Carin Dackman, Stefan Olsson, Hans-B6rje Jansson, Bj6rn Lundgren, and. Birgit Nordbring-Hertz. Department of Microbial Ecology, Ecology Building, University ...
Mierob Ecol (1987) 13:89-93
MICROBIAL ECOLOGY 9 Springer-VerlagNew York Inc. 1987
Quantification of Predatory and Endoparasitic Nematophagous Fungi in Soil Carin D a c k m a n , Stefan Olsson, Hans-B6rje Jansson, Bj6rn Lundgren, and Birgit N o r d b r i n g - H e r t z Department of Microbial Ecology,EcologyBuilding, University of Lund, S-223 62 LUND, Sweden
Abstract. M e t h o d s were d e v e l o p e d to quantify predatory and e n d o p a r asitic fungi in soil. T h e m e t h o d s w e r e based on previously developed detection techniques and c o m b i n e d with a most probable n u m b e r estimation. The m e t h o d s were applied to an agricultural soil fertilized with f a r m y a r d manure. Large a m o u n t s o f f a r m y a r d m a n u r e resulted in increased a m o u n t s o f organic matter, n u m b e r s o f propagules o f predatory and endoparasitic fungi, and n u m b e r s o f bacteria and nematodes. Introduction Nernatophagous fungi are usually d i v i d e d into two different groups: fungi that capture n e m a t o d e s with specialized h y p h a l devices (predatory fungi), and those that infect n e m a t o d e s with their spores, the so-called endoparasitic fungi [1]. These fungi frequently occur in m a n y types o f soil [1, 2]. T o detect them, a n u m b e r o f m e t h o d s have been used--e.g., the sprinkled plate m e t h o d originally devised by Drechsler [6] and further d e v e l o p e d by D u d d i n g t o n [7], the Baerm a n n funnel technique described by G i u m a and Cooke [ 10], and the differential centrifugation technique [1, 2], as well as modifications o f these [3]. Living nematodes, either present in the soil sample or added as bait for the fungi, are a prerequisite for the detection o f nernatophagous fungi with these methods. Some semiquantitative m e t h o d s have also been described to e n u m e r a t e both predatory and endoparasitic fungi f r o m soil [8, 14, 16, 20]. Since there is still a need for techniques to estimate these fungi in soil, we decided to develop procedures to quantify both p r e d a t o r y and endoparasitic species. T h e m e t h o d s we used were c o m b i n a t i o n s o f detection techniques and most probable n u m b e r (MPN) estimations.
Material and Methods The quantification of predatory fungi was based on the method of Eren and Pramer [8]. The recovery of predatory fungi was performed in two different ways. In the earlier experiments the sprinkled plate technique was used, where one gram of soil was sprinkled on a water agar plate (15 replications per soil sample) and 500 nematodes (Panagrellus redivivus) were added. In later experiments we found that a soil dilution technique gave approximately 10 times as many prop-
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Table 1.
Soil characteristics
Soil treatment NPK fertilizer Farmyard manure I Farmyard manure 2
Nitrogen addition 80 kg ha -j yr -~ 500 kg ha -~ yr -) 1,000 kg ha -~ 2 yr ~
Water content % H20 o f dry weight
Organic content % LOP o f dry weight
pH
23.8 31.2 32.5
5.5 7.1 7.8
7.1 7.2 7.3
~ LOI, loss on ignition at 650~ agules as the sprinkled plate method, when similar soils were compared. Furthermore, a prerequisite for the MPN estimation is that some of the replicates give a negative result. Since there is a higher probability to obtain this condition with soil dilutions rather than with sprinkled plates, the soil dilution method was preferred for the estimation of predatory fungi. The soil dilution method was performed in the following manner: 15 g soil was shaken in 15 ml 0.01% (w/v) sodium hexametaphosphate (Calgon, BDH Chemicals, Lid.) in a 100-ml Erlenmeyer flask for I 0 rain. Heavy material was allowed to settle before the soil suspension was decanted into a graduated cylinder and the volume adjusted to 15 ml with water. A dilution series was prepared giving I, 0.1, 0.01, and 0.001 g soil ml -~. From each dilution a sample of 0.5 ml was spread on a water agar plate (1.5~ w/v) in such a way that a star was formed [I], and the plates were left with the lid half open to allow excess water to evaporate. Five replicates were made from each dilution. On the following day, the plates were baited with approximately 500 nematodes per plate. The plates were incubated in the dark at approximately 22"C and examined for growth of predatory fungi after 2 and 6 weeks. More frequent examination was found unnecessary. Plates showing growth of predatory fungi, manifested in the formation of trapping organs, were recorded as positive. The proportions o f positive plates in each dilution were used to calculate the most probable numbers from an appropriate MPN table. The method for quantification o f endoparasitic species is a combination o f MPN and the dif= ferential centrifugation technique described by Barton [I, 3]. A mixture of 15 g fresh soil and 15 ml 0.01 o/oCalgon solution was shaken with 20 glass beads (5 m m diameter) in a i 00-ml Erlenmeyer flask for 5 min. The soil suspension was sieved twice (2= and 0.5=ram sieve, respectively) to remove the beads and course organic debris, and the volume of the suspension was adjusted to 50 ml with water. The suspension was diluted 1:10 and I:I00, and 5 replicates of each concentration were prepared. Of the original suspension and of each dilution, 5 ml were transferred to three separate 50-ml centrifuge tubes and water was added to a total volume of about 40 ml per tube. The tubes were centrifuged at 150 x g for 3 rain to remove heavy particles and spores o f predatory fungi. The supernatants were transferred to new tubes and centrifuged a second time, now at 12,000 x g for 5 min. The pellets were resuspended in 0.5-I ml water and spread on water agar plates to form stars. The plates were baited with nematodes as described above and examined for endoparasitic fungi after 3-5 days and again after 4 weeks. The most probable number of propagules per g o f soil was calculated from a MPN table as described for predatory fungi. The use o f a MPN estimation gives a better quantification of propagules o f both predatory and endoparasitic fungi in a soil than just recording the percentage o f positive plates. The soil sprinkle technique and the differential centrifugation, with the appropriate MPN estimations, were tested on plots o f a Danish agricultural soil (Askov) treated with different amounts of farmyard manure for 6 years. These plots were chosen since their densities of nematophagous fungi were expected to differ greatly. Large amounts of farmyard manure give high numbers o f bacteria and especially bacteria-feeding nematodes. The nematodes in their turn would stimulate the activities of the nematophagous fungi. The soil characteristics are given in Table I. One plot was fertilized with inorganic fertilizer (NPK, 80 kg N ha-t yr-1), the second plot was fertilized with
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91
A.
B.
20'
} O
NPK
MANUB[I
MANURE 2
c.
NPK
UANUBE
1
WANUR~ 2
D.
~oo --
~ FnA r " - " 3 Ao pt D
~0
~ NPK
e" 2
0 MANUB[ i
MANURE
~, NPK
MANUBF I
MANUBE 2
Fig. I. Numberof propagulesof predatoryand endoparasiticnematophagousfungi, and number of nematodes and bacteria in an agricultural soil fertilized with N P K (80 kg N ha-' yr) and farmyard manure (500 kg N ha -~ yr -t and 1,000 kg N ha -t 2 yr -~, respectively). (A) Predatory fungi estimated with the sprinkled plate + M P N method. (B) Endoparasitic fungi estimated with the differential centrifugation + M P N method. ((3) N u m b e r of nematodes extracted with the Seinhorst elutriator. (D) Bacterial numbers determined with the fluorescein diacetate (FDA) and the acridine orange (AO) methods. farmyard'manure at a rate of 100 tons ha -~ once a year, and the third plot with the double amount of farmyard manure every second year. Thirty subsamples of soil were taken in each plot, to a depth of 5 cm, in the autumn one year after the last addition of fertilizer. Dry weights of the soil were obtained gravimetrically (105~ and organic matter content was estimated as loss on ignition (LOI, 650*(3). Total bacterial numbers were estimated using the acridine orange (AO) method [19], as modified by Clarholm and Rosswall [4], and metabolically active bacteria with the fluorescein diacetate (FDA) staining method [ 15]. Nematodes present in the soil samples were extracted using a Seinhorst elutriator.
Results and Discussion Figures I A and B show that there were increased levels o f both predatory and endoparasitic fungi with increased levels of organic material (Table 1). The increased numbers of propagules were parelleled with increased numbers of nematodes (Fig. 1C). There was a significant increase in FDA-active bacterial numbers along with increasing nematode levels (Fig. ID). The soils rich in nematodes also contained high total numbers of bacteria (Fig. 111)). Thus, as
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expected, large a m o u n t s o f f a r m y a r d m a n u r e resulted in increased a m o u n t s o f organic matter, a n d n u m b e r s o f bacteria, n e m a t o d e s , a n d b o t h p r e d a t o r y a n d endoparasitic fungi. T h e r e are considerable p r o b l e m s in d e t e r m i n i n g the levels o f n e m a t o p h a g o u s fungi in soil. M e t h o d s n o r m a l l y used for e s t i m a t i o n o f fungal b i o m a s s c a n n o t be used for these fungi because o f their c o m p a r a t i v e l y low s a p r o p h y t i c ability [5]. Even if it would be possible to estimate the h y p h a l lengths, this would give a p o o r picture o f the n e m a t o d e - d e s t r o y i n g activity o f the fungi in soil, since a h e a v y mycelial d e v e l o p m e n t is not necessarily correlated with a high predaceous activity [11, 12, 13] or a high m o r p h o g e n i c response [9, 17, 181. This lack o f correlation between n e m a t o d e - d e s t r o y i n g activity a n d h y p h a l lengths is p e r h a p s m o s t o b v i o u s in the e n d o p a r a s i t e s t h a t spend their entire vegetative lives within the n e m a t o d e body. In the detection plates, therefore, living n e m a todes are a necessary addition [7, 20], since they stimulate the f o r m a t i o n o f the particular d e v e l o p m e n t a l p h a s e responsible for the n e m a t o d e - d e s t r o y i n g activity. T h e m e t h o d s described here are no d o u b t v e r y t i m e - c o n s u m i n g because o f the intense m i c r o s c o p y work. T h e scoring o f plates for presence or absence o f n e m a t o p h a g o u s fungi only, w i t h o u t identification o f individual genera a n d species, however, reduced the e x a m i n a t i o n t i m e considerably. Nevertheless, the m e t h o d s described here to quantify n e m a t o p h a g o u s fungi in soil should give new insight into the ecology o f these fungi. Acknowledgments. We thank Drs. E. B~t~tthand B. S~Sderstrrm for helpful discussions. This study
was supported by grants (to BN-H) from the Swedish Natural Science Research Council.
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12. Hayes WA, Blackburn F (1966) Studies on the nutrition ofArthrobotrys oligospora Fres. and A. robusta Dudd. II. The predaceous phase. Ann Appl Biol 58:51-60 13. Jansson HB (1982) Predacity by nematophagous fungi and its relation to the attraction of nematodes. Microb Ecol 8:233-240 14. Klemmer HW, Nakano RY (1964) A semiquantitative method of counting nematode-trapping fungi in soil. Nature 203:1085 15. Lundgren B (1981) Fluorescein diacetate as a stain of metabolically active bacteria in soil. Oikos 36:17-22 16. Mankau R (1975) A semiquantitative method for enumerating and observing parasites and predators of soil nematodes. J Nematol 7:119-122 17. Nordbring-Hertz B (1973) Peptide-induced morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora. Physiol Plant 29:223-233 18. Pramer D, Kuyama S (1963) Symposium on biochemical bases of morphogenesis in fungi. II. Nemin and the nematode-trapping fungi. Bacteriol Rev 27:282-292 19. T.roUdenier G (1972) Fluoreszenz mikroskopische Z~ihlungyon Bodenbakterien. I. Historischer Uberblick und Beschreibung eines Verfahrens zur Z~hlung yon Bodenbakterien in Trockenpr~iparaten nach F~irbung mit Acridinorange. Zentrbl Bakt Hyg Abt II 127:25-40 20. Wyborn CHE, Priest D, Duddington CL (1969) Selective technique for determination of nematophagous fungi in soils. Soil Biol Biochem 1:101-102
