Chemical stimulation of sexual reproduction in Phytophthora and Pythium



	Bot. Bull. Acad. Sin. (1998) 39: 81_86
	Ko — Sexual reproduction in pythiaceous fungi

	(Invited review paper) Chemical stimulation of sexual reproduction in Phytophthora and Pythium Wen-Hsiung Ko Department of Plant Pathology, Beaumont Agricultural Research Center, University of Hawaii at Manoa, Hilo, Hawaii 96720, U. S. A. Keywords: Fatty acids; Oospore formation; Phospholipids; Phytophthora; Pythiaceous fungi; Pythium; Sexual reproduction; Sterols; Terpenoids; Vitamin A. Contents Introduction 81 Discovery of Sterol Requirement for Sexual Reproduction in Pythiaceous Fungi 82 Reevaluation of Essentiality of Sterols for Sexual Reproduction 83 Presence of Inhibitory Substances in Highly Purified Commercial Products 83 Stimulation of Sexual Reproduction by Fatty Acids and Related Compounds after Removal of the Inhibitory Substances 83 Do Pythiaceous Fungi Really Need an Exogenous Stimulatory Substance for Sexual Reproduction? 84 Perspective 84 Literature Cited 85

	Introduction The genera of Phytophthora and Pythium are two of the very destructive groups of plant pathogens in the world (Plaats-Niterink, 1981; Erwin and Ribeiro, 1996). They attack mainly mature plants and young seedlings, respectively, of forest, fruit, vegetable, flower, and ornamental species. These two groups of fungi are classified in the family Pythiaceae of the Oomycetes, which is excluded from the traditional "true fungi" of the kingdom Myceteae and included along with brown algae in the kingdom chromista according to the newer classification (Erwin and Ribeiro, 1996). One of the main reasons for such separation is that the major part of their life history is diploid whereas the other fungi are haploid. Sexual reproduction in Phytophthora and Pythium results in production of thick-walled oospores each in an oogonium attached by single or multiple antheridia. The process is very important in the life cycle of these fungi because it provides not only a means of propagation and survival in nature but also a potential source of genetic variation. Some species of Phytophthora are homothallic and are capable of producing oospores by single isolate while others are heterothallic and require the presence			of opposite mating types, known as A1 and A2, for sexual reproduction. The sexual behavior of heterothallic species of Phytophthora is different from all other known organisms in that sexual reproduction readily occurs even between morphologically and physiologically distinct species (Savage et al., 1968; Ko, 1980). Extensive research eventually revealed that the opposite mating type is needed for the production of a mating-type specific hormone to initiate sexual reproduction by selfing (Ko, 1978). Subsequently it was also found that, in Phytophthora, species are homothallic because they carry receptor(s) for hormone(s) produced by themselves (Ko, 1980a). In the interspecific crosses in Phytophthora, all the progeny are resulted from selfing induced by hormones (Chang and Ko, 1993), while in the intraspecific crosses both selfed and hybrid progeny are produced (Chang and Ko, 1990). Such hormonal beterothallism and homothallism represent a novel mode of sexual reproduction in the biological world (Ko, 1988). Recent evidence suggests that a similar phenomenon may also exist in Pythium (Guo and Ko, 1991). This review focuses on the nutritional requirement for sexual reproduction in Phytophthora and Pythium. These chemicals may be needed for hormone production and/or oospore formation after hormonal induction.



			Botanical Bulletin of Academia Sinica, Vol. 39, 1998

	Discovery of Sterol Requirement for Sexual Reproduction in Pythiaceous Fungi As early as 1937, Leonian and Lilly found that many species of Phytophthora and Pythium were not able to produce oospores on a defined medium consisting of essential salts and glucose unless an extract from peas was added (Leonian and Lilly, 1937). The stimulatory activity was also found in other seeds and various kinds of oils from plants and animals (Haskins et al., 1964; Klemmer and Lenney, 1965). In 1964, scientists from several institutes independently reported that sterols are required for sexual reproduction in Phytophthora (Harnish et al., 1964; Elliott et al., 1964; Hendrix, 1964; Leal et al., 1964) and Pythium (Haskin et al., 1964; Hendrix, 1964). Their observations have since been confirmed and expanded by researchers from other laboratories (Hendrix, 1970; Elliott, 1983). Although a number of species of Phytophthora and Pythium tested were able to produce oospores on basal medium supplemented with cholesterol or b-sitosterol (Tables 1 and 2), in both genera several species tested failed to produce oospores in the presence of sterols (Table 3). Different species appear to have different sterol requirements. Wilkinson and Millar (1981) reported the production of oospores by Phytophthora megasperma on synthetic medium supplemented with stigmasterol but not with cholesterol or b-sitosterol. However, for Phytophthora cactorum cholesterol, b-sitosterol and stigmasterol were all very effective in supporting oospore formation (Elliott, 1972). There are some indications that even among isolates of the same species, the ability to produce oospores in the presence of sterols may differ markedly. Among 10 isolates of Phytophthora fragariae tested, only three isolates were able to produce oospores on bean meal agar, and only one isolate showed an increase in oosproe production with b-sitosterol (Maas, 1972). However, it is not known if this isolate will produce oospores on basal medium amended with b-sitosterol. Response of sexual reproduction to sterols in a chemically defined medium among isolates of the same species remains to be investigated. Nutrient contaminants in agar or carried over from the inoculum have a strong effect on the activity of sterols. Addition of 1.5% Bacto agar to liquid basal media containing b-sitosterol or cholesterol increased production of oospores by P. cactorum more than 7 and 11 fold, respectively (Ko and Ho, 1983). When highly purified SeaKem HGT-P agarose (Ho and Ko, 1980) was used to solidify the liquid media, the increased activity of sterols disappeared (Ko and Ho, 1983). A number of pythiaceous species reported to show sterol stimulation of sexual reproduction (Hunter et al., 1965; Hendrix, 1965) needs to be reevaluated because it was based on tests with agar media. To prevent the carry-over of nutrient contaminants, the inoculum should be obtained from culture grown on a basal agarose medium (Ko, 1985; 1986; Jee et al., 1997).				Table 1. Species of Phytophthora reported to have a sterol requirement for sexual reproduction. Species Reference Homothallic P. cactorum Leal et al., 1964; Elliott et al., 1964; Harnish et al., 1964 P. heveae Leal et al., 1964; Harnish et al., 1964 P. sojae Hendrix, 1964; Harnish et al., 1964 P. boehmeriae Harnish et al., 1964 P. erythroseptica Harnish et al., 1964 P. megasperma Hunter et al., 1965 P. citricola Hunter et al., 1965 P. ilicis Hunter et al., 1965 P. porri Hunter et al., 1965 Heterothallic (A1 + A2) P. capsici Harnish et al., 1964 P. drechsleri Harnish et al., 1964 P. palmivora Harnish et al., 1964 P. parasitica Harnish et al., 1964 P. cambivora Leal et al., 1967 P. cinnamomi Leal et al., 1967

					Table 2. Species of Pythium reported to have a sterol requirement for sexual reproduction. Species Reference Homothallic Py. periplocum Hendrix, 1964 Py. acanthicum Haskins et al., 1964; Child and Haskins, 1976 Py. ultimum Kerwin and Duddles, 1989 Py. aphanidermatum Hendrix, 1965 Py. paroecandrum Hendrix, 1965 Py. spinosum Hendrix, 1965 Py. vexans Ko, 1965 Py. catenulatum Child and Haskins, 1991 Py. sylvaticum Child and Haskins, 1971 Heterothallic (paired) Py. catenulatum Child and Haskins, 1971 Py. sylvaticum Child and Haskins, 1971

					Table 3. Species of Phytophthora and Pythium with sexual reproduction non-responsive to sterols. Species Reference *Phytophthora* Homothallic P. fragariae Harnish et al., 1964 P. colocasiae Harnish et al., 1964 P. hibernalis Harnish et al., 1964; Leal et al., 1967 P. syringae Hunter et al., 1965; Leal et al., 1967 Heterothallic (A1 + A2) P. cinnamomi Hunter et al., 1965 P. cryptogea Hunter et al., 1965 P. parasitica Ko and Ho, 1983 P. capsici Ko, 1985 *Pythium* Homothallic Py. polytylum Hendrix, 1965 Py. proliferum Hendrix, 1965



	Ko — Sexual reproduction in pythiaceous fungi

			nificant role in the stimulation of sexual reproduction in P. cactorum in these experiments. Presence of Inhibitory Substances in Highly Purified Commercial Products Kerwin and Duddles (1989) and Nes (1988) reported that the ability of lecithin to induce oospore formation in P. cactorum diminished after passage through aminopropyl or alumina columns, respectively, and assumed that such an effect was due to removal of sterol contaminants. However, when we used an aminoproyl column to remove neutral lipids such as sterols and fatty acids from the highly purified commercial phospholipids, instead of reduction in activity these compounds became more stimulatory to sexual reproduction of P. cactorum compared to untreated control (Jee et al., 1997). The activities of lecithin (99% pure) and cephalin (98% pure) were increased 47 and 2.8 fold, respectively, by this treatment. When highly purified commercial phospholipids were developed with a solvent system of acetone-chloroform-acetic acid-water on a thin layer chromatograph (TLC) plate, the major spot on TLC was R_F 0.2 for lecithin and R_F 0.35 for cephalin as indicated by the iodine stain. There was also a faint stain of unknown component at the origin in each case. When the unknown component was isolated from the TLC plate and added to the basal medium supplemented with purified lecithin or cephalin, growth of P. cactorum was completely inhibited (Jee et al., 1997). Addition of the unknown component to the phospholipid fraction of lecithin and cephalin caused a 50% and 100% inhibition of oospore production by P. cactorum, respectively. The 99% pure commercial lecithin from one of the shipments was partially inhibitory to the growth of P. cactorum on basal medium and was not stimulatory to oospore formation of the fungus. When it was dissolved in ether and then washed with deionized water or NaCl solution, the inhibitory effect disappeared, and the compound became strongly stimulatory to oospore formation of P. cactorum. It is considered possible that the decrease of the stimulatory effect of lecithin after purification by column chromatography as reported by Kerwin and Duddles (1989) and Nes (1988) is due to the introduction of inhibitory substances during column treatment. The nature of the inhibitory substances present in the commercial products remains to be investigated. The inhibitors appear to be very polar. They could be the by-products formed during manufacture or could result from degradation or oxidation of the products. Stimulation of Sexual Reproduction by Fatty Acids and Related Compounds after Removal of the Inhibitory Substances The discovery of substances inhibitory to oospore formation of P. cactorum in highly purified commercial lecithin and cephalin suggests that there may be other
	Reevaluation of Essentiality of Sterols for Sexual Reproduction Since the discovery of the stimulatory effect of sterols on oospore formation of Phytophthora and Pythium, these compounds have been considered essential for the production of sexual propagules. Moreover, the alleged essentiality of sterols for sexual reproduction in pythiaceous fungi has been frequently cited as factual (Smith and Berry, 1974; Barnett, 1976; Webster, 1980). Subsequently, Ko and Ho (1983) found that sterols were stimulatory to oospore formation of P. cactorum but not Phytopthora parasitica (A1 + A2), while phosphatidylcholin (lecithin) was stimulatory to the sexual reproduction of both species. According to the criteria of essentiality, an essential substance can not be replaced by any other substance (Bidwell, 1974; Naggle and Fritrz, 1976). It was, therefore, concluded that sterols are stimulatory to, but not essential for, sexual reproduction of P. cactorum. At that time it was not known whether lecithins are essential to P. parasitica as other chemicals were not tested. Chemical stimulation of sexual reproduction of another heterothallic species of Phytophthora, P. capsici (A1 + A2), was similar to that of P. parasitica. The process was stimulated by lecithins but not sterols (Ko, 1985). In the genus Pythium, sterols were also found to be stimulatory to, but not essential for, sexual reproduction in Pythium aphanidermatum because, in addition to sterols, lecithin, phosphatidylethanolamine (cephalin), and certain glycerides tested (such as dipalmitin and trilinolein) were also effective in supporting oospore formation (Ko, 1985; 1986). Another species of Pythium tested, Pythium vexans, behaved quite differently. It produced oospores in the presence of sterols but not lecithins (Ko, 1985). Whether sterols are essential for sexual reproduction in this fungus remains to be tested. Nes (1987), and Kerwin and Duddles (1989) suggested that the stimulatory effect of phospholipids on sexual reproduction in pythiaceous fungi was caused by sterol contamination. However, sterols alone did not stimulate sexual reproduction in P. parasitica and P. capsici, but these fungi were stimulated by lecithin (Ko and Ho, 1983; Ko, 1985). Even when sterol-responsive P. cactorum was used as the test organism, sterol contamination still could not account for the stimulatory effect of lecithin. Assuming that sterols constitute the 1% of impurities in the natural lecithin used in these experiments, it has been estimated that these compounds would account for less than 2% of oospores produced in the presence of lecithin (Ko, 1995). Moreover, our recent GC-MS analysis of phospholipids failed to detect the presence of sterol contaminants, and further purification of phospholipids by column chromatography increased rather than decreased their activity (Jee et al., 1997). When cholesterol, at the concentration detected by Kerwin and Duddles (1989) in their phospholipid sample was added to basal medium containing purified phospholipid, the numbers of oospores produced by P. cactorum did not significantly increase or decrease (Jee et al., 1997). Therefore, sterols did not play any sig



			Botanical Bulletin of Academia Sinica, Vol. 39, 1998

					thesize the substances required for sexual reproduction (Elliott, 1983; Hendrix, 1970; Nes, 1987). Although incapable of synthesizing sterols, pythiaceous fungi are known to be able to synthesize various types of terpenoids, fatty acids, phospholipids, and other lipid compounds (Losel, 1988). Phytophthora cinnamomi has been shown to be capable of synthesizing geraniol and squalene from acetate (Wood and Gottlieb, 1978a; 1978b). The synthesis of phospholipids including lecithin and cephalin by P. parasitica and of fatty acids by P. cactorum have been reported by Hendrix and Rouser (1976) and Nes (1988), respectively. Pythium ultimum has also been reported to synthesize lecithin, cephalin, and fatty acids (Bowman and Mumma, 1967). Since these compounds are all stimulatory to the sexual reproduction of P. cactorum (Jee and Ko, 1997), it was considered possible that pythiaceous fungi are capable of synthesizing substances needed for their own sexual reproduction and that a certain stress factor is needed to trigger the process of sexual reproduction in these fungi growing on basal medium. We, therefore, grew P. cactorum in liquid basal medium and obtained extracts from mycelium, culture filtrate, and basal medium separately. Although extracts from culture filtrate and basal medium were not stimulatory, the extract from mycelium was highly stimulatory to oospore formation of P. cactorum and P. parasitica (Jee and Ko, unpublished data). When mycelium of P. cactorum produced in liquid basal medium was transferred to nutrient-free water agarose, oospores were produced. The fungus also produced oospores on solid basal medium after a prolonged incubation period (Jee and Ko, unpublished data). These results show that the hypothesis is well founded. Contrary to the common belief, P. cactorum is capable of synthesizing substances needed for its sexual reproduction. However, a stress factor such as nutrient deprivation or aging is required to trigger the sexual process. This may explain why oospore formation by pythiaceous fungi on basal medium had not been reported previously. Perspective The discovery of stimulation of sexual reproduction in homothallic P. cactorum and heterothallic P. parasitica by non-sterol substances and the new revelation of P. cactorum's ability to synthesize substances needed for sexual reproduction broaden the research scope of the physiology of sexual reproduction in pythiaceous fungi. The exogenous stimulatory substances may serve as a signal, just like the stress factors, for initiation of the sexual reproduction process. If this is the case, addition of these substances should only shorten the time for oospore production and not change the amount of oospores produced on basal medium. On the other hand, these substances may serve as nutrients needed for the sexual reproduction process per se. In this case, adding these substances should increase the amount of oospores produced on basal medium. Each substance should then be tested to determine if it is needed for hormone production and/or oospore formation after hormone induction. Because of the disclo
	chemicals stimulatory to sexual reproduction of Phytophthora and Pythium if they are purified just before the biosassay. A number of fatty acids and related compounds were, therefore, tested. We were pleasantly surprised to find that eight out of nine tested fatty acids stimulated oospore formation in P. cactorum after purification by TLC (Jee and Ko, 1997). From 243 to 10,250 oospores per 12 mm-disc of basal medium containing 100 mg of the fatty acid were produced. Among the fatty acids tested, palmitoleic acid was the most stimulatory to oospore formation followed by oleic acid, palmitic acid, and linoleic acid. Lauric acid, myristic acid, stearic acid, and linoleic acid were moderately stimulatory. Only arachidonic acid was not stimulatory. Without pre-purification none of the tested fatty acids was stimulatory to oospore formation. Purified palmitoleic acid also stimulated P. parasitica to produce 2,700 oospores per basal medium disc containing 100 mg of the test chemical. The other purified fatty acids had no effect on oospore formation of P. parasitica. Three hydrocarbons and five derivatives of hydrocarbons tested become slightly stimulatory to oospore formation in P. cactorum after purification by TLC (Jee and Ko, 1997). With or without TLC purification these compounds did not stimulate oospore formation of P. parasitica. After TLC purification, geranial and squalene were stimulatory to oospore formation of P. cactorum but not P. parasitica, while phytol, retinol (vitamin A) and vitamin A esters were stimulatory to oospore formation by both fungi. The presence of inhibitory substances in commercially available fatty acids and terpenoids may explain the negative results previously reported on the effect of squalene (Elliott et al., 1964; Harnish, 1968; Nes et al., 1982), and certain fatty acids (Harnish, 1968; Ko, 1985) on sexual reproduction in P. cactorum. The reported inhibitory effect of linoleic acid on Py. aphanidermatum (Ko, 1986), and that of palmitic acid and oleic acid on P. cactorum (Ness, 1988), likewise could be caused by contaminants rather than the fatty acids themselves. The presence of inhibitory substances in highly purified commercial chemical products coupled with the unique ability of cholesterol and b-sitosterol even without purification to stimulate oospore formation by P. cactorum (Jee et al., 1997) may explain the early discovery of sterols and the near two-decade delay in the finding of other substances stimulatory to sexual reproduction in pythiaceous fungi. It is considered possible that more physiological activities may be found if the test chemicals are purified just before bioassay. Do Pythiaceous Fungi Really Need an Exogenous Stimulatory Substance for Sexual Reproduction? Prior to our discovery of the stimulatory effect of non-sterol substances on oospore formation, failure of pythiaceous fungi to produce sexual progeny on basal medium was believed to be due to their inability to syn



	Ko — Sexual reproduction in pythiaceous fungi

			Ho, W.H. and W.H. Ko. 1980. Agarose medium for bioassay of antimicrobiol subtances. Phytopathology 70: 764_766. Hunter, J.L., L.A. Berg, W.N. Harnish, and W.G. Merz. 1965. The nutritional requirements for sexual reproduction in some species of Phytophthora. W. Virg. Acad. Sci. 37: 75_82. Jee, H.J. and W.H. Ko. 1997. Stimulation of sexual reproduction of Phytophthora cactorum and P. parasitica by fatty acids and related compounds. Mycol. Res. 101: 1140_1144. Jee, H.J., C.S. Tang, and W.H. Ko. 1997. Stimulation of sexual reproduction in Phytophthora cactorum by phospholipids is not due to sterol contamination. Microbiology 143: 1631_1638. Kerwin, J.L. and N.D. Duddles. 1989. Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi. J. Bacteriol. 171: 3831_3839. Klemmer, H.W. and J.F. Lenney. 1965. Lipids stimulating sexual reproduction and growth in pythiaceous fungi. Phytopathology 55: 320_323. Ko, W.H. 1978. Heterothallic Phytophthora: evidence for hormonal regulation of sexual reproduction. J. Gen. Microbiol. 107: 15_18. Ko, W.H. 1980a. Hormonal regulation of sexual reproduction in Phytophthora. J. Gen. Microbiol. 116: 459_463. Ko, W.H. 1980b. Sexuality, evolution and origin of Phytophthora. Plant Prot. Bull. (Taiwan) 22: 141_151. Ko, W.H. 1985. Stimulation of sexual reproduction of Phytophthora cactorum by phospholipids. J. Gen. Microbiol. 131: 2591_2594. Ko, W.H. 1986. Sexual reproduction of Pythium aphanidermatum: stimulation by phospholipids. Phytopathology 76: 1159_1160. Ko, W.H. 1988. Hormonal heterothallism and homothallism in Phytophthora. Annu. Rev. Phytopathol. 26: 57_73. Ko, W.H. and W.C. Ho. 1983. Reassessment of apparent sterol requirement for sexual reproduction in Phytophthora. Ann. Phytopathol. Soc. Japan 49: 316_321. Leal, J.A., J. Friend, and P. Holliday. 1964. A factor controlling sexual reproduction in Phytophthora. Nature 203: 545_546. Leal, J.A., M.E. Galleghy, and V.G. Lilly. 1967. The relation of the carbon-nitrogen ratio in the basal medium to sexual reproduction in species of Phytophthora. Mycologia 59: 953_964. Leonian, L.H. And V.G. Lilly. 1937. Partial purification of a vitamin-like substance which stimulates sexual reproduction in certain fungi. Am. J. Bot. 24: 700_702. Losel, D.M. 1988. Fungal lipids. In C. Ratledge and S.G. Wilkinson (eds.), Microbial Lipids. vol. 1, Academic Press, London. Maas, J.L. 1972. Growth and reproduction in culture of ten Phytophthora fragariae races. Mycopathol. Mycol. Appl. 48: 323_334. Naggle, G.R. and G.J. Fritz. 1976. Introductory Plant Physiology. Prentice-Hall, Englewood Cliffs. Nes, W.D. 1987. Biosynthesis and requirement for sterols in the growth and reproduction of Oomycetes. In G. Fuller and W.D. Nes (eds.), Ecology and Metabolism of Plant Lipids, American Chemical Society, Washington D.C., pp. 304_328. Nes, W.D. 1988. Phytophthorols-novel lipids produced by
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	Phytophthora cactorum. Lipids 23: 9_16. Nes, W.D., G.A. Saunders, and E. Heftmann. 1982. Role of steroids and triterpenoids in the growth and reproduction of Phytophthora cactorum. Lipids 7: 178_183. Plaats-Niterink, A. J. van der. 1981. Monograph of the genus Pythium. Stud. Mycol. 21: 1_242. Savage, E.J., C.W. Clayton, J.H. Hunter, J.A. Brenneman, C Laviola, and M.E. Gallegly. 1968. Homothallism, heterothallism and interspecific hybridization in the genus Phytophthora. Phytopathology 58: 1004_1021. Smith, J.A. and D.R. Berry. 1974. An Introduction to Biochemistry of Fungal Development. Academic Press. New York.				Webster, J. 1980. Introduction to Fungi. Cambridge University Press, London. Wilkinson, H.T. and R.L. Millar. 1981. Reproduction and growth of Phytophthora megasperma var. megasperma on a defined medium. Phytopathology 71: 265. Wood, S.G. and D. Gottlieb. 1978a. Evidence from mycelial studies for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi. Biochem. J. 170: 343_354. Wood, S.G. and D. Gottlieb. 1978b. Evidence from cell-free systems for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi. Biochem. J. 170: 355_363.