Bot. Bull. Acad. Sin. (2003) 44: 323-328

Wu et al. Sexual reproduction in pythiaceous fungi

Effect of culture origin on chemical stimulation of sexual reproduction in Phytophthora and Pythium

Hau Wu1, Xiau-Bo Zheng1, and Wen-Hsiung Ko2,*

1Department of Plant Protection, Nanjing Agricultural University, Nanjing 210095, P.R. China

2Department of Plant and Environmental Protection Sciences, Beaumont Agricultural Research Center, University of Hawaii at Manoa, Hilo, Hawaii 96720-4037, USA

(Received January 27, 2003; Accepted May 27, 2003)

Abstract. Lecithin washed with NaCl solution was stimulatory to oospore formation of Phytophthora boehmeriae, P. sojae, P. cactorum, and Pythium aphanidermatum. Commercial glycerides of palmitic acid with 99% purity were found to contain a substance inhibitory to oospore formation. Purification with florisil column chromatography was more effective in removing the inhibitory substance than thin-layer chromatography or NaCl washing. After purification, monopalmitin, dipalmitin and tripalmitin were found to be stimulatory to the sexual reproduction of Phytophthora for the first time. These compounds were stimulatory to oospore formation of P. boehmeriae and P. sojae but not P. medicaginis, and were stimulatory to isolate S317 of P. sojae but not isolate CN-1 of the same species. Pythium aphanidermatum cultured on basal medium grew sparsely but steadily and produced oospores after being transferred to nutrient-free water agarose. Extracts from mycelia of P. boehmeriae, P. cactorum, and P. sojae grown in liquid basal medium were stimulatory to their own sexual reproduction. Results support the hypothesis that pythiaceous fungi can synthesize substances needed for sexual reproduction but require a stress factor to trigger the process.

Keywords: Lecithin; Glycerides; Phytophthora spp.; Pythium spp.; Sexual reproduction.

Introduction

Phytophthora and Pythium species are two of the very destructive groups of plant pathogens in the world (Van der Plaats-Niterink, 1981; Erwin and Ribeiro, 1996). 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. The family is instead included with brown algae in the kingdom Chromista because the major part of their life history is diploid whereas other fungi are haploid (Erwin and Ribeiro, 1996).

Sexual reproduction in Phytophthora and Pythium 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. 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). 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).

During the 1980's, it was found that lecithin was also stimulatory to the sexual reproduction of Phytophthora cactorum and Pythium aphanidermatum (Ko and Ho, 1983;

Ko, 1985, 1986). Moreover, Phytophthora parasitica and Phytophthora capsici, whose sexual reproduction was not responsive to sterols, were also stimulated by lecithin to produce oospores (Ko, 1985; Jee et al., 1997). According to the criteria of essentiality, an essential substance can not be replaced by any other substance (Bidwell, 1974; Naggle and Fritz, 1976). It was, therefore, concluded that sterols are not essential for sexual reproduction in pythiaceous fungi (Ko, 1988, 1998). Our subsequent study revealed the common presence of inhibitory substances in the highly purified commercial compounds (Jee et al., 1997; Jee and Ko, 1997). After removal of these inhibitory substances, a number of fatty acids and related compounds were found to be stimulatory to the oospore formation of P. cactorum, and some of them were also stimulatory to P. parasitica (Jee et al., 1997; Jee and Ko, 1997).

Prior to our discovery of the stimulatory effect of non-sterol substances on oospore formation, the failure of pythiaceous fungi to produce sexual progeny on basal medium was attributed to their inability to synthesize the substances required for sexual reproduction (Elliot, 1983; Hendrix, 1970; Nes, 1987). This hypothesis was subsequently refuted as extracts from mycelia of P. cactorum grown in liquid basal medium were shown to be capable of inducing oospore formation in P. cactorum and P. parasitica (Jee and Ko, 1998). Our results show that P. cactorum can synthesize substances needed for sexual reproduction, but it requires a stress factor, such as nutrient deprivation, to trigger the process.

*Corresponding author. Fax: +1-808-974-4110; E-mail: kowh@hawaii.edu


Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Since only three species of Phytophthora and one species of Pythium were used in the above study, it was not known if other species or isolates of the same species of pythiaceous fungi would respond similarly to chemical stimulation of sexual reproduction. The objectives of this study were to determine if non-sterol substances were also stimulatory to sexual reproduction in other species or isolates of the same species of Phytophthora and Pythium, and to test the ability of other Phytophthora species to synthesize substances needed for their sexual reproduction. Whether nutrient deprivation can also trigger the process of sexual reproduction in other pythiaceous fungi was also investigated.

Materials and Methods

Organisms and Chemicals

Fungi used in this study included three isolates of Phytophthora boehmeria, two isolates of P. sojae, and one isolate each of P. cactorum, P medicaginis, Pythium aphanidermatum, and Py. myriotylum (Table 1).

Soybean lecithin, monopalmitin, dipalmitin, and tripalmitin (purchased from Sigma) were all 99% pure. The lecithin in chloroform was evaporated to dryness and then dissolved in ether before use to prevent it from becoming mucilaginous.

Bioassay

The basal medium of Jee and Ko (1997) was used for maintenance of test organisms and for bioassay. It consisted of 0.1 g KNO3, 0.2 g K2HPO4, 0.1 g MgSO4, 0.1 g CaCl2, 1 ml trace elements, 0.1 g L-asparagine, 0.05 g L-serine, and 4 g glucose in 1 l of distilled water. The trace element solution contained 200 mg FeEDTA, 10 mg CuSO4, 10 mg MnCl2, 10 mg Na2MoO4, 10 mg Na2B4O7, and 20 mg ZnSO4, and 100 mg thiamine hydrochloride in 100 ml distilled water. Highly purified SeaKem agarose (HGT-P Agarose, FMC, BioProducts, Rockland, ME, USA.), which contained no detectable nutrient contaminants (Ho and Ko, 1980; Ko, 1985), was used to solidify the basal medium at a concentration of 0.8% (w/v). The medium was adjusted to pH 6.2 with 0.5 N KOH. For each test organism, a piece (ca 333 mm) of culture grown on 10% V-8 agar (10% V-8 juice, 0.02% CaCO3, and 2% agar) was placed in the center of a basal medium plate and incubated at 25C in darkness. After the colony reached about 4 cm in diameter, the same size of culture was cut from the advancing margin and placed in the center of a plate containing fresh basal medium. The procedure was repeated three times before use to avoid possible contamination of nutrients from the original inoculum. Under these conditions, none of the test organisms formed oospores on the basal medium unless a stimulatory substance, such as lecithim, was added, indicating that stimulatory substances were not carried over from the inoculum.

Three basal medium blocks (10105 mm) were evenly placed in a 6-cm Petri plate, and 20 l of ether containing 500 g of lecithin or ethyl acetate containing 100 to 500

g of other substances was spread over the surface of a basal medium block. After evaporation of the organic solvent in a fume hood for 30 min, the basal medium blocks were each inoculated with a small cube (ca 222 mm) of a test organism. Inoculated blocks in Petri plates were incubated for 7 days at 25C in darkness in a moist chamber for oospore production. To determine the number of oospores produced, each block was triturated with 5 ml of water in a mixer at 4,000 rpm for 1 min. Oospore concentration in the suspension was determined by counting the number of oospores in a 100 l sample (Ko et al., 1973).

Washing with NaCl Solution

The method developed by Jee and Ko (1997) was used. Ten mg of a test chemical dissolved in 50 ml of ether was vigorously shaken with 100 ml of 1% NaCl solution adjusted to pH 8 with 0.5 N KOH in a 500 ml separatory funnel for 1-2 min. After 2-3 h standing, the ether layer was collected and dried on a rotary evaporator. Ten ml of ether was added to the evaporation flask to dissolve the residue, and 10 g of anhydrous sodium sulphate was added to remove remaining water molecules. Ether containing the residue was transferred to a test tube and evaporated to dryness. The residue was redissolved in 1 ml of ethyl acetate for bioassay.

Thin-Layer Chromatography

Ten mg of the test chemical dissolved in 0.5 ml of ether was spotted on a TLC plate (1020 cm, 60 A silica gel, layer 1 mm thick, Whatman adsorption plate, Fisher Scientific, Pittsburgh, PA, USA.) using 7 l per spot (Jee and Ko, 1997). The loaded plate was developed with a mixture of hexane-ethyl ether-acetic acid (70:30:1), dried and visualized with iodine vapor to locate sample bands. After evaporation of iodine, the sample band was placed in a 25 ml centrifuge tube, and extracted with two aliquotes of 20 ml of ether. The combined ether solution was washed with 80 ml of 1% NaCl solution as described above.


Wu et al. Sexual reproduction in pythiaceous fungi

Washed samples were dissolved in ethyl acetate for bioassay.

Florisil Column Chromatography

The method of Carroll (1961) was used to purify palmitic glycerides by Florisil column chromatography. Non-treated Florisil (30 g, 60-100 mesh; Fisher Scientific, Pittsburgh, PA, USA.) was packed in a column (2030 cm) with 60 ml of hexane. Excess hexane was drained from the bottom before 200 mg of palmitin in 2 ml of hexane was loaded on top of the Florisil in the column. The Florisil column was washed with 50 ml of hexane and with 150 ml of 2% methanol in ether at the flow rate of 3 ml/min to remove non-target compounds. The palmitin was eluted by passing 150 ml of 4% acetic acid in ether through the column. The ether fraction was evaporated in the rotary evaporator, and the residue was dissolved in 10 ml of ethyl acetate for bioassay.

Effect of Nutrient Deprivation on Oospore Formation

A culture block (11115 mm) obtained from a 7-day-old culture established on basal medium was placed in a 6-cm Petri plate containing 5 ml of 0.8% water agarose (SeaKem HGT-P agarose). After incubation for 7 days at 25C in darkness in a moist chamber, oospores were counted directly under a microscope. Three plates were used for each test organism, and the experiment was done

twice. One of the two experiments with similar results was presented.

Extraction of Stimulatory Substance from Mycelia

Two 6-cm plates of 14-day-old culture grown on basal agarose medium were triturated with 25 ml sterile distilled water in a mixer. One ml of culture suspension was added to a 250-ml flask containing 50 ml double strength liquid basal medium. After incubation for 14 days at 25C in darkness, mycelia were harvested by filtration through filter paper, washed with 200 ml distilled water, and dried overnight at 60C. Extraction was carried out following the method described previously (Jee and Ko, 1998). About 10 g dried mycelia was ground in a mortar and added to a 500-ml boiling flask containing 210 ml 95% (v/v) ethanol and 35 ml 50% (w/v) KOH. After gentle boiling under a reflux condenser for 2 h, the mixture was adjusted to 500 ml with deionized water. Non-saponifiables were extracted from the mixture by three aliquotes of 35 ml petroleum ether. The saponifiable fraction was obtained by adjusting the aqueous portion to pH 2 with 6 M HC1 and extracting the acidified solution with petroleum ether. Both non-saponifiable and saponifiable fractions were evaporated, and the residues were dissolved in 1 ml ethyl acetate for bioassay.

Results

After being washed with NaCl solution, lecithin was stimulatory to the oospore formation of P. boehmeriae, P. cactorum, P. sojae, and Py. aphanidermatum (Table 2). These four species of pythiaceous fungi were similar in responsiveness to the stimulation of lecithin. Among the three methods tested for purification of glycerides, FCC was most effective, followed by NaCl and TLC (Table 3). Without treatment, monopalmitin, dipalmitin, and tripalmitin with 99% purity were not effective in inducing sexual reproduction of P. cactorum. However, after purification by FCC all three glycerides of palmitic acid were stimulatory to oospore formation at concentrations of 200 g and 500 g/block (Table 3). Monoplamitin was most effective in inducing the sexual reproduction of P. cactorum, followed by dipalmitin and tripalmitin. After treatment with NaCl or TLC, these compounds were stimulatory to


Botanical Bulletin of Academia Sinica, Vol. 44, 2003

oospore formation only at 500 g/block. These glycerides purified by FCC were, therefore, studied for their effect on other test organisms. The result showed that different species and even different isolates of the same species responded differently to stimulation by glycerides. The palmitic glycerides were stimulatory to oospore formation of P. boehmeriae and P. sojae, but not P. medicaginis or Py. myriotylum (Table 4). For P. sojae, although all these glycerides were stimulatory to oospore formation of isolate S317, none of them had any stimulatory effect on isolate CN-1. Among three isolates of P. boehmeriae tested, isolate Js-2 was most responsive to stimulation by all three glycerides followed by isolate Je-20 and isolate Ec-8. In general, monopalmitin was most effective in inducing sexual reproduction, followed by dipalmitin and tripalmitin (Table 4).

When Py. aphanidermatum growing on basal medium was subjected to nutrient deficiency by transfer to water agarose, the mycelia grew sparsely but steadily and reached the edge of the Petri plates and formed oospores in 7 days (Table 5). However, the growth of P. boehmeriae, P. cactorum and P. sojae was very weak and slow after being transferred to water agarose. The mycelia extended less than 5 mm away from the inocula after 2 months, and no oospores were produced.

Extracts from mycelia of each organism grown in liquid basal medium were tested for ability to stimulate its own sexual reproduction. The non-saponifiables, excluding saponifiables, extracted from the mycelia of P. boehmeriae, P. cactorum and P. sojae, were stimulatory to its own oospore formation (Table 6). Phytophthora boehmeriae produced the largest amount of stimulatory substances followed by P. cactorum and P. sojae.

Discussion

Lecithin was known to be stimulatory to the sexual reproduction of only P. cactorum, P. parasitica, and Py. aphanidermation prior to the present study (Ko, 1985; Jee et al., 1997). In this study, lecithin purified by washing with NaCl was also found to be stimulatory to P. boehmeriae and P. sojae, and isolates of P. cactorum and Py. aphanidermatum different from those tested previously. These results suggest that lecithin has a broad spectrum of stimulatory effect on the sexual reproduction of pythiaceous fungi. The only tested organism not responsive to lecithin was Pythium vexans (Ko, 1985). Since commercial lecithin is known to contain a substance inhibitory to oospore formation (Jee et al., 1997), and the lecithin used during that time was not pre-washed, oospore


Wu et al. Sexual reproduction in pythiaceous fungi

formation of Py. vexans was probably suppressed by the inhibitor in the lecithin rather than being non-responsive to lecithins per se.

In this study, commercial palmitic glycerides of 99% purity were found to contain a substance inhibitory to oospore formation. Purification with FCC was more effective in removing the inhibitory substance than purification with TLC or washing with NaCl solution. After purification with FCC, palmitic glycerides were found to be stimulatory to sexual reproduction of Phytophthora for the first time. The stimulatory effect of these compounds appeared to be very specific. They were stimulatory to oospore formation of P. boehmeriae and P. sojae but not P. medicaginis. Moreover, these compounds were stimulatory to isolate S317 of P. sojae but not isolate CN-1 of the same species.

Pythium aphanidermatum cultured on basal medium grew sparsely but steadily and produced oospores after being transferred to nutrient-free water agarose. This is the first time a species of Pythium was shown to have the ability to produce oospores without the presence of any stimulatory substances. However, growth of P. boehmeriae, P. cactorum, and P. sojae was extremely slow and limited, and no oospores were produced after transfer from basal medium to water agarose. The amount of basal medium used in this study was apparently not enough for mycelia of Phytophthora to obtain nutrients sufficient for continuation of growth and production of oospores after being transferred to nutrient-free medium. There was a direct relationship between the amount of basal medium used for mycelial growth and the amount of oospores produced after being transferred to water agarose (Jee and Ko, 1998). In this study, extracts from mycelia of P. boehmeriae, P. cactorum, and P. sojae grown in liquid basal medium were stimulatory to their own sexual reproduction. Our results support the hypothesis of Jee and Ko (1998) that pythiaceous fungi can synthesize substances needed for sexual reproduction but require a stress factor to trigger the process.

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Botanical Bulletin of Academia Sinica, Vol. 44, 2003