Journal of Nematology 22(4):567-573. 1990. © The Society of Nematologists 1990.

Antagonists of Plant-parasitic Nematodes in Florida Citrus 1

DAVID EVANS WALTER AND DAVID T. KAPLAN 2

Abstract: In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were re- covered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyceslilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and DactyleUa ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous also were detected. Nematode-trapping fungi and nematophagous ar- thropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare. Key words: biological control, burrowing nematode, citrus nematode, nematophagous , nematophagous fungus, Pasteuria, Streptomyces.

Nematode control in citrus has relied ida for the presence of antagonists to plant- heavily upon nematicides that are highly parasitic nematodes. water soluble, toxic to vertebrates, and persistent (I 3). In addition to nematicides, MATERIALS AND METHODS resistant rootstocks have been used to limit Sampling: Randomly selected 10 x 10 losses associated with citrus (Tylenchulus tree blocks in 27 citrus groves, each with semipenetrans Cobb) and burrowing (Ra- a history of citrus nematode infestation, in dopholus citrophilus Huettel et al.) nema- Marion, Lake, Seminole, Orange, Brevard, todes. These rootstocks are not always hor- Polk, Hardee, Highlands, St. Lucie, and ticulturally acceptable, however, and some Dade counties in Florida were sampled. have limited tolerance to cold and disease. The presence of citrus nematode was con- Furthermore, occurrence of resistance- firmed in 25 of the groves surveyed. Nine breaking nematode populations in the field groves were sampled by bulking cores from and variability in citrus rootstock germ- three to five trees. In each of 18 groves, plasm suggest that existing rootstocks re- five individual trees older than 10 years quire improvement before they can be re- were sampled. Each sample consisted of lied upon to limit nematode-related yield three cores (7 cm d, 15-30 cm deep) of losses (13,16). citrus fibrous roots exposed by removing Improved understanding of the identity surface soil at equidistant locations midway and function of antagonists will be neces- between the trunk and dripline of each tree. sary if they are to be used as management Tools were disinfected with 0.5% NaOCI tools of plant-parasitic nematodes (14,24). between samples. Samples were placed in This paper reports the results of a survey polyethylene bags in ice chests and pro- of citrus groves and other habitats in Flor- cessed the same or the next day. Seventeen noncitrus habitats in Orange, Seminole, Lake, and Brevard counties also Received for publication 23 January 1990. were sampled, including soil and litter from Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. De- a composted garden, rotted horse manure, partment of Agriculture and does not imply its approval to beach wrack, a rotted palm tree, a carrot the exclusion of other products or vendors that may also be field muck soil, and rhizosphere soil from suitable. U.S. Department of Agriculture, Agricultural Research stands of live oak, pine, palmetto, banana, Service, 2120 Camden Road, Orlando, FL 32803. We thank D. Johnson for maintaining cultures of burrow- broomsedge, anthurium, and impatiens. ing nematode. Samples of citrus leaf litter (normally not 567 568 Journal of Nematology, Volume 22, No. 4, October 1990 included in samples) from two sites (Ona 1% WA in 9-cm petri dishes that were in- and Apopka, FL) also were evaluated for cubated at 25 C. Roots from bulked sam- the presence of nematode antagonists. ples and noncitrus habitats were not pro- Isolation of nematophagous microbes: Citrus cessed. roots from each tree or bulked sample were Bait and root incubation plates were ex- gently rolled to dislodge rhizosphere soil, amined with a compound inverted light mi- root fragments, and citrus nematode egg croscope at 100-400 magnification at 1- masses. The dislodged material was passed 3-day intervals for up to 6 weeks. Fungi through a I-ram-pore sieve and collected were identified using keys (1,5,11,17,18, on a 0.25-mm-pore sieve. The screens were 20,23) and original descriptions. Spores or then tapped to remove very fine sand grains infected nematodes were transferred with that interfered with observation. The a flamed wire to an appropriate medium. screened rhizosphere material (0.25 g) was Most Eumycota were cultured on corn divided into equal subsamples and sprin- meal, yeast malt agar, or potato dextrose kled on two 9-cm petri dishes, each con- agar. Streptomycetes were cultured on taining 4 ml solidified 1.0% water agar tryptic soy agar. Chytridiomycetes were (WA). Between samples, sieves were washed cultured on YpSs agar (9). Most other en- in soapy water, disinfected with 1.0% doparasites could be maintained in bur- NaOCI, rinsed with tap water, and dried rowing or rhabditid nematodes on 1% WA with compressed air. Soil, litter, and root plates at 25 C. Nematode cadavers infected fragments from the noncitrus samples and with microbes that could not be identified two citrus litter samples were plated on four were transferred with a flamed wire to 1% replicate WA plates. Because these samples WA plates with fresh burrowing nema- were not comparable in composition and todes from monoxenic cultures to deter- moisture content to the citrus samples, ma- mine if further infection occurred. terial was added to each plate to cover a Isolation of predators: Ten citrus groves surface area similar (by visual estimation) were surveyed for nematophagous arthro- to that obtained with citrus root samples. pods. Five citrus groves and six noncitrus Each dish was inoculated with 5,000 Ra- sites were surveyed for predatory nema- dopholus citrophilus (sometimes supple- todes. Nematophagous arthropods were mented with R. similis) in 0.5 ml water and extracted from 100-200 cm ~ soil in stan- then incubated at 25 +- 1 C. Bait nema- dard TuUgren funnels over 70% ethanol. todes were obtained from monoxenic car- Predatory nematodes were obtained from rot disk cultures extracted enzymatically 180 cm s soil in Baermann funnels. Other (15). At five sites the efficacy of burrowing predatory including amoebae, nematode as a bait organism was evaluated nematodes, and arthropods were obtained by baiting one of each pair of plates with from bait plates and root incubations. Live 5,000 burrowing nematodes, and the other arthropods were tested for predation on R. plate in the pair with 5,000 Rhabditis sp., citrophilus in small arenas (27). which had been isolated from citrus roots, cultured on pea soup agar, and extracted RESULTS by Baermann funnel (2). Microbial antagonists: Approximately 24 After removal of rhizosphere material microbial species antagonistic to vermi- for the sprinkle-bait plates, citrus roots form stages of burrowing nematode were from 18 groves (five samples per grove) isolated from bait plates, and 12 microbial were incubated in jars at 25 C (28) for 5- species were observed attacking citrus 7 days. Subsequently, the roots were rinsed nematodes from root incubations (Table in tap water and the nematode suspension 1). In addition, Paecilomyces lilacinus was centrifuged at 175 g for 5 minutes. All (Thom.) (Samson), P. marquandii (Massee) but 1 ml supernatant was aspirated, and (Hughes), and VerticiUium chlamydosporium the pellet was dispersed and pipetted onto Goddard (also isolated from vermiform Antagonists of Phytonematodes: Walter, Kaplan 569

TABLE 1. Microbial antagonists of vermiform stages of phytonematodes recovered from the rhizospheres of 27 citrus groves and from 19 samples of soil and litter from other habitats in Florida.

Infection']" Tylen- Radoph- chulus Number of positive sites:[: olus semi- citroph- pene- Frequency§ Species Mode of infection ilus trans Citrus Other (Mean + SE)

Arthrobotrys dactyloides Drechsler Constricting ring + + 20 3 0.44 + 0.06 Catenaria anguiUulae Sorokine Zoospore + + 19 10 0.68 + 0.08 Arthrobotrys oligospora Fresenius Adhesive network + + 19 3 0.59 + 0.07 Streptomyces sp. ?Spore + + 19 3 0.35 + 0.08 Dactylella eUipsospora Grove Adhesive knob + + 18 11 0.41 + 0.10 DactyleUa cionapaga Drechsler Adhesive hyphae + + 18 4 0.57 + 0.08 Rhizophidium sp. Zoospore + + 12 7 0.37 - 0.08 Arthrobotrys musiformis Drechsler Adhesive network + + 12 1 0.32 + 0.08 Dactylaria eudermata Drechsler Adhesive network + - 9 6 0.10 + 0.05 Myzocytium spp. Zoospore + + 5 4 0.14 + 0.06 Unidentified lagenidioid Zoospore + - 5 3 0.13 + 0.06 Dactylaria nr, candida (Nees) Sacc. Adhesive knob & + + 4 0 0.02 + 0,02 nonconstricting ring Dactylaria haptospora Drechsler Adhesive knob & + - 1 1 0.01 _+ 0.01 spore Haptoglossa heterospora Drechsler Spore + - 1 1 0.01 + 0.01 Pasteuria (Thorne) Sayre & Starr Spore - + 1 0 0.06 + 0.17 Dactylaria sclerohypha Drechsler Adhesive knob + - 1 0 0.01 _+ 0.01 VerticiUium spp. Spore + - 0 4 Gonimochaete horridula Drechsler Adhesive spore + - 0 2 Gonimochaete latitubus Newell, Adhesive spore + - 0 1 Cefalu & Fell Dactylella phymatopaga Drechsler Adhesive knob + - 0 1 DactyleUa bembicoides Drechsler Constricting ring + - 0 4 Dactylaria brochopaga Drechsler Constricting ring + - 0 2 t Host response: + = infections observed; - = infections not observed, For all 27 citrus groves and 19 other samples irrespective of sampling effort. § Mean proportion citrus rhizospheres positive for antagonist, based on combined bait plate and root incubation data from 18 groves from which five replicate samples were taken. burrowing nematodes) were observed Nematode-destroying fungi were com- growing from citrus nematode egg masses mon in the citrus rhizosphere, but only 17 that included hypha-filled eggs. Strepto- species were collected from 27 citrus sites myces sp., Arthrobotrys oligospora Fresenius, as opposed to 26 species from 19 noncitrus and DactylelIa ellipsospora Grove also were rhizosphere sites (including two samples of observed growing from citrus nematode citrus litter). Within-site diversity (based on egg masses, but only vermiform stages sprinkle-bait plates only) averaged 5.8 + within the egg masses appeared to be at- 0.4 species/site in the citrus rhizosphere, tacked. Harposporium anguilullae Lohde, H. but only 4.1 + 0.4 species/site in other helicoides Drechsler, and Nematoctonus lepto- habitats (P < 0.01, Mann-Whitney test). sporus Drechsler were isolated from rhab- Plots of cumulative species number against ditid nematodes, but never from burrow- cumulative sample number (species area ing nematodes. Mean number of species of curve) indicated that the isolation of new microbial antagonists detected on plates species reached an asymptote in the citrus baited with burrowing nematodes (3.0 +- groves, but not in the noncitrus habitats. 0.3) was greater than on plates baited with Twelve species of nematode-trapping Rhabditis sp. (1.6 __+ 0.3) (P < 0.05, Wil- fungi, representing all major trap types, coxon signed ranks test). were isolated during this study. Every cit- 570 Journal of Nematology, Volume 22, No. 4, October 1990

TABLE 2. Predators of nematodes recovered from citrus rhizosphere soil in Florida citrus groves.

Congeners or closely related species reported Fed on to be nema- nematodes tophagous Nematodes in in vitro (22,26,27) gut contents Parasitidae Gamasiphis (two species) X Rhodacaridae Afrodacarellus sp. X Afrogamasellus citri Loots X Rhodacarus denticulatus Berlese X Ascidae Protogamasellus mica (Athias) X Protogamasellus nr. brevicornis Genis, Loots, & Ryke X Protogamasellus n. sp. X Protogamasellus nr. massula (Athias) X Protogamasellopsis nr. corticalis Evans & Purvis X Asca duosetosa (Fox) X Cheiroseius nr. browningi (Evans & Hyatt) X Gamasellodes vermivorax Walter X Gamasellodes rectiventris Lindquist X Lasioseius dentatus Fox X Laelapidae Cosmolaelaps nr. weeversi Oudemans X Cosmolaelaps nr. vacua (Michael) X Cosmolaelaps nr. carvalhoi (van Aswegen & Loots) X Euandrolaelaps sp. X Geolaelaps nr. aculeifer Can. X Geolaelaps nr. angusta (Karg) X Hypoaspis nr. similiseta Karg X Hypoaspis (2 species) X Macrochelidae Holostaspella bifoliata (Trag.)' X Pachylaelapidae Zygoseius furciger Berlese X Astigmata Acaridae Schwiebia rocketti Woodring X Prostigmata Alicorhagiidae Alicorhagia sp. X Symphyla Scutigerellidae Symphylellopsis arvernorum Ribaut X Sarcodina Theratromyxa weberi Zwillenberg X Thecamoeba sp. X Nematoda Mononchida Mononchus sp. X Antagonists of Phytonematodes: Walter, Kaplan 571

T.~LE 2. Continued.

Congeners or closely related species reported Fed on to be nema- nematodes tophagous Nematodes in in vitro (22,26,27) gut contents

Dorylaimida Dorylaimus sp. X Discolaimus sp. X Labronema sp. X Diplogastevidae Fictor sp. X rus grove sampled had 1-6 species of trap- served predatory nematodes. The vampy- ping fungi, and 174 of the 187 individual rellid Theratromyxa weberi Zwillenberg, a samples (93%) from citrus roots contained commonly isolated amoeba, was observed at least one trapping fungus. Two Chytri- feeding on small rhabditid nematodes. diomycetes, Catenaria anguiluUae Sorokin Another amoeba, resembling a species of and a species of Rhizophidium, were among Thecamoeba that is known to prey on nema- the most frequently collected antagonists todes in Florida (6), also was observed en- in citrus samples (Table 1). Other zoo- gulfing small rhabditid nematodes. sporic and related fungi were relatively rare Nematophagous arthropods were pres- and difficult to identify with certainty. Sev- ent in all of the citrus groves sampled. Me- eral forms of Myzocytium-like oomycetes sostigmatic (: Mesostigmata) have been combined in Table 1. An un- were the most diverse (26 species) and identified fungus that appeared similar to abundant. Mean densities were estimated Nematophthora gynophila Kerry & Crump, at three groves and ranged from 0.5 to but which produced oospores within the 11.8 Mesostigmata/100 cm 3 soil. Sixteen sporangia rather than laterally, also was of the species were obtained alive, and recovered. they readily fed on burrowing nematodes Spores similar to those of Pasteuria pen- in small laboratory arenas. The gut con- etrans (Thorne) Sayre & Starr were ob- tents of a symphylan, Symphylellopsis ar- served 0nly on citrus nematode juveniles vernorum Ribaut (Scolopendrellidae), col- and males from Ona. Several juveniles from lected from six citrus groves, included this grove were filled with Pasteuria spores. dorylaims with large odontostyles. A No females with clear indications of infec- springtail, Onychiurus sp., and an oribatid tion by Pasteuria could be found. mite, Galumna sp., fed on burrowing nema- An actinomycete, Streptomyces sp., was re- todes in the laboratory, but no systematic covered from dead burrowing nematodes attempt was made to determine the prev- and from citrus nematode egg masses. alence of nematophagy in other mycopha- Nematode bodies were filled with a fine gous soil arthropods from citrus groves. mycelium, and numerous spiral conidio- phores that divided basipetally into small DISCUSSION round conidia were produced from the ca- All surveys are highly dependent on the davers. techniques used to recover antagonists. We Predators: Thirty-six species of preda- sampled citrus roots or rhizosphere soil tors that fed on nematodes were collected from groves with a history of citrus nema- (Table 2). Dorylaimida (including species tode infestation and used plant-parasitic of Labronema, Discolaimus, Dorylaimus, and nematodes as bait. In this way we hoped to Mononchus) were the most frequently ob- isolate antagonists with a high potential for 572 Journal of Nematology, Volume 22, No. 4, October 1990 use as biological control agents. Every grove gOUS arthropods, especially mesostigmatic and almost every sample from the citrus mites, were common and occasionally rhizosphere contained one or more micro- abundant in citrus rhizospheres. Many me- bial antagonists of plant-parasitic nema- sostigmatic mites have rapid generation todes. Nematode-trapping fungi and en- times and high reproductive outputs, and doparasites such as C. anguillulae, which they feed voraciously on nematodes, indi- obtain nutrition from both detritus and cating that they may have potential as bi- nematodes, were the most common antag- ological control agents (26,27). onists in the citrus rhizosphere. More than Noncitrus habitats in Florida also con- 20 species of antagonists to plant-parasitic tain organisms that can attack burrowing nematodes, mostly generalist trapping fun- nematodes, yet several of these antagonists gi and predators, have been reported from were not detected in adjacent citrus groves. citrus groves in California (10,12,19,24). The manner in which nematode predators In our survey, specialized nematode par- and parasites become dispersed is un- asites were rare. For example, species of known, but our results indicate that citrus Pasteuria are thought to be species-specific, groves have relatively predictable antago- or even strain-specific, parasites of nema- nist fauna and flora dominated by gener- todes (21). The Pasteuria sp. we found in- alist predators (Mesostigmata, Dorylaimi- fecting citrus nematode was very abundant da) and fungi (nematode-trapping fungi and (present in 19 of 20 samples) at one site chytrids). Factors contributing to the for- but was not recovered at any of the 26 mation of this assemblage of generalists, other sites. Fattah et al. (8) also reported and limiting the establishment of more spe- a strain ofP. penetrans from Iraq that spor- cialized antagonists, require investigation. ulates within the juvenile. Obligate para- sites might have been detected more fre- LITERATURE CITED quently in this study if techniques that 1. Barron, G. L. 1976. Nematophagous fungi: emphasize recovery of moribund or dead Protascus and its relationship to Myzocytiura. Canadian hosts had been used more extensively (B. Journal of Botany 55:819-824. Jaffee, pers. comm.). 2. Barron, G. L. 1977. The nematode destroying fungi. Topics in mycobiology, vol. 1. Guelph: Cana- Without extensive study, it is often dif- dian Biological Publications. ficult to determine if a microbial organism 3. Boosalis, M. G., and R. Mankau. 1965. Para- is in fact a nematode antagonist. Procar- sitism and predation of soil microorganisms. Pp. 374- yotes are not often reported as nematode 389 in K. F. Baker and W. C. Snyder, eds. Ecology of soil-borne plant pathogens. Los Angeles: Univer- antagonists, but 70% of the citrus groves sity of California Press. we sampled contained a Streptomyces sp. that 4. Cohn, E., and M. Mordechai. 1974. Experi- appears to be a parasite of both burrowing ments in suppressing citrus nematode populations by and citrus nematode. A species of Strepto- use of a marigold and a predacious nematode. Ne- matologia Mediterranea 1:43-53. myces has been reported to attack tylenchid 5. Cooke, R. C., and B. E. S. Godfrey. 1964. A and aphelenchid nematodes by means of key to the nematode-destroying fungi. Transactions sticky spores (21). Isolates of the citrus of the British Mycological Society 47:61-74. Streptomyces sp. have been sent to R. Cha- 6. Esser, R. P. 1983. Amoebic predation upon nematodes. Nematology Circular No. 98, Division of rudatan (University of Florida, Gainesville) Plant Industry, Florida Department of Agriculture for further e~aluation. and Consumer Services, Gainesville. Generalist predators of nematodes have 7. Esser, R. P. 1987. Biological control of nema- not been studied extensively, but at least todes by nematodes. II. Seinura (Nematoda: Aphelen- choididae). Nematology Circular No. 147, Division of nine species of predatory nematodes have Plant Industry, Florida Department of Agriculture been reported to feed on citrus nematode and Consumer Services, Gainesville. and at least three species feed on burrow- 8. Fattah, F. A., H. M. Saleh, and H. M. Aboud. 1989. Parasitism of the citrus nematode, Tylenchulus ing nematode (3,4,7,22). Predatory nema- semipenetrans, by Pasteuria penetrans in Iraq. Journal todes, however, appeared to be relatively of Nematology 21:431-433. rare in the groves sampled. Nematopha- 9. Fuller, M. S., and A.Jawarski. 1987. Zoosporic Antagonists of Phytonematodes: Walter, Kaplan 573 fungi in teaching and research. Athens, GA: South- 20. Newell, S. Y., R. Cefalu, andJ. W. Fell. 1977. eastern Publishing Co. Myzocytium, Haptoglossa, and Gonimochaete (Fungi) in 10. Gaspard, J. T., and R. Mankau. 1986. Nema- littoral marine nematodes. Bulletin of Marine Sci- tophagous fungi associated with Tylenchulus semipe- ences 27:177-207. netrans and the citrus rhizosphere. Nematologica 32: 21. Sayre, R. M., and M. P. Starr. 1988. Bacterial 359-363. diseases and antagonists of nematodes. Pp. 70-101 in 11. Gray, N. F. 1988. Fungi attacking vermiform G. O. Poinar, Jr., and H. B. Jansson, eds. Diseases of nematodes. Pp. 3-38 in G. O. Poinar, Jr., and H. B. nematodes, vol. 2. Boca Raton, FL: CRC Press. Jansson, eds. Diseases of nematodes, vol. 2. Boca Ra- 22. Small, R. W. 1987. A review of the prey of ton, FL: CRC Press. predatory soil nematodes. Pedobiologia 30:179-206. 12. Imbriani, I. L., and R. Mankau. 1983. Studies 23. Sparrow, F. K. 1960. Aquatic phycomycetes. on Lasioseius scapulatus, a mesostigmatid mite preda- Ann Arbor: University of Michigan Press. ceous on nematodes.Journal of Nematology 15:523- 24. Stirling, G. R., and R. Mankau. 1977. Biolog- 528. ical control of nematode parasites of citrus by natural 13. Kaplan, D. T. 1986. Variation in population enemies. Proceedings of the International Society of densities in the citrus rootstock Carrizo citrange. Citriculture 3:843-847. Journal of Nematology 18:31-34. 25. Tarjan, A. C., and J. H. O'Bannon. 1977. 14. Kaplan, D. T. 1988. Future considerations for Nonpesticidal approaches to nematode control. Pro- nematode management in citrus. Proceedings of the ceedings of the International Society of Citriculture Sixth International Citrus Congress. Pp. 35-41. 3:848-853. 15. Kaplan, D. T., and E. L. Davis, 1990. Im- 26. Walter, D. E. 1988. Nematophagy by soil ar- proved nematode extraction from carrot disk culture. thropods from the shortgrass steppe, Chihuahuan Journal of Nematology 22:399-406. desert and Rocky Mountains of the Central United 16. Kaplan, D. T., andJ. H. O'Bannon. 1985. Oc- States. Agriculture, Ecosystems and Environment 24: currence of biotypes in Radopholus citrophilus. Journal 307-316. of Nematology 17:158-162. 27. Walter, D.E.,andE. K. Ikonen. 1989. Species, 17. Karling, J. S. 1977. Chytridiomycetarium guilds, and functional groups: and behav- iconographia. Monticello, NY: J. Cramer. ior in nematophagous arthropods. Journal of Nema- 18. Karling, J. S. 1981. Predominantly holocarpic tology 21:315-327. and eucarpic simple biflagellate phycomycetes. Mon- 28. Young, T. W. 1954. An incubation method ticello, NY: J. Cramer. for collecting migratory endoparasitic nematodes. 19. Mankau, R., and O. F. Clark. 1959. Nema- Plant Disease Reporter 38:794-795. tode-trapping fungi in southern California citrus soils. Plant Disease Reporter 43:968-969.