Reassessment of Apparent Sterol Requirement for Sexual Reproduction in Phytophthora*

Transcription

1 Reassessment of Apparent Sterol Requirement for Sexual Reproduction in Phytophthora* Wen-hsiung Ko** and Wang-thing Ho** Abstract Moderate numbers of oospores were produced by cross-inducing Phytophthora parasitica on chemically defined media with agar. However, even in the presence of sterols very few oospores were produced in liquid media or when agar was replaced by highly purified agarose. Elimination of agar from defined media also reduced the numbers of oospores produced by self-inducing Phytophthora cactorum in defined media. A new chemically defined medium was developed and used to assess sterol requirement for sexual reproduction in Phytophthora. Addition of lecithin to liquid basal medium greatly increased the numbers of oospores produced by P.parasitica, whereas ƒà-sitosterol at 20ƒÊg/ml was ineffective. Lecithin in basal medium also increased oospore production by P.cactorum 550 times over basal medium only, much more than the increase by ƒà-sitosterol. Addition of ƒà-sitosterol at the concentrations of 1,5,10 and 20 ƒêg/ml to basal medium supplemented with lecithin did not increase the numbers of oospores produced by both P.parasitica and P.cactorum. Vegetative growth of both fungi in basal medium with lecithin was fast and similar to that in V-8 juice medium, but was slow in basal medium with ƒà-sitosterol. Chromatographically purified lecithin from soybean was still very effective in promoting oospore formation, but lecithin from egg yolk and two synthetic lecithins tested were ineffective. The results show that sterols are not essential for sexual reproduction in both P.parasitica and P. cactorum. (Received October 20, 1982) Key Words: Lecithin, sterol, sexual reproduction, Phytophthora prasitica, P.cactorum. Introduction Sexual reproduction in species of Phytophthora has been shown to be induced by a hormones produced by itself ( ehomothallic f) or by the opposite mating type ( eheterothallic f) 13,14. In 1964, scientists from four different institutes independently reported that sterols were required for sexual reproduction in both cross-inducing and self-inducing species of Phytophthora including P.parasitica and P.cactorum 7,8,9,18). The conclusion was based on observations that addition of sterols to basal agar media induced sexual reproduction. Their observations have since been confirmed and * Journal Series Paper No.2632 of the Hawaii Institute of Tropical Agriculture and Human Resources ** Department of Plant Pathology, University of Hawaii, Beaumont Agricultural Research Center, Hilo, Hawaii 96720, U.S.A. Present address of the junior author: Taiwan Seed Service, Shinshieh, Taichung, Taiwan

2 Ann. Phytopath. Soc. Japan 49(3). July, expanded by many researchers from other countries, and the alleged essentiality of sterol for sexual reproduction in Phytophthora has been cited as factual in mycology textbooks 2,23,25). During our investigation of the role of sterols in hormone production and oospore formation after hormone induction, it was found that sterols are not essential for sexual reproduction in Phytophthora. This paper reports the detailed account of experimentations leading to such conclusion. A brief report has been published 16). Materials and Methods Isolates P 991 (A1) and P 731 (A2) of the cross-inducing P.parasitica Dastur were supplied by G.A. Zentmyer, and the self-inducing P.cactorum (Lebert et Cohn) Schroeter by D.L. McIntosh. Chemically defined media developed by Ribeiro et al. 22) and by Elliott 3) for oospore production of Phytophthora were used to study the effect of agar or sterols on oospore formation. Media were supplemented with 1.5% Bacto agar or 0.6% SeaKem agarose (HGT-P Agarose, Marine Colloids, Rockland, Maine). Cholesterol (Calbiochem- Behring, La Jolla, California) and ƒà-sitosterol (NBCo., ICN Pharmaceuticals, Cleveland, Ohio) in water were emulsified by homogenization in a tissue homogenizer before being added to the basal media. Twenty-five ml of a solid medium in a 100mm plate and 30ml of a liquid medium in a 250-ml flask were used. A piece (ca. 5mm diam.) of inoculum was placed on the center of a V-8 agar (10% V-8 juice, 0.02% CaCO3, 1.5% Bacto agar) plate and 20 to 30 sterilized discs (6mm) of polycarbonate membrane (CPR, 0.2ƒÊm; Nuclepore Corporation, Pleasanton, California) were placed around the inoculum. After incubation at 24 C for 5 days, discs covered with mycelia were removed, washed by dipping in sterile distilled water for 30 min, and blotted on a sterile petri plate. Solid media were inoculated with a disc of P.cactorum at the center or a disc each of isolates P 991 and P 731 of P.parasitica at 10mm apart. Liquid media were shaken three times by hand following inoculation as described above. After incubation 24 C for 10 days in darkness, cultures were placed in boiling water to melt solidifying agents and mycelia were recovered with a pair of forceps. Oospores were detached from mycelia by grinding mycelial mats suspended in 50 ml of water at 4,500 rpm for 5 min with an Omni mixer. Spore concentration in the suspension was determined with a Pipetman digital microliter pipet l5). A chemically defined medium recently developed by us for oospore production of Phytophthora in liquid culture was used to assess sterol requirement for sexual reproduction. The basal medium contained: KNO3, 0.1g; KH2PO4, 0.1g; MgSO4 7H2O, 0.05g; CaCO3, 0.1g; trace elements, 10ml; thiamine hydrochloride, 1mg; asparagine, 0.1g; glucose, 2g; and distilled water, 1l. Trace elements contained: FeEDTA, 100mg; CuSO4 5H2O, 10mg; MnCl2 4H2O, 10mg; NaMoO4 2H2O, 5mg; Na2B4O7 10H2O, 10mg; ZnSO4 7H2O, 10mg; and distilled water, 1l. Emulsified lecithin (1mg/ml soybean, refined, NBCo.) or ƒà-sitosterol (20ƒÊg/ml NBCo.) was added to the basal medium. Two replicates were used for each treatment and all the experiments were repeated at least twice. Data were analyzed using Duncan's multiple range test.

3 Moderate numbers of oospores were produced by P.payasitica in chemically defined agar media containing either ƒà-sitosterol or cholesterol (Table 1). However, even in the presence of sterols very few oospores were produced in liquid media or when Bacto agar was replaced by highly purified SeaKem agarose 10). Elimination of agar also reduced the number of oospores produced by P.cactoyum in defined media, and by both fungi in V-8 juice medium. The new chemically defined basal medium without agar was poor in supporting oospore formation by P.payasitica (Table 2). Addition of lecithin to basal medium at 1mg/ml greatly increased the numbers of oospores produced, whereas ƒà-sitosterol at 20ƒÊg/ml was not effective. ƒà-sitosterol was used because it has been shown to be the most effective one among various sterols tested 4), and has been the most commonly used sterol, usually at the concentration of 10 to 30ƒÊg/ml 21). Lecithin in basal medium also increased oospore production by P.cactorum 550 times over basal medium only, much more than the increase by ƒà-sitosterol. Addition of ƒà-sitosterol at the concentrations of 1,5,10 and 20ƒÊg/ml to basal medium supplemented with lecithin did not significantly increase the numbers of oospores produced by either P.payasitica or P.cactorum (Table 3). When lecithin was eliminated from the medium the numbers of oospores produced by both fungi Table 1. Effect of agar on oospore production by Phytophthora in culture media a) Numbers followed by the same letter for each medium under each fungus do not differ significantly at P=0.05. Table 2. Comparison of the effect of sterol and lecithin on oospore production by Phytophthora in liquid defined medium a) Numbers followed by the same letter in each column do not differ Significantly at P=0.05.

4 Ann. Phytopath. Soc. Japan 49(3). July, Table 3. Effect of sterol on oospore production by Phytophthora in liquid medium containing lecithin a) Numbers followed by the same letter in each column do not differ significantly at P=0.05. Table 4. Effect of source of lecithins on oospore production by Phytophthorn in linquid defined medium a) Numbers followed by the same letter in each column do not differ significantly at P=0.05. were greatly decreased even in the presence of ƒà-sitosterol at 20ƒÊg/ml. Vegetative growth of both P.parasitica and P.cactorum in basal medium with lecithin was fast, similar to that in V-8 juice medium, but was slow in basal medium with ƒà-sitosterol. In 5 days colonies of both fungi were about 80mm in basal medium plus lecithin, but only about 44mm in basal medium plus ƒà-sitosterol, and 25mm in basal medium alone. Lecithins from other sources were also tested at 1mg/ml to determine their effect on oospore production. Chromatographically purified lecithin from soybean (Sigma Chemical Company, Saint Louis, Missouri) was still very effective in promoting oospore formation of both P.parasitica and P.cactorum (Table 4). Lecithins from egg yolk and two synthetic Lecithins tested were ineffective in inducing oospore formation. Discussion The results show that without the unknown substance(s) in agar sterols are not stimulatory to oospore formation by P.parasitica. The same substance is probably

5 also responsible for the increase in oospore production of P.cactorum in agar media. Honour and Tsao 12) also failed to induce oospore production of P.parasitica in several liquid chemically defined media supplemented with sterols. Uchida and Aragaki 24) also demonstrated the presence in Bacto agar of the substance stimulatory to oospore formation by Phytophthora capsici, although Elliott et al. 6) reported that the oosporeinducing substance was present in Oxoid agar No.3, but not in Bacto agar. Our results show that both P. parasitica and P.cactorum are capable of producing oospores in a chemically defined liquid medium containing lecithin, but no sterols. The possibility that stimulation is due to sterols existing in lecithin as an impurity has been ruled out because of the observations that (i) Ĉ-sitosterol did not induce oospore formation by P.parasitica in liquid basal medium, but lecithin did, (ii) the stimulatory effect of lecithin on oospore production by P.cactorum was 6-9 times greater than that of Ĉ-sitosterol, (iii) addition of Ĉ-sitosterol to basal medium containing lecithin did not increase oospore formation by P.parasitica and P.cactorum, (iv) vegetative growth of both fungi on basal medium supplemented with lecithin is different from that on basal medium supplemented with Ĉ-sitosterol and (v) chromatographically purified lecithin was still much more effective than sterols in promoting oospore formation by both fungi. According to criteria of essentiality, an essential substance cannot be replaced by any other substance 1,9,20) We, therefore, conclude that sterols are stimulatory to, but not required for sexual reproduction in P.parasitica and P.cactorum. Since lecithin can be replaced partially by sterols alone or in combination with the factor in agar, it is also not considered an essential substance for sexual reproduction in Phytophthora. Recently, Elliott and Glen 5) reported that sterol is not required for production of a hormones which regulate the sexual reproduction in Phytophthora 13,14). Stimulation of vegetative growth of Phytophthora by lecithin has been reported recently by Hohl 11) who also showed that effectiveness of lecithin in promoting vegetative growth of Phytophthora was affected by source of lecithin used. Our basal medium containing sterol was more effective in inducing oospore formation by both P.Parasitica and P.cactorum than the other two media tested (Table 1 and 2). This may be due to the use of lower concentrations of minerals in our medium. Inhibition of fungi by minerals present in media has been retorted nreviouslv 17). Literature cited 1. Bidwell, R.G.S. (1974). Plant Physiology. Macmillan Publishing Co., New York. pp Burnett, J.H. (1976). Fundamentals of Mycology. Crane Russak & Co., New York. pp Elliott, C.G. (1972). Trans. Br. Mycol. Soc. 58: Elliott, C.G. (1972). J.Gen. Microbiol. 72: Elliott, C.G. and Glen, B. (1982). Ibid. 128: Elliott, C.G., Hendrie, M.R. and Knights, B.A. (1966). Ibid. 42: Elliott, C.G., Hendrie, M.R., Knights, B.A, and Parker, W. (1964). Nature 203: Harnish, W.N., Berg, L.A. and Lilly, V.G. (1964). Phytopathology 54: 895 (Abstr.). 9. Hendrix, J.W. (1964). Science 144: Ho, W.C. and Ko, W.H. (1980). Phytopathology 70: Hohl, H.R. (1975). Phytopathol. Z. 84: Honour, R.C. and Tsao, P.H. (1974). Mycologia 66:

6 Ann. Phytopath. Soc. Japan 49(3). July, Ko, W.H. (1978). J.Gen. Microbiol. 107: Ko, W.H. (1980). Ibid. 116: Ko, W. H., Chase, L.L. and Kunimoto, R.K. (1973). Phytopathology 63: Ko, W.H. and Ho, W.C. (1982). Ibid. 72: 995 (Abstr.). 17. Ko, W.H., Kliejunas, J.T. and Shimooka, J.T. (1976). Ibid. 66: Leal, J.A., Friend, J. and Holliday, P. (1964). Nature 203: Mertz, W. (1981). Science 213: Naggle, G.R, and Fritz, G.J. (1976). Introductory Plant Physiology. Prentice-Hall, Englewood Cliffs. pp Ribeiro, O.K. (1978). A Source Book of the Genus Phytophthora. J. Cramer, Germany. pp Ribeiro, O.K., Erwin, D.C. and Zentmyer, G.A. (1975). Mycologia 67: Smith, J.E. and Berry, D.R. (1974). An Introduction to Biochemistry of Fungal Development. Academic Press, New York, pp Uchida, J.Y. and Aragaki, M. (1980). Mycologia 72: Webster, J. (1980). Introduction to Fungi. Cambridge University Press, London. pp.669.