indicates that the number was below the detection limit of.100 cells g+'.

results (Table 3) demonstrate that in the pres- ence of D. coniospora the flagellate showed a lateral dispersion of at least 12 mm during 3 weeks of incubation. This is a clear indication that the organism is able to move easily through the soil under these conditions. Hence, factors other than a lack of dispersion of the flagellate were responsible for the high concentration of conidia that remained in the soil.

5. DISCUSSION

The flagellate described above was found in three different soils, which indicates that it is probably a fairly common soil-borne organism.

The characteristics of the organism made it difficult to identify it. The presence of two flag- ella, which were used for movement, clearly places the organism into the order of Mastigophora. Morphologically, the flagellate had certain prop- erties in common with the genus Bod0 [171 but differed from it by the absence of the kinetoplast characteristic for bodonoids. It also showed some resemblance to Cercomonas but it contained no contractile vacuole. The most striking characteris- tic of this flagellate, which makes it different from all flagellates described so far, is its obligate mycophagous way of living (Table 2). Other my- cophagous protozoa have been described before, most of which are amoebae belonging to several

genera [ l ] and ciliates [2]. A more detailed de- scription of the mycophagous flagellate, including its classification, will be published elsewhere.

The food choice of the flagellate was wide (see Table 2): every food particle with a minimum length between 2 and 16 pm was ingested. The degradation process did not involve perforation of conidia, as is the case in some mycophagous amoebae [18,19], but involved engulfment of the food particles followed by intracellular digestion.

Some ingested particles did not result in flagel- late growth, presumably because these particles could not be digested. Among the latter were both pigmented and non-pigmented conidia, in- cluding which the heavily melanized P. melunii. Melanization is often thought to confer resistance to microbial attack [20]. Several mycophagous protozoa, such as the Thecamoeba isolate of Esser et al. [21] and Ripidommyxu uustruliensis [22] were indeed not able to attack pigmented fungal struc- tures. However, melanization is certainly not the only factor important in digestion, since there are several examples of mycophagous protozoa feed- ing on both melanized and non-melanized fungal conidia or hyphae [ 1,23,24].

Old and Darbyshire [23] suggested production of toxic products by fungal conidia as a possible explanation for the absence of growth of giant vampyrellid amoebae on Stachybotrys atru.

Growth of the flagellate on conidia of D. coniospora occurred readily in liquid culture. Be-

264

cause in liquid culture the flagellate reduced the conidia number to almost zero at the end of the exponential growth phase, we concluded that the flagellate could potentially reduce D. coniosporu conidia to low numbers in soil. In soil, flagellate growth also occurred, although with lower growth rates and less effective with respect to conidia reduction compared to liquid. In soil, 9 conidia were needed for the reproduction of 1 flagellate. The expected reduction in number of conidia in soil, as based on flagellate production, is then approximately 20%. So, the reduction found is in the same order of magnitude as expected.

It is not clear why so many conidia remained in the soil after flagellate growth. Several possible explanations exist.

First of all, a lack of dispersion of the flagel- lates in the soil could protect many conidia from predation. This explanation can be ruled out. The mycophagous flagellate could move over a dis- tance of at least 12 mm in soil during 3 weeks. This distance is sufficient to colonize the entire microcosm when 10 inoculation sites were used. Losina-Losinsky and Martinov [25] and Biczok [26] also showed that protozoa could easily move in soil. Their and our observations are in contrast with those of Kuikman et al. [27], who suggested that protozoa showed only very limited movement through the soil.

A second explanation, auto-inhibition of flag- ellate growth above a certain density in the soil, is not very likely either because of the linear corre- lation between the number of flagellates and the inoculum density of conidia in the soil.

Thirdly, production of secondary conidia by D. coniosporu could partly compensate for the loss of conidia upon predation. Normally, conidia can only be formed after infection of a host nematode [S] , or axenically in cultivation media [28-311. However, during our experiments germination of conidia in PJ was repeatedly observed, resulting in the formation of germ tubes with 1-4 new conidia. Whether or not this phenomenon also occurred in y-sterilized soil is unknown, but it provides an explanation for the increase in coni- dia number in the control experiment (Fig. 7B). Still, this does not fully explain the survival of so many conidia in the microcosms with flagellates.

In conclusion, the soil matrix somehow provides a protection for the conidia against predation by the flagellate.

Thus, it seems unlikely that this mycophagous flagellate will play a dominant controlling role in regulating the population density of D. co- niosporu in soil. However, the ability of this flag- ellate to reduce the number of D. coniosporu to near zero in liquid culture, combined with its wide food choice, make this organism interesting for application in biological control of plant- parasitic fungi in nutrient-film plant cultures.

ACKNOWLEDGEMENTS

The authors thank J. Dijksterhuis for prepara- tion of the EM photographs. The technical assis- tence of G. de Rink with some of the experiments is gratefully acknowledged.

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