FEMS Microbiology Ecology 85 (1991) 207-210 © 1991 Federation of European Microbiological Societies 0168-6496/91/$03.50 Published by Elsevier ADONIS 016864969100074W

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FEMSEC 00329

Direct observation of trapping activities of nematode-destroying fungi in the soil using fluorescence microscopy

Chr i s t i an Jensen and G e r n o t L y s e k

Institut ]'fir Systematische Botanik und Pflanzengeographie, Freie Universitdt Berlin, Berlin, F.R. G.

Received 15 August 1990 Revised versions received 3 December 1990 and 4 January 1991

Accepted 4 January 1991

Key words: Nematophagous fungi; Trapping activity; Soil; Formation of trapping organs; 3-Dimensional sticky networks; Fluorescence microscopy; Fluorescein-diacetate

1. S U M M A R Y

The nematophagous fungi (Arthrobotrys oligo- spora and Monacrosporium cionopagum ) were ob- served under semi-natural conditions by fluores- cence microscopy after fluochroming, using fluo- rescein-diacetate. The formation of 3-dimensional sticky networks and the capture of nematodes inside the soil could be demonstrated.

2. I N T R O D U C T I O N

For a long time mutual effects between nema- tophagous fungi and soil nematodes have been studied to obtain more insight into the dynamics of the antagonists with the aim of controlling parasitic nematodes by biological means [1]. How- ever, promising nematophagous competence of

Correspondence to: G. Lysek, Institut fiir Systematische Botanik und Pflanzengeographie, Freie Universitiit Berlin, Alten- steinstr. 6, D-1000 Berlin-33, F.R.G.

some predacious fungi in laboratory experiments was usually opposed by disappointing results in the field [2,3]. This was partially due to the lack of observations of fungal growth, and the formation of t rapping organs, including the t rapping processes. Such studies are hardly possible under natural conditions, as the optically nontransparent substrate of soil and sand excludes direct observa- tion. Thus, the fluochroming of fungi [4] was at tempted as an aid to prove and to observe trapping activities in the soil. Such a method could contribute to a better understanding of nema- t o d e / fungi interactions and could also be used to study other processes in the soil. Fluorescein-di- acetate was chosen as it stains living cells exclu- sively and thus.allows the staining of the vital, i.e., active parts in this system only.

3. MATERIALS A N D M E T H O D S

3.1. Fungi The Deuteromycete fungi Arthrobotrys oligo-

spora Fres. and Monacrosporium cionopagum

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(Drechsler) Subramanian (syn. Dactylella cionopa- ga Drechsler) were used in the experiments.

3.2. Culture The fungi were grown in sterile culture on a

solid medium (2% malt extract, 2% agar-agar) or in a shaker in a 2% malt extract solution. The test cultures were inoculated with material from these precultures. Material from liquid cultures was cut into small pieces in a Waring Blendor according to Lysek [5] and mixed into the soil. From solid cultures conidia and mycelial fragments were transferred to the soil by cutting out small agar blocks with a sterile lancet. Petri dishes (glass, 9 cm diameter) filled with purchased garden soil and then autoclaved served as experimental cul- tures. After incubation at room temperature in continuous darkness for 7 days, nematodes from a culture of Turbatrix aceti (about 1000 per Petri dish) were added as a bait to induce trap forma- tion.

3.3. Staining After 2 further days the cultures were ex-

amined: about 0.1 g soil was transferred to a cover slide (18 x 72 mm), sprinkled with a few drops of the staining solution and then spread out and smoothed with a lancet. The cover slide was then turned upside down and placed on two matches fixed on a microscopic slide to allow the micro- scopic observation of the soil through the glass in a ' hanging drop'.

After the microscopic observation some of the prepared specimens were kept in a moist chamber and again stained and controlled after a further 2 or 3 days.

3. 4. Staining solution A stock solution of 2 m g / m l fluorescein-di-

acetate (FDA) in acetone was kept in the dark in the refrigerator. I ml of this solution was added to 200 ml of a 60 m M phosphate buffer pH 7.5 to obtain a final concentration of 10 /~g F D A / m l . This 'working solution' lasts for a few hours only and thus it always had to be prepared freshly for every experiment. The FDA itself does not fluo- resce; the fluorescein only develops after taking up the FDA into metabolically active cells, where

the acetate is split off by hydrolysis [4,6]. Thus, only vital, i.e., actively metabolizing cells or hy- phae develop the fluorescence. Beside FDA the following dyes were tested: acridin orange, ethidium bromide, fluorescein and rhodamin B. Among these FDA gave the best results.

3.5. Microscopic observation A Leitz epifluorescence microscope 'Laborlux '

was used to examine the 'hanging drops'. It was equipped with 4.0 to 40.0 NPL Fluotar objectives, giving a total magnification between 40- and 400- fold.

The photographs were taken with a Wild-KB- stage and ISO 1000/31 ° black-and-white films.

4. RESULTS A N D DISCUSSION

Against the black background of soil particles the living hyphae show yellow green fluorescence, the intensity of which depends on the metabolic activity of the cells (Fig. 1). A slight background fluorescence can be avoided to a great extent by removing the surplus of the staining liquid. At greater magnifications (250 X and 400 x ) a fade- out occurs (after about 30-40 s), due to the stimu- lation by the high light intensities required. In this m a n n e r bo th Arthrobotrys oligospora and Monacrosporium cionopagum revealed the forma- tion of 3-dimensional sticky networks as trapping organs in the soil. A similar number of traps developed to full size as known from agar cultures (Fig. 2). In Monacrosporium cionopagum sticky knobs could occasionally be identified. Nema- todes adhering to such knots were not observed. In contrast, numerous eelworms appeared fixed to the sticky networks which, in a later stage, were penetrated and digested by trophic hyphae.

The method described once again demonstrates the nematophagous capacity of these two fungal species in sterile soil, i.e., under more natural conditions than in agar media. As the formation of trapping organs could not always be observed it is not yet possible to decide on their dependence on additional factors, such as method of cultiva- tion, soil type or bait.

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Irrespective of these possibilites, the induction of trapping organs did require the addition of nematodes. In this respect the fungi responded in the same way in soil as when grown on agar media [1,7,8].

This is also true for nematodes: in spite of the high trapping effectiveness all nematodes were never caught. In fact they outlived the fungi. This agrees with the observations by Cooke [2] who reported a significant increase in soil nematodes in the presence of trapping organ-forming fungi. Liv- ing or disintegrating mycelium apparently served as a source of food for these animals [9].

The method described here may also be useful for the observation of other microbial activities inside soil-like ecosystems, as already pointed out by S~Sders'tr~Sm [4].

On the other hand, the rapid fading of the dye still represents a major problem for continuous observation of long-term developments, as is the case with the entire cycle of nematode adhesion, the formation of the infection bulb by the fungi and the final digestion. Further improvements in equipment and fluorescent stains will be required for a further extension of the period available for observation by microscope.

Fig. 1. Two completely developed sticky networks of Arthrobotrys oligospora. The light line running through both the traps is the corpse of a previously caught and almost entirely digested nematode.

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Fig. 2. Large sticky network of Monacrosporium cionopagum with a trapped nematode. The high fluorescence inside the animal is due to the metabolic activity of the digestion hyphae.

R E F E R E N C E S

[1] Dowe, A. (1987) R~tuberische Pilze im Boden, Die neue Brehm-Btieherei, Wittenberg-Lutherstadt; 2rid Edn.

[2] Cooke, R.C. (1963) The predaceous activity of nematode- trapping fungi added to soil. Ann. Appl. Biol. 51, 295-299.

[3] Mankau, R. (1980) Biological control of nematode pests by natural enemies. Ann. Rev. Phytopathol. 18, 415-440.

[4] S~Sderstr~Sm, B.E. (1977) Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol. Biochem. 9, 59-63.

[5] Lysek, G. (1972) Rhythmic mycelial growth in Podospora

anserina III. Rhythmic dry weight production in liquid culture. Arch. Mikrobiol. 81,221-233.

[6] Cohen, S.D. (1984) Detection of mycelium and oospores of Phytophtora megasperma forma spezialis Glycinea by vital stain in soil. Mycologia 76, 38-39.

[7] Nordbring-Hertz, B. (1977) Nematode-induced morpho- genesis in the predacious fungus Arthrobotrys oligospora. Nematologica 23, 443-451.

[8] Lysek, G. and Nordbring-Hertz, B. (1983) Die Biologie nematodenfangender Pike. Forum Mikrobiologie 6, 201 208.

[9] Cooke, R.C. (1963) Ecological characteristics of nematode trapping Hyphomycetes. Ann. Appl. Biol. 52, 431-437.