Journal of Nematology 26(3):304-307. 1994. © The Society of Nematologists 1994. Effect of Hirsutella rhossiliensis on Infection of by

P. TIMPER AND B. B. BRODIE I

Abstract: We evaluated the ability of the -pathogenic fungus Hirsutella rhossiliensis (Deu- teromycotina: Hyphomycetes) to reduce root penetration and population increase of Pratylenchus penetrans on potato. Experiments were conducted at 24 C in a growth chamber. When were placed on the soil surface 8 cm from a 14-day-old potato cutting, the fungus decreased the number entering roots by 25%. To determine the effect of the fungus on population increase after the nematodes entered roots, we transplanted potato cuttings infected with P. penetrans into Hirsu- tella-infested and uninfested soil. After 60 days, the total number of nematodes (roots and soil) was 20 -+ 4% lower in Hirsutella-infested than in uninfested soil. Key words: biological control, Hirsutella rhossiliensis, migratory endoparasite, nematode, nematoph- agous fungus, potato, Pratylenchus penetrans, root lesion nematode, Solanum tuberosum.

Migratory endoparasites, such as Praty- jective of this study was to determine the lenchus spp., are considered difficult tar- effectiveness ofH. rhossiliensis when a plant gets for biological control (16). These is present. We determined the effect of the nematodes spend most, and sometimes all, fungus on nematode penetration of potato of their life cycle within plant roots, where roots and population increase after initial they are believed to be protected from in- root penetration. fection by antagonists that inhabit soil. Consequently, the effectiveness of an an- MATERIALS AND METHODS tagonist as a biological control agent will depend, in part, on the amount of time the General methods: We obtained juveniles nematodes are outside roots. and adults of P. penetrans from alfalfa cal- In annual crops, many Pratylenchus spp. lus culture (15) using the Baermann pie- leave the roots when the plants begin to pan method (9). The nematodes were senesce or are harvested. At the end of the stored at 10 C for 1-3 days before an ex- growing season, from 50-96% of the pop- periment. We reared the entomopatho- ulation has been found in the soil (12,14). genic nematodes Steinernema glaseri and When a new susceptible crop is planted, Heterorhabditis bacteriophora in Galleria mel- the nematodes migrate to and penetrate loneUa (5) and stored them after harvesting the roots. Once within the roots, most in- in distilled water at 23 -+ 5 C. dividuals remain there during the growing A loamy sand (88.7% sand, 4.6% clay, season. MacGuidwin et al. found that ca. and 6.6% silt; pH 5.4 in water) was moist- 80% ofP. scribneri were within the roots of ened, heated to 60 C for 30 minutes to corn and potato during plant growth (12). eliminate most organisms, and air dried. We have been examining the nematode- In one experiment, the loamy sand was pathogenic fungus HirsuteUa rhossiliensis as mixed (1:1) with a medium-fine sand (98% a possible biological control agent of Pra- of grains between 100-500 txm). Dauerju- tylenchus penetrans on potato. In a previous veniles of S. glaseri were infected with H. study, the fungus killed 40% ofP. penetrans rhossiliensis by adding healthy individuals in soil without a host plant (17). The ob- to a 3- to 5-week-old sand culture of the fungus (6) and extracting them 4 days later. A portion of the heat-treated soil was Received for publication 21 May 1993. infested with H. rhossiliensis by mixing the t Research Affiliate and Research Plant Pathologist, USDA ARS, U.S. Plant, Soil and Nutrition Laboratory, Tower Road, infected nematodes into moistened (8% Cornell University, Ithaca, NY 14853. w/w) soil (18). The authors thank M.A. Arevalo-Guerra, B. A. Jaffee, H.K. Kaya, P. MuIlin, and A.J. Sawyer for helpful com- The relative amount of conidia in the ments on the manuscript. soil was determined at least once per ex- 304 Control of Pratylenchus by Hirsutella: Timper, Brodie 305

periment. We assayed conidia in the soil by plant. The plants were watered from be- adding either 3,246 -+ 109 (root penetra- low to avoid dislodging conidia from the tion experiment) or 8,074 -+ 1,098 (popu- conidiophores (13). We removed the lation increase experiment) H. bacterio- plants after 6 days and counted the num- phora to pots containing Hirsutella-infested ber of nematodes in the roots. HirsuteUa soil. The nematodes were extracted 24 conidia were assayed 10 days after potato hours later, and 40/pot were examined at was transplanted into the fungus-infested random for adhering conidia. Acquisition soil. The experiment was repeated three of H. rhossiliensis conidia by this nematode times with 12-13 pots per treatment (in- is correlated with the density in soil (13). fested and uninfested soil). We compared Conidial-assay pots were identical to ex- the number of P. penetrans entering roots perimental pots. The percentage of H. bac- in HirsuteUa-infested and uninfested soil teriophora with conidia was used to com- using analysis of covariance (11). Root pare fungal concentrations among trials weight was considered a covariate because (repetitions of the experiment). of its potential influence on nematode We used Solanum tuberosum clone NY85, penetration. Within a cultivar, root weight a known host for P. penetrans, in all exper- is directly related to root size. More nema- iments (3). Potato cuttings were dipped in todes may penetrate larger root systems RootTone and placed in moist vermiculite than may penetrate smaller root systems. for 4-10 days before being transplanted to Effect of H. rhossiliensis on population in- the experimental pots. Plants were kept in crease: Potato cuttings were grown in 150- a growth chamber at 24 C under 15 hours cm 3 pots filled with medium-fine sand for light/day (318 p~E/second/m2). We applied 10-15 days before 1,925 -+ 282 P. penetrans deionized water when the soil surface was were pipetted onto the sand surface. After dry and liquid fertilizer (23-19-17) once 4 days, we removed the plants, rinsed the per week. Deionized water was used be- roots, and transplanted them into 13-cm-d cause we were concerned that the heavily pots containing 925 cm 3 of HirsuteUa- chlorinated tap water would suppress infested (5.0 -+ 0.1 infected S. glaseri/cm ~) sporulation of H. rhossiliensis conidia. At or uninfested loamy sand. The number of the end of an experiment, roots were P. penetrans in the roots at the time of rinsed free of soil, weighed, stained with transplanting was 800 + 142 (N = 6 acid fuchsin-lactoglycerol (2), and homog- plants/trial). At 30 days, we added 3,050 + enized in a blender at high speed for 30 180 healthy S. glaseri to HirsuteUa-infested seconds (1). Root debris was separated pots to maintain the fungus at a high level. from the nematodes using a 250-p,m sieve. Healthy S. glaseri were also added to unin- We adjusted the nematode suspension to a fested pots to serve as a control for any volume of 50 ml and averaged the number influence they might have on the popula- of stained P. penetrans counted in three tion of P. penetrans. At 60 days, we ex- 1-ml aliquots. Extraction efficiency of tracted P. penetrans from 100 cm 3 of bulk nematodes from roots was 80%. soil, and from the total rhizosphere soil Effect of H. rhossiliensis on root penetra- (trials 2 and 3) and roots. The rhizosphere tion: We added 520 cm 3 of a sand:soil mix- soil was collected by first gently shaking ture (1:1) to 1,055-cm 3 rectangular pots (L excess soil from the roots and then soni- x W × H = 15 × 10.5 × 6.7 cm). The soil cating the roots with the adhering soil in a was either infested with Hirsutella (4.3 +- plastic bag containing 250 ml water for 1 0.2 infected S. glaseri/cm 3) or uninfested. minute. Nematodes were extracted from We transplanted a 5-day-old potato cutting bulk and rhizosphere soil by wet sieving 4 cm from one end of the pot and, after a through 250- and 38-p.m sieves followed 9-day incubation, added 2,944 -+ 113 P. by centrifugal flotation (8). Extraction ef- penetrans to the pots. The nematodes were ficiency of P. penetrans from soil was 42%. applied on the soil surface 8 cm from the The soil was assayed for H. rhossiliensis 306 Journal of Nematology, Volume 26, No. 3, September 1994

conidia at 10, 30, and 60 days. There were 7000 five pots per assay day, except for the final assay, when weadded the H. bacteriophora '~ [] Hirsutella to all Hirsutella-infested pots 1 day before [] No Hirsutella ~b 5000 harvest. The experiment was repeated I three times with 10-13 replicate pots per ~- treatment (infested and uninfested soil), a/ We performed a two-way analysis of vari- ~ 3000 ance to determine the effects of H. rhossi- E liensis and trial on the population of P. pe- ~, netrans. 1000

RESULTS Soil Root Total Effect of H. rhossiliensis on root penetra- FIG. 1. Pratylenchuspenetrans in soil and roots of tion: The results from the three trials were potato after 60 days in the presence and absence of combined into a single analysis because the Hirsutella rhossiliensis. Columns are the means ± SE per pot (925 cms soil) of 35 replicates per treatment difference between Hirsutella-infested and (pooled from three experimental trials). Columns uninfested soil did not vary among trials with an asterisk are different from the control (P (treatment x trial interaction, P = 0.42). 0.05). The number of nematodes within roots at the There were 25% fewer nematodes (P = start of the experiment was 800 -+ 142. The percent- age of H. bacteriophorawith at least one H. rhossiliensi.~ 0.01) in roots from HirsuteUa-infested (403 conidium adhering to their cuticle was 59 ± 12 (day -+ 33, ~ + SE) compared with uninfested 10), 56 ± 13 (day 30), and 30 ± 8 (day 60). pots (539 -+ 38). At the conclusion of the experiment, the potato roots had reached from pots without the fungus. We found the end of the pot opposite the plant. Root no Hirsutella-infected nematodes within weight influenced penetration (P = the roots. The percentage of assay nema- 0.003), with more nematodes entering todes with at least one conidium adhering larger than entering smaller root systems. to their cuticle was 59 +- 12 (day 10), 56 +- Only a few Hirsutella-infected nematodes 13 (day 30), and 30 + 8 (day 60). (2 + 1) were observed within the roots. In- fected nematodes were identified by the DISCUSSION presence of hyphae protruding from their cuticle. The percentage of assay nema- Older plants are generally more tolerant todes with at least one conidium adhering to nematode damage than are younger to their cuticle was 75 -+ 5. plants (4); therefore, the critical time to Effect of H. rhossiliensis on population in- protect plants from nematode infection is crease." Because the difference between the when they are young. We found that H. Hirsutella-infested and uninfested pots was rhossiliensis reduced the number of P. pe- similar among the trials (treatment × trial netrans entering roots of young potato interaction, P = 0.6), we combined the tri- plants by 25%. The effect of the fungus on als into a single analysis. The fungus re- root penetration is dependent on the dis- duced (P = 0.04) the total number of tance the nematode has to move through nematodes per pot by 20 + 4% after 60 HirsuteUa-infested soil to reach a root and days. Most P. penetrans were in the roots on the density of infective conidia. In our (Fig. 1); only 23 +- 2% were in the soil (bulk study, the distance the nematodes moved + rhizosphere soil). The number of nema- to penetrate the roots is unknown. The todes in soil was similar in pots with and roots grew continuously throughout the without the fungus. However, fewer experiment and had extended past the nematodes were found in roots from pots point of nematode application by the end containing H. rhossiliensis (P = 0.05) than of the experiment. The density of H. rhos- Control of Pratylenchus by Hirsutella: Timper, Brodie 307 siliensis that we tested is within the high penetrans. M.S. thesis, Cornell University, Ithaca, New range of densities found in naturally in- York. 2. Bridge, J., S. Page, and S. Jordon 1982. An im- fested field soils (13). Higher densities may proved method for staining nematodes in roots. Re- be achieved by adding formulated hyphae port of the Rothamsted Experiment Station for 1981 along with the potato-seed piece at plant- Part 1:171. ing. Alginate pellets containing macerated 3. Brodie, B. B., and R.L. Plaisted. 1993. Resis- tance in potato to Pratylenchus penetrans. Journal of H. rhossiliensis hyphae suppressed the Nematology 25:466-471. number of schachtii infecting 4. Dropkin, V.H. 1989. Introduction to plant cabbage roots by up to 95% in small vials nematology. New York: Wiley. 5. Dutky, S. R., J. V. Thompson, and G. E. Cant- (10). well. 1964. A technique for the mass propagation of Reduction in the population of P. pene- the DD-136 nematode. Journal of Insect Pathology trans during the growing season of the cur- 6:417-422. rent crop will result in lower nematode 6. Jaffee, B. A., A. E. Muldoon, R. Phillips, and M. Mangel. 1990. Rates of spore transmission, mortality, densities for the next susceptible crop. and production for the nematophagous fungus Hir- When we tested the effect of H. rhossiliensis sutella rhossiliensis. Phytopathology 80:1083-1088. on the nematode population after they 7. Jaffee, B. A., and E. I. Zehr. 1985. Parasitic and had entered potato roots, we documented saprophytic abilities of the nematode-attacking fun- gus Hirsutella rhossiliensis. Journal of Nematology 17: a 20% suppression of the nematode pop- 341-345. ulation by the fungus after 60 days. A 8. Jenkins, W.R. 1964. A rapid centrifugal- small proportion of P. penetrans were flotation technique for separating nematodes from found in the soil. Nematodes that leave soil. Plant Disease Reporter 48:692. 9. Kable, P. F., and W. F. Mai. 1968. Influence of roots during plant growth may eventually soil moisture on Pratylenchus penetrans. Nematologica reenter the roots or remain outside and 14:101-122. feed ectoparasitically (19). Some of the in- 10. Lackey, B. A., A. E. Muldoon, and B. A. Jaf- dividuals that entered the soil in Hirsutella- fee. 1993. Alginate pellet formulation of HirsuteUa rhossiliensis for biological control of plant-parasitic infested pots may have become infected by nematodes. Biological Control 3:155-160. the fungus and died before reentering the 11. Lentner, M., and T. Bishop. 1993. Experimen- roots. An alternative explanation may be tal design and analysis. Blacksburg, VA: Valley Book that the fungus killed some of the nema- Co. 12. MacGuidwin, A. E. 1989. Distribution of Pra- todes within the roots. However, no in- tylenchus scribneri between root and soil habitats. Jour- fected nematodes were observed within nal of Nematology 21:409-4 15. the roots, and H. rhossiliensis can not grow 13. McInnis, T. M., and B. A. Jaffee. 1989. An as- saprophytically on or in unsterilized roots say for HirsuteUa rhossiliensis spores and the impor- tance of phialides for nematode inoculation. Journal (7). of Nematology 21:229-234. Our results demonstrate the potential of 14. Olthof, T. H. 1986. Reaction of six Solanum tu- H. rhossiliensis for reducing root penetra- berosum cultivars to Pratylenchus penetrans. Journal of tion and population increase of P. pene- Nematology 18:54-58. trans on potato. A weakness of our study 15. Riedel, R. M., and J. G. Foster. 1970. Mon- oxenic culture of Ditylenchus dipsaci and Pratylenchus was that we killed most of the resident or- penetrans with modified Krusberg's and White's me- ganisms in the soil before adding the fun- dia. Plant Disease Reporter 54:251-254. gus. We don't know what influence these 16. Stirling, G. R. 1991. Biological control of plant organisms may have had on H. rhossiliensis. parasitic nematodes: progress, problems and pros- pects. Wallingford, England: C.A.B. International. Further research should be done to deter- 17. Timper, P., and B. B. Brodie. 1993. Infection mine the ability of the fungus to control of Pratylenchus penetrans by nematode-pathogenic the nematode under field conditions and fungi. Journal of Nematology 25:297-302. to enhance its effectiveness. 18. Timper, P., H. K. Kaya, and B. A.Jaffee. 1991. Survival of entomogenous nematodes in soil infested with the nematode-parasitic fungus Hirsutella rhossi- liensis (Deuteromycotina: Hyphomycetes). Biological LITERATURE CITED Control 1:42-50, 19. Zunke, U. 1990. Ectoparasitic feeding behavior 1. Arevalo-Guerra, M. A. 1990. Effects of selected of the root lesion nematode Pratylenchus penetrans on marigold cultivars on penetration, development, re- root hairs of different host plants. Revue de N6ma- production, infectivity and egression of Pratylenchus tologie 13:331-338.