Journal of Nematology 26(2): 152-161. 1994. © T h e Society of Nematologists 1994. Parasitism of Nematodes by the Fungus Hirsutella rhossiliensis as Affected by Certain Organic Amendments 1 g. A. JAFFEE,2 H. 17ERRIS, 2 J . J . STAPLETON, 3 M. V. K. NORTON, 4 AND A. E. MULDOON 2 Abstract: Experiments were conducted to determine whether the addition of organic matter to soil increased numbers of bacterivorous nematodes and parasitic activity of the nematophagous fungus HirsuteUa rhossiliensis. In a peach orchard on loamy sand, parasitism of the plant-parasitic nematode Criconemella xenop~ by H. rhossiliensis was slightly suppressed and numbers of C. xenoplax were not affected by addition of 73 metric tons of composted chicken manure/ha. In the laboratory, numbers of bacterivorous nematodes (especially Acrobeloides spp.) and fungivorous nematodes increased but parasitism of nematodes by H. rhossiliensis usually decreased with addition of wheat straw or cornposted cow manure to a loamy sand naturally infested with H. rhossiliensis. These results do not support the hypothesis that organic amendments will enhance parasitism of nematodes by H. rhossiliensis. Key words: bacterivorous nematode, biocontrol, biological control, CriconemeUa xenoplax, densityd e p e n d e n t parasitism, fungivorous nematode, HirsuteUa rhossiliensis, nematode, nematophagous fungus, organic amendment. Linford et al. (18) reported that addition of c h o p p e d pineapple plants to soil suppressed root-knot nematodes in Hawaiian pineapple fields. They suggested that the a m e n d m e n t s u p p o r t e d s e q u e n t i a l increases in bacteria, bacterivorous nematodes, and nematode-trapping fungi. T h e presumed increase in density of trapping fungi in response to total nematode density was thought to suppress all hosts, including root-knot n e m a t o d e s (trapping fungi generally possess broad host ranges). Although Linford et al. (18) did not use the term d e n s i t y - d e p e n d e n t parasitism, that concept dearly was central to their und e r s t a n d i n g o f the system. Densityd e p e n d e n t parasitism occurs w h e n the probability of a host being parasitized increases with host density (10,22). Cooke (7) and Pramer (19) noted that Linford et al. (18) were too hasty in concluding that parasitism by trapping fungi was influenced chiefly by nematode density. Cooke (8) found little relationship beReceived for publication 23 November 1993. 1 Supported in part by a grant from the California Cling Peach Advisory Board. 2 Department of Nematology, University of California, Davis CA 95616-8668. Statewide Integrated Pest Management Project, Kearney Agricultural Center, University of California, Parlier, CA 93648. 4 University of California, Cooperative Extension, Merced, CA 95340. The authors are grateful to E. M. Noffsinger for identification of nematodes in the field experiment. 152 tween parasitism by trapping fungi and nematode density and concluded that capt u r e d n e m a t o d e s s u p p l e m e n t saprophytism. Linford et al. (18) and Drechsler (9) had assumed that these fungi behave largely as obligate parasites in soil, saprophytism being a laboratory artifact. Cooke (8) p r o p o s e d that endoparasitic f u n g i (fungi that parasitize nematodes with adhesive or ingested spores rather than with traps) d e p e n d e d more on nematodes as a food source than did trapping fungi and thus were more likely to exhibit densitydependent parasitism. His prediction with respect to endoparasitic fungi was supported by a recent study with the fungus Drechmeria coniospora (1). Field observations and laboratory experiments (10,12,13,15) indicate that another endoparasitic fungus, HirsuteUa rhossiliensis, also parasitizes nematodes in a densityd e p e n d e n t manner. T h e spores o f H . rhossiliensis adhere to vermiform nematodes, which are directly penetrated and subsequently killed by the fungus. New spores are produced from the cadaver. T h e fungus has a broad host range, including species of Heterodera and Meloidogyne (4,24, 25), Pratylenchus (24,27), Criconemella (11), Anguina (5), Ditylenchus (4,24), Xiphinema (6), Aphelenchoides (4,24), Steinernema (26), Heterorhabditis (26), Anaplectus (24), and Cephalobus (24). Our objective in the present study was to H. rhossiliensis and Organic Amendments: Jaffee et al. 153 examine the concept of Linford et al. (18) as applied to H. rhossiliensis. We asked, "Does addition of organic matter to soil increase numbers o f bacterivorous nematodes and thus the parasitic activity of H. rhossiliensis ?" MATERIALS AND METHODS Field experiment: The field site was a mature peach (Prunus persica) orchard on 'Nemaguard' rootstock in Merced County, California. T h e characteristics o f the loamy sand, designated M in previous studies, were described elsewhere (11). The soil contained H. rhossiliensis and the plant-parasitic nematode CriconemeUa xenoplax. In previous studies in this orchard, C. xenoplax composed more than 95% of the nematodes parasitized by H. rhossiliensis (11-14). For the current study, 20 trees in one c o r n e r o f the o r c h a r d were selected. Eleven kg of composted chicken manure plus rice hulls (30% moisture and 1.5% nitrogen based on dry weight), equivalent to 73 metric tons of chicken manure/ha, were added to a rectangular plot (1.5 × 1.0 m) on one side of each of 10 trees in April 1988. Each plot was considered a replicate. Composted chicken manure from Foster Farms, Livingston, California, was selected as the a m e n d m e n t because it is commercially available in the San Joaquin Valley of California. The closest side and center of each plot were 30 and 80 cm from the trunk, respectively. After addition to the surface of each plot, the compost was mixed into the soil with a shovel 25-30 cm deep. Plots adjacent to the 10 remaining trees were treated in the same manner, but compost was not added. The experimental design was completely random, with two treatments replicated 10 times. Soil samples were collected 70-80 cm from the trunk and 0-33 cm or 34-66 cm deep with a soil auger (4.5 cm d) before addition o f compost (week 0), and at weeks 3, 10, 30, and 45. Two 700-cm 3 samples were combined per depth and plot. Nematodes were extracted from a 500-cm 3 sub- sample by elutriation (2) and centrifugation in water and then in sucrose (454 g sucrose/liter) (16); a 38-p~m-pore-d sieve was used. Numbers of parasitized and nonparasitized nematodes (almost entirely C. xenoplax and microbivores) were determ i n e d with an agar plate assay using NaOCI, as d e s c r i b e d e l s e w h e r e (11). Nematodes not added to agar plates were fixed in formalin, and the most common nematodes (other than C. xenoplax) in subsamples from three replicate plots per treatment, depth, and sample time were identified to species. The number of colony-forming units of the plant-pathogenic fungus Pythium ultimum was determined in each soil sample (23). The concentrations of NH~ and NO 3 (3,17) and the percentage organic matter were determined in one composite sample per treatment, depth, and sample period by the DANR Laboratory at the University of California at Davis. General procedures for laboratory experiments: The effect of organic amendments on nematodes and parasitism by H. rhossiliensis was examined in three experiments with soil in vials. The soil had properties similar to the loamy sand in the field experiment and was collected from the rhizospheres of twelve 30-year-old almond trees adjacent to the peach trees used in the field e x p e r i m e n t . This soil c o n t a i n e d about 500 H. rhossiliensis-parasitized C. xenoplax/lO0 cm ~ soil (corrected for extraction efficiency), which represented about 50% of the total C. xenoplax population. The soil was screened (2-ram-pore d) to remove large roots, leaves, and almond hulls and stored at 10 C for less than 1 month before use. Additional soil was collected for each experimental trial, because H. rhossiliensis declines in storage at 10 C (11). Composted cow manure plus rice hulls, leaves of woolypod vetch (Vicia pillosa), and wheat straw were obtained from the Student Experimental Farm at the University of California at Davis. Composted chicken manure plus rice hulls was obtained from Foster Farms, Livingston, C a l i f o r n i a . 154 Journal of Nematology, Volume 26, No. 2, June 1994 Amendments were dried and heated at 80 C in a drying oven, ground in a Wiley Mill, and screened (0.83-ram-pore d) before addition to soil. The vetch and cow manure were moist when collected and were dried and heated for 2 days, but the wheat straw and chicken manure, already dry, were heated for only 2 hours. Nematodes were extracted from soil by decanting and sieving (25-v~m-pore d) followed by centrifugation in water and then in sucrose (454 g/liter); nematodes in sucrose were collected on a 25-1xm-pore-d sieve and suspended in 10 ml of water. Laboratory experiment 1: To determine which of the amendments caused the largest increase in microbivorous nematodes, soil was moistened to about 11.0% with distilled water and divided into 20-g samples (18.0 g dry weight soil). One of the four amendments was mixed into each sample (800-mg a m e n d m e n t per sample, equivalent to approximately 70 metric tons/ha). Samples that received no amendment were controls. Addition of dry organic matter affected the water status of the soil, and we attempted to standardize matric potential (based on subjective assessment) by adding distilled water; the percentage moisture was 24, 24, 19, 17, and 14% for the vetch, straw, cow manure, chicken manure, or control, respectively. Each sample was packed (bulk density = 1.2) into a 25-ml plastic vial, which was sealed with a lid having a 0.2-mm-d hole. The vials were placed in a moisture chamber at 20 C. On day 0, 10, 27, 40, and 55, the nematodes from four vials per treatment were extracted, the suspension was adjusted to 10 ml, and 1 ml of suspension was placed in a Hawksley counting slide and examined at 140x magnification. Nematodes with and without spores of H. rhossiliensis attached were counted and separated into trophic groups: bacterivores, fungivores, predators, plant-parasites, and others, but were not identified to species or genus (in contrast to the field experiment and other laboratory experiments). The numbers of C. xenoplax and other nematodes parasitized by H. rhossiliensis also were deter- mined by incubating aliquots of the suspension on agar as described for the field experiment, except that the nematode suspensions were not treated with NaOC1. There were four replicates per treatment per sample time, and the experiment was not repeated. Laboratory experiment 2: Based on the results from the previous experiment, the effect of straw and cow manure on parasitism of nematodes by H. rhossiliensis was exa m i n e d . N o n a m e n d e d soil o r soil amended with straw or cow manure was packed into 25-ml plastic vials and incubated at 20 C as described. Soil moisture was 15, 20, or 25% for the control, manure-amended, or straw-amended soil, respectively. To quantify the parasitic activity of H. rhossiliensis, each vial was inoculated with 600 h e a l t h y s e c o n d - s t a g e juveniles (J2) of Heterodera schachtii in 0.5 ml of 4.5 mM KC1. The J2 were added to the soil surface of four vials per treatment on day 21, 42, 63, 85, and 105, and soil was extracted 66 hours later as in laboratory experiment 1. Because H. schachtii was not naturally present in the soil, all of the H. schachtii recovered were added to the soil and exposed to the fungus for 66 hours. On day 0, four vials per treatment that did not receive H. schachtii were extracted to determine initial nematode numbers. The nematode suspension was adjusted to 10 ml, and 1 ml was examined at 140× magnification to determine the number of nematodes by trophic group and the percentage of H. schachtii J2 and microbivorous nematodes with spores. The numbers of C. xenoplax and other nematodes parasitized by H. rhossiliensis were determined by incubating aliquots of the suspension on agar as in the first laboratory experiment. In addition, the first 20 specimens of microbivorous nematodes in each sample were identified to genus and in some cases to species on day 85 and 105. This experiment was repeated (trials 1 and 2). Laboratory experiment 3: T h e effect of quantity of organic amendment was examined. In the first trial, soil amended with 0, 50, 100, 200, 400, or 800 mg of cow ma- H. rhossiliensis and Organic Amendments: Jaffee et al. 155 nure or straw per vial was packed and incubated at 20 C as in the first laboratory experiment. Soil moisture was 11.0% in u n a m e n d e d soil and 13.6% or 16.3% in soil amended with the highest levels of man u r e a n d s t r a w , r e s p e c t i v e l y . Vials amended with intermediate levels of manure or straw had intermediate levels of soil moisture. On day 65, all vials were inoculated with H. schachtii J2 and extracted 66 hours later. The numbers of microbivorous nematodes and the percentage of H. schachtii J2 and microbivorous nematodes with spores of H. rhossiliensis were determined as in the second laboratory experiment. Twenty randomly selected nematodes per treatment were identified to genus and sometimes to species. There were five replicate vials per treatment. Experiment 2 was repeated (trial 2), except that the levels of amendment were 0, 25, 50, 100, 200, or 400 mg per vial. Statistical analysis: Field data (log transf o r m e d for counts o f n e m a t o d e s a n d fungi, and arcsine transformed for percentages) were subjected to repeatedmeasures analysis of variance with the general linear model procedures of SAS (21). Treatment, time, and the interaction of t r e a t m e n t and time were i n d e p e n d e n t variables for each depth; the effect of depth was determined in a separate analysis. For the laboratory experiments, inferences were based on means and standard errors. RESULTS Field experiment: Numbers of C. xenoplax were unaffected (P > 0.09) by treatment (+ compost), time, or treatment x time, but were fewer (P < 0.01) in the top soil (0-33 cm deep) than at the greater depth (34-66 cm) (Fig. 1A). Numbers of microbivorous nematodes were unaffected (P > 0.08) by treatment, time, or treatment x time but were greater (P < 0.01) in the top soil than below (Fig. 1B). The percentage of C. xenoplax parasitized by H. rhossiliensis was smaller (P = 0.05) in amended soil than in nonamended soil in the top soil but not below; parasitism was greater (P < 0.01) in soil from the lower depth than in the top (Fig. 1C). Parasitized nematodes other than C. xenoplax were rare. In the top soil, numbers of colony-forming units of P. ultimum were suppressed by the amendment. Suppression was affected by time, as indicated by a significant (P < 0.01) interaction of treatment x time (Fig. 1D). In soil from the lower depth, neither treatment nor treatment x time affected P. ultimum (P > 0.16), but time was marginally significant (P = 0.06). In treated plots, NH 4 peaked at week 3 and then declined (Fig. 1E); NO 3 increased to much higher concentrations in the topsoil than in the lower depth and decreased only slowly (Fig. 1F). In untreated plots, NH 4 and NO s concentrations r e m a i n e d low, but NH 4 increased slightly on week 3 (Fig. 1E,F). At week 45, the percentage organic matter in the topsoil was 1.0 and 1.3 in untreated and treated plots, respectively; in the lower depth, the percentage organic matter was 0.5, regardless of treatment. The most common species of microbivorous nematodes were Acrobeloides bodenheimeri and Acrobeles complexus. Other common species were Acrobeloides buetschlii, A. uberrinus, Cervidellus serratus, P anagrolaimus subelongatus, Eudorylaimus monohystera, and Rhabditis spp. Neither treatment nor time greatly affected the species observed, although Panagrolaimus subelongatus, A. ubergnus, and a Rhabditis sp. were encountered s o m e w h a t m o r e c o m m o n l y in p l o t s amended with manure. Laboratory experiment 1: Few bacterivorous and fungivorous nematodes were present on day 0, and they remained low in u n a m e n d e d soil (Fig. 2A,B). In soil a m e n d e d with vetch, no bacterivorous nematodes were observed after day 27. A substantial increase in bacterivorous nematodes occurred between day 27 and 55 in cow manure- and straw-amended soil; a m o d e r a t e i n c r e a s e o c c u r r e d in soil amended with chicken manure. Fungivorous nematodes increased in some vials but not in others amended with straw and cow manure, as indicated by the high variances (Fig. 2B), but remained few in other treatments. Predaceous n e m a t o d e s with or without spores and microbivorous nema- Journal of Nematology, Volume 26, No. 2,June 1994 156 = O 6000 ' ' 4000 0 • -compost +compost 0 -- 33' c m r=_ 8 o ~4 2000 o o 0 6000 I i i i i I 34 - i 34 - 66 cm i 66 cm c. 4000 x 2000 nl 0 I I I I 4 I - 2000 40 1000 "E'20 E o r\ cm 33 () - 33' cm o, Cu 0 o I 0 [ I 34 - 66 c m v z 0 I :Z40 2000 > z 20 1ooo o 0 i I I I ! i i i ' 60 w 0 - I 33' c m 80 "~" 40 × ¢D 20 "o .~ ~ " 4O Q. Q_ 0 I I f I I 34 - S0 66 c m .-- ~ 0 34 - 66 c m 30 40 I,,,.)80 0 Z 40 0 I ~ z 4O 20 I 0 I I I I I 0 10 20 30 40 0 0 10 Week 20 Week F[~. 1. Effect o£ composted chicken manure on numbers of nematodes and fungi and on nitrogen status in a peach orchard. Manure was mixed into the top 25-30 c m o f soil on 14 April 1988. Soil samples were collected from 0-33 cm or from 34-66 cm; the week 0 sample was collected 1 h o u r before incorporation of the manure. A) Numbers of Criconemellaxenoplax(Cx). B) Numbers of microbivorous nematodes. C) Percentage of C. xenoplax parasitized by Hirsutella rhossiliensi~(Hr). D) Numbers of colony forming units of Pythium ultimum. E) Concentrations of NH4-N. F) Concentrations of NO3-N. Values are the means of 10 replicate plots. t o d e s w i t h a d h e r e n t s p o r e s o f H. rhossiliens~ were observed rarely. N u m b e r s o f C. xenoplax p a r a s i t i z e d b y H . rhossilien,sis t e n d e d t o b e g r e a t e r i n t h e c o n trol than in the other c l i n e d i n all t r e a t m e n t s treatments and deover time (Fig. 2C). H. rhossiliens# and Organic A m e n d m e n t s : Jaffee et al. 157 800 -5 °~ CO 600 k_ 0 > 400 "r" 0 o co i v i i i i i straw • C O W manure /• chicken m a n u r e / / [] vetch / / 0 control ~ ~ / ± i i 200 0 600 ~ 63 5oo i i ~ B 400 V L. o > 300 ._ (2n r- 200 b._ 100 0 = : i •-20O > C X O 150 "IO ~9 N .-D L. 100 Q_ 5O I L_ t 0 0 10 20 30 40 50 Day FIG. 2. Numbers of bacterivorous, fungivorous, and parasitized nematodes in soil amended with four kinds of organic matter. A loamy sand naturally infested with Hirsutella rhossiliensis was amended with wheat straw, cow manure, chicken manure, or vetch leaves and was incubated in vials (18 g dry weight soil, 800 mg dry weight organic matter, and 15 cm s soil per vial) at 20 C. A) Numbers of bacterivorous nematodes. B) Numbers of fungivorous nematodes. C) Numbers of H. rhossiliensis- parasitized Criconemella xenoplax (Hr-parasitized Cx). Each value is the mean of four replicate vials; vertical bars = 1 SE. A l t h o u g h species o f n e m a t o d e s o t h e r t h a n C. xenoplax were parasitized by H. rhossiliensis, the n u m b e r was n e v e r g r e a t e r t h a n five p e r vial. Laboratory experiment 2: Results o f b o t h trials were similar, a n d data were combined for presentation. N u m b e r s o f bacterivorous a n d f u n g i v o r o u s n e m a t o d e s rem a i n e d small in u n a m e n d e d soil but increased in soil a m e n d e d with straw or cow m a n u r e (Fig. 3A,B). P r e d a c e o u s n e m a todes were observed rarely. Most ( > 8 4 % ) o f the bacterivorous n e m a t o d e s at day 85 or 105 in straw- or cow m a n u r e - a m e n d e d soil were Acrobeloides spp. (A. bodenheimeri, A. buetschlii, and A. tricornis); the r e m a i n d e r were species o f Cervidellus and Plectus. Fungivorous n e m a t o d e s included Aphelenchus sp. and Aphelenchoides sp. T h e n u m b e r o f C. xenoplax parasitized by H. rhossiliensis was g r e a t e r in u n a m e n d e d than in a m e n d e d soil at time 0 and declined in all treatments over time (Fig. 3C). No species o t h e r t h a n C. xenoplax was parasitized by H. rhossiliensis. T h e p e r c e n t a g e o f H. schachtii J2 with spores o f H. rhossiliensis was greatest in una m e n d e d soil, i n t e r m e d i a t e in strawa m e n d e d soil, and least in cow m a n u r e a m e n d e d soil (Fig. 3D). This p e r c e n t a g e t e n d e d to d e c l i n e with t i m e in strawa m e n d e d soil. Microbivorous n e m a t o d e s with spores were seldom observed. Laboratory experiment 3: In b o t h trials, the n u m b e r s o f bacterivorous n e m a t o d e s after 65 days were greater in soil a m e n d e d with cow m a n u r e t h a n in u n a m e n d e d soil (Fig. 4A,D); Acrobeloides tricornis, A. buetschlii, and A. bodenheimeri c o m p o s e d m o r e t h a n 50% o f these n e m a t o d e s across all treatments. Also present were Diploscapter sp., Panagrolaimus sp., Rhabditis sp., Acrobeles sp., Cruznema sp., Cylindrolaimus sp., Mononchus sp., Tylenchus davainei, a n d Eurodorylaimus sp. N u m b e r s o f f u n g i v o r o u s n e m a t o d e s (Aphelenchoides sp. and Aphelenchus sp.) also were greater with addition o f cow m a n u r e , especially in trial 1 (Fig. 4B,E). Numbers of bacterivorous nematodes usually w e r e less in soil a m e n d e d with straw than in soil a m e n d e d with cow man u r e but were g r e a t e r than in u n a m e n d e d soil (Fig. 4 A , D ) ; as in cow m a n u r e a m e n d e d soil, A. tricornis, A. buetschlii, and A. bodenheimeri c o m p o s e d m o r e than 50% 158 Journal of Nematology, Volume 26, No. 2, June 1994 1000 i 0 -6 •>- i i i i I I control 750 I I I X 60 0 ID N t.. o I 8O > 500 t~ 40 > o-- k_ o L 250 ~O.. 20 o3 I 7- I 200 i i i i i 5O o~ 15o -9 100 i i i V i t D 4-0 ~ 30 I -i- 2O ~D E u_ ~ O4 ID k. o > V i B "6 ~ 0 r" 50 ~10 0 0 0 20 40 80 80 I00 120 DGy 0 20 40 60 80 100 20 Ooy FIG. 3. Effect o f straw and cow m a n u r e on n u m b e r s o f microbivorous nematodes and parasitism o f nematodes by Hirsutella rhossiliensis over time. A loamy sand naturally infested with H. rhossiliensis was a m e n d e d with organic matter and incubated in vials (18 g dry weight soil, 800 nag dry weight organic matter, and 15 cm 3 soil p e r vial) at 20 C. A) N u m b e r s o f bacterivorous nematodes. B) N u m b e r s o f fungivorous nematodes. C) N u m bers o f H. rhossiliensis-parasitized Criconemella xenoplax (Hr-parasitized Cx). D) Percentage o f Heterodera schachtii juveniles (Hs J2) with spores o f H. rhossiliensis. Data were combined from two trials. Each value is the m e a n o f eight replicate vials; vertical bars = 1 SE. o f the bacterivorous n e m a t o d e s across all t r e a t m e n t s receiving straw. O t h e r species p r e s e n t were similar to those in cow man u r e - a m e n d e d soil, except that species o f Acrobeles, P anagrolaimus, and Cylindrolaimus were not observed. N u m b e r s o f fungivor o u s n e m a t o d e s (Aphelenchoides sp. a n d Aphelenchus sp.) were correlated with the a m o u n t o f straw a d d e d in trial 2 (Fig. 4E) but not in trial 1 (Fig. 4B). In trial 1, the p e r c e n t a g e o f H. schachtii J2 with spores increased with addition o f low quantities o f cow m a n u r e o r straw and t h e n d e c r e a s e d as either a m e n d m e n t increased (Fig. 4C). In trial 2, only a marginal increase in parasitism o c c u r r e d with low levels o f a m e n d m e n t (Fig. 4F); as in trial 1, parasitism was inversely correlated with quantity o f a m e n d m e n t w h e n level o f a m e n d m e n t e x c e e d e d 100 m g for straw or 50 m g f o r cow m a n u r e . M i c r o b i v o r o u s n e m a t o d e s with spores were seldom observed. N e m a t o d e s may have b e e n i n t r o d u c e d to the soil with the organic a m e n d m e n t s , even t h o u g h the a m e n d m e n t s were h e a t e d to 80 C. Based on observation o f nematodes f r o m u n a m e n d e d soil in e x p e r i ments 2 and 3, the following n e m a t o d e s were naturally present: Criconemella xeno- plax, Acrobeloides bodenheimeri, A. buetschlii, A. tricornis, Aphelenchoides sp., Aphelenchus sp., Cruznema sp., Cylindrolaimus sp., Mononchus sp., Rhabditis sp., and Tylenchus davainei. DISCUSSION Organic amendments usually suppressed r a t h e r than stimulated parasitism o f n e m a t o d e s by H. rhossiliensis in the field H. rhossiliensis a n d O r g a n i c A m e n d m e n t s : Jaffee et al. trial i 600 i trial 1 i i i i A i 159 2 i i i i i i D 400 "~ 500 300 "~ 400 o 300 200 monure o ¢~ 2 0 0 c~ m~ 1 0 0 100 0 I I 100 400 200 80 o 60 1 I I I I 100 200 300 40 4o a_ 2o ~'~'~ T 0 ,I I 100 i i T ~ i I i i i 20 0 100 F C 80 80 u] L. c~ o ' 60 °_ ~ I 80 "~ ~o 0 T 50 60 CO 40 Z c- ~ 2O 20 O 0 200 400 Straw 600 or 800 manure , 0 added 400 (rag/vial) Fzo. 4. Effect of quantity of straw and cow manure amendments on numbers of microbivorous nematodes and parasitism of nematodes by Hirsutella rhossiliensis. A loamy sand naturally infested with H. rhossiliensis was amended with organic matter and incubated in vials (18 g dry weight soil and 15 cm 3 soil per vial) for 65 days at 20 C. The experiment was performed twice (trials 1 and 2). A,D) Numbers of bacterivorous nematodes. B,E) Numbers of fungivorous nematodes. C,F) Percentage of Heteroderaschachtiijuveniles (Hs J2) with spores of H. rhossiliensis (Hr). Each value is the mean of five replicate vials; vertical bars = 1 SE. a n d in t h e l a b o r a t o r y . T h e m e c h a n i s m o f s u p p r e s s i o n was n o t c l e a r , h o w e v e r , a n d e x p e r i m e n t s l i k e t h e s e a r e d i f f i c u l t to interpret because the amendments can affect m a n y p r o c e s s e s . I n a d d i t i o n to s t i m u l a t i n g antagonists of nematodes via densityd e p e n d e n t p a r a s i t i s m (18), o r g a n i c m a t t e r may be the source of ammonia and other nematicidal and fungicidal compounds (20); m a y a l t e r soil p o r o s i t y a n d soil w a t e r and thus influence several aspects of the b i o l o g y o f n e m a t o d e s a n d H. rhossiliensis (25); and may stimulate fungivorous nematodes and other antagonists of fungi. 160 Journal of Nematology, Volume 26, No. 2, June 1994 Suppression of P. ultimum in the field experiment demonstrated that the suppression was not specific to H. rhossiliensis. Low quantities of organic amendments did appear to stimulate parasitism in one laboratory trial (Fig. 4C). The stimulation, however, was not reproducible (Fig. 4E). Perhaps by affecting several processes simultaneously, the organic amendments both enhanced and suppressed parasitism, and the net effect was suppression unless levels o f organic matter were low. In two laboratory experiments, amendments decreased the number of parasitized C. xenoplax detected with the agar plate assay at time 0, suggesting a direct effect on extraction. Many parasitized C. xenoplax detected with this assay had hyphae growing from their bodies, and such external hyphae could become entwined or otherwise interact with organic matter during extraction. External hyphae would not be a problem with the second and more important quantification procedure (in which healthy H. schachtii J2 were added to soil and recovered and examined for spores after 66 hours) because these nematodes are not in soil long enough for growth of external hyphae. We have no evidence that organic matter differentially affected the extraction of H. schachtii J2 with and without spores. Inadequate information on the many possible influences of organic matter and on the host status ofbacterivorous nematodes limits understanding of the results. Amendments enhanced numbers of bacterivorous nematodes in the laboratory experiments, but even though the host range of H. rhossiliensis is broad, one cannot assume that all bacterivorous nematodes are hosts. Some species o f bacterivorous nematodes acquire spores but fail to become infected (P. Timper, pers. comm.). Thus, the probability of suppression would be reduced. In the present study, however, we observed few microbivorous nematodes with spores. Organic matter may have stimulated H. rhossiliensis, had experimental conditions been different. For example, the soil used in this study already contained high levels of H. rhossiliensis, and enhancement of this fungus may have occurred had the initial levels been lower. Other kinds of organic matter and different soils also may have provided more encouraging results. 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