Species of Phytophthora and as -destroying Fungi S. S. Tzean and R. H. Estey 1 Abstract: Pythium monospermum,, P. aphanidermatum, and Phytophthora palmivora were found to be capable of destroying certain nonstylet-bearing through endozoic para- sitism by hyphae from ingested zoospores. Hyphae of P. monospermum parasitized nematode eggs but could not capture or otherwise prey upon living nematodes. We suggest that endo- of free-living nematodes may be common among in nature. Key words: Pythiurn monospermum, Pythium aphanidermatum, Phytophthora palmivora, endozoic para- sitism, nematode control.

The literature of mycology has few, if oratories, Detroit, ) on which a any, references to either descriptive or ex- pure culture was established. perimental research on nematode parasitiza- The isolated was grown on half- tion by an . Duddington (5) men- strength cornmeal agar at room temperature tioned having seen a species of Phytoph- for 2 wk before morphological studies were thora in a dead nematode. He did not de- made under a light microscope. The fungus scribe the fungus but suggested it may have was identified with keys proposed by Hen- been identical with a fungus, described as drix and Papp (6), Middleton (7), and Pythium anguillulae, that Sadeheck had Waterhouse (8). found in vinegar eels. To induce production of zoospores by In the examination of nematodes iso- the fungus from the dead nematode, the lated from garden at Macdonald fungus was first cultured at room tempera- Campus at McGill University, protoplasm ture on hemp-seed broth. A portion of the was seen streaming toward the apices of resulting mycelial mat was transferred to short hyphal branches that originated from sterile pond water which was cooled for a dead nematode. Within minutes the hy- 24 h at 5 C and then brought to room tem- phal tips bulged out to form numerous perature. This treatment induced the for- zoosporangium-like vesicles. After a short mation of zoosporangia, from which many period of differentiation, zoospores were zoospores were discharged. Phytophthora discharged in all directions. Eventually a palmivora produced zoospores when it was pure culture of this fungus was established, grown for 7 d on V-8 juice-agar at room and it was found to be an Oomycete. The temperature, then flooded with sterile dis- question then arose, do other Oomycetes tilled water, and shaken vigorously for parasitize nematodes in this manner? In 30 s. Zoospores were collected by passing order to determine the answer, Pythium tile fluid through a 43-#m screen. To induce aphanidermatum (Edson) Fitz. and Phyto- zoospore production by Phythium aph- phthora palmivora Butler, two well known anidermatum, the fungus was first grown for plant-pathogenic fungi, were selected for 7 d on yeast-extract at room temperature. experimentation and for comparison with -bearing agar blocks cut form the the unknown Oomycete. periphery of the resulting colonies were floated in 10 ml of distilled water in a petri MATERIALS AND METHODS dish and incubated for 48 h at 10 C. This treatment usually induced the formation of A fungus-infected nematode was trans- inflated filamentous sporangia, but zoo- ferred to 2% water-agar in a petri dish and spores were not produced until some root incubated for 24-48 h at room temperature segments of Vigna sp. were added and the (23-28 C). A hyphal tip growing from a culture chilled for 24 h at 5 C and then re- dead nematode was excised and transferred turned to room temperature. to half-strength cornmeal agar (Difco Lab- Two inoculation methods were used for studying the mode of parasitism on nema- Received for publication 9 October 1979. 1Department of Plant Science, McGill University, Ste- todes by P. aphanidermatum, Phytophthora Anne-de-Bellevue, Quebec, Canada H9X ICO. Present ad- paImivora, and the fungus from the dead dress of senior author: Institute of Botany, Academia Sinlca, Taiwan, China. nematode. In the first method, either stylet 160 Phytophthora and Pythium as Nematode Destroyers: Tzean, Estey 161 bearing nematodes (Alphelenchus avenae biflagellate. After encysting, the zoospores Bastian, MeIoidogyne hapla Chitwood) or germinated by means of germ tubes. free-living nematodes (species of Cephalo- Filamentous -like hyphae bus, Plectus, or Rhabditis) were added to were formed on an agar medium but did zoospore suspensions of the fungi. In the not discharge zoospores even when irrigated second method, the nematodes were added with water. Sexual reproduction by this to the fungus cultures as they grew on half- fungus was characterized by a fusion of dis- strength cornmeal agar. Infected nematodes tinctive antheridia and oogonia. There was were transferred to either water or lacto- usually only one antheridium per oogon- phenol on glass slides for viewing under a ium, but rarely there were two. The globose light microscope. Appropriate lighting and oogonia were smooth walled, terminal, and camera equipment were used for time-lapse intercalary; each was about 19-23 ~m d. cinematography. Each contained only one oospore. The antheridia were usually clavate at RESULTS AND DISCUSSIONS points of contact with oogonia. Young The formation of zoosporangia and the hyphae of this fungus were without septa, discharge of zoospores from hyphae that and they commonly branched at right grew from a naturally infected nematode angles to the parent . Septate hyphae indicated that this endozoic fungus was an occasionally formed bundles of coils. Micro- Oomycete. A subculture of the fungus, in sclerotium-like lateral outgrowths were seen axenic liquid culture, formed filamentous occasionally. The general morphology of sporangia, produced evacuation vesicles, the fungus isolated from the nematode and discharged zoospores in a manner iden- cadaver indicated that it was a strain of tical to those of the fugus growing from the Pythium monospermum Pringsheim, which, naturally infected nematode. As a spor- according to Middleton (7), was originally angium matured, protoplasm flowed rather described as a saprohyte on insect cadavers rapidly to its apex, which then extended to in water. form a spherical vesicle. The protoplasm P. monospermum did not produce any within the vesicle trembled vigorously and specialized trapping devices in response to differentiated into zoospores within 5 rain. the presence of nematodes in the solid agar The crowded, moving zoospores soon broke medium, nor did it secrete adhesive sub- the [ragile wall of the vesicle, from which stances in the manner of such phycomy- they scattered in all directions. The zoo- ceteous nematophagous fungi as Stylopage spores were reniform to ovate and laterally hadra Drechlser or Cystopage lateralis

Figs. 1-3. Invasion and consumption of nematodes, and an egg, by Phythiurn monospermum. 1) Nema- todes filled with the hyphae of the fungus. X660.2) A nematode egg. infected and deformed. XS00.3) Zoo- spores clustered around the head of an infected nematode. ×600. 162 Journal o] Nematology, Volume 13, No. 2, April 1981 Drechsler (4). Hyphae of P. monospermum monspermum. Whether the ingested zoo- did not attach themselves to adult or juv- spores of P. aphanidermatum lodged and enile nematodes even when the inoculated germiuated in the stoma (Fig. 4) or in the petri dishes were kept at low temperatures esophagus (Fig. 5), infection hyphae soon (5-15 C) to make the nematodes very slug- grew throughout the infected nematode and gish. On the other hand, dead nematodes of protruded out of the body cavity, as illus- all the test species were penetrated and con- trated in Fig. 6. Nematodes that were lightly sumed by this fungus (Fig. 1). P. mono- infected remained motile for several hours, spermum did parasitize apparently viable but as infection increased they gradually nematode eggs by unspecialized hyphae. became nonmotile and soon died. Hundreds The infected eggs were eventually con- of zoospores or cysts of P. palmivora were sumed and destroyed (Fig. 2). Barton (2) commonly seen in individual nematodes commented that very few fungi are known (Fig. 7). Hyphae sometimes grew through to specialize as parasites of nematode eggs. tile stoma of the infected nematode and ex- Although P. monospermum could not en- tended to the surrounding medium (Fig. 8), snare nematodes by trapping or by ad- and occasionally there was a secondary in- hesion, it could parasitize species of free- vasion of a dead or moribund nematode by living nematodes from the inside if its zoo- zoospores that developed from attached spores were swallowed. The ingested zoo- sporangia (Figs. 9, 10). It may be note- spores, some of which may have been worthy that the internal infection hyphae of swallowed as cysts, were not digested by the P. aphanidermatum and P. palmivora did nematodes; instead, they encysted, germi- not develop to the extent of those of P. nated, and initiated infection. Some zoo- rnonospermum during the same infection spores lodged and germinated in the stoma time. of the host nematode, whereas others germ- Also seen in Fig. 7 were spherical inated in the esophagus. Trophic hyphae bodies whose identity and function were usually grew throughout the body of the unknown to Capstick et al. (3) who re- nematode (Figs. 3, 4, 5, 6), and zoosporangia ported seeing them on the cuticle of in- formed and zoospores were released within fected nematodes isolated from forest litter. 48 h after the zoospores or cysts had germi- We assumed them to be encysted zoospores nated, thus completing the cycle. because of their morphological similarity The mode of parasitism of Phytophthora to ones that were known to be cysts. An- palmivora and Pythium aphanidermatum other species of Pythium, probably P. dis- was found to be the same as that of P. sotocum Drechsler (7), also had the same

Figs. 4--6. Free-living nematodes infected with Pythiurn aphanidermatum. 4) Ingested zoospore located in the buccal cavity of a nematode (arrow). X840. 5) Ingested zoospore germinating in the esophagus of a nematode (arrow). >(625.6) Hyphae protruding from from an infected nematode. ×550. Phytophthora and Pythium as Nematode Destroyers: Tzean, Estey 163

Figs. 7-10. Free-living nematodes infected with Phytophthora palmivora. 7) Ingested zoospores or their cysts in the body of a nematode. X670. 8) Hapha from an ingested zoospore protruding from the stoma of a nematode. × 720.9) A zoosporangium extending from an infected nematode (arrow). The spherical struc- tures inside the nematode are cysts and zoospores. X890. 10) An enlarged view of a zoosporangium that grew from inside a nematode. )<920. mode of parasitism as the above mentioned LITERATURE CITED fungi (personal observations). 1. Barron, G. L. 1970. Nematophagous Hyphomy- None of the stylet-bearing nematodes cetes: observations on Harposporium helicoides. Can. were parasitized during these trials. J. Bot. 48:329-831. 2. Barron, G. L. 1977. The nematode-destroying fungi. Canadian Biological Publications, Guelph. DISCUSSION 3. Capstick, C. K., D. C. Twinn, and J. S. Waid. The tested species of Phytophthora and 1957. of natural populations of free-living nematodes by fungi. Nematologica 2:194-201. Pythium parasitized nematodes by a method 4. Drechsler, C. 1945. Several additional phy- similar to that described by Barron (1) for comycetes subsisting on nematodes and amoebae. Harposporium anguillulae Lohde and H. Mycologia 37:1-13. helicoides Drechsler. These fungi are un- 5. Duddington, C. L. 1968. Fungal parasites of likely to be of use in biological control of invertebrates. 2 Predaceous fungi. Pp. 239-251 in G. C. Ainsworth and A. S. Sussman, eds. The Fungi. plant-parasitic nematodes because practi- Vol. 3. Academic Press, New York. cally all such nematodes have a stylet or 6. Hendrix, F. F., Jr., and K. E. Papp. 1974. spear-like mouthpart with a lumen too and genetics of Pythium. Proc. Am. narrow for ingesting zoospores. Phytopathol. Soc. 1:200-207. 7. Middleton, J. T. 1943. The taxonomy, host Our observations suggest that endopara- range, and geographic distribution of the genus sitism of nematodes by the zoospores of Pythium. Bull. Torrey Bot. Club 20:1-171. Oomycetes may be a common phenomenon, 8. Waterhouse, G. M. 1968. The genus Pythium and one is tempted to speculate that en- I'ringsheim. Commonwealth Mycological Institute. Myco. Paper 110:1-71. dozoic parasitism may have some signif- icance for the survival of these fungi.