W. H. Ko

Heterothallic Phytophthora: Evidence for Hormonal Regulation of Sexual Reproduction

SUMMARY: Both A1 and A2 mating types of Phytophthora cinnamomi, Phytophthora parasitica and Phytophthora palmivora formed oospores by selfing when they were paired with different mating types on opposite sides of polycarbonate membranes. The selfing of one mating type in the presence of the other mating type demonstrates the production of diffusible substances like plant hormones as found in related fungi. Young cultures and A2 isolates were better hormone producers, whereas old cultures and A1 isolates were more responsive to hormones in both intra- and interspecific pairings. The polycarbonate membrane method should facilitate identification and genetic studies of heterothallic species of Phytophthora.

Reversible Change of Mating Type in Phytophthora parasitica

In addition to the original mating type, the opposite mating type appeared in the zoospore population of single isolates of Phytophthora parasitica after long-term storage or chloroneb treatment. Occasionally self-fertile zoospores were also detected, but their self-induction nature was transitory and unstable. Both hormone production and hormone reception were changed in the sexual variants. The results explain the apparent change from cross-induction (‘heterothallic’) to self-induction (‘homothallic’) in P. parasitica after long-term storage and chloroneb treatment, and suggest one possible evolutionary origin of sex in the lower fungi.

A simplified method for sporangial production by Phytophthora cinnamomi.

Fischer, G. W., and C. S. Holton. 1957. Biology and control of the smut fungi. Ronald Press Co., New York. 622 p. Leach, J. G., and Mary A. Ryan. 1946. The cytology of Ustilago striiformis forma poae-pratensis in artificial culture. Phytopathology 36: 876-886. Raghunath, T. 1969. Nuclear behavior in the germinating teliospores of the smut Georgefischeria riveae. Caryologia 22: 223-228. Ward, E. W. B., and Kathleen W. Ciurysek. 1961. Somatic mitosis in a basidiomycete. Canad. J. Bot. 39: 1497-1503.

Isolates of Phytophthora colocasiae from Hainan Island in China: evidence suggesting an Asian origin of this species.

The 280 isolates of Phytophthora colocasiae obtained from Hainan Island consisted of 136 A1, 102 A2 and 42 A0 mating types. The three mating types tested were all pathogenic to taro leaves and simi...

Segregation following interspecific transfer of isolated nuclei between Phytophthora parasitica and P. capsici.

Nuclei isolated from metalaxyl-resistant (MR) protoplasts of Phytophthora parasitica were transferred into chloroneb-resistant (CnR) protoplasts of Phytophthora capsici and vice versa, with an average success rate of 2.6 x 10(-4) (protoplasts with donor nuclei/regenerated protoplasts), using a selective medium containing only the fungicide tolerated by the nuclear donor. No colonies appeared when self-fusion products of donor nuclei or recipient protoplasts were exposed to the selective medium. Colonies produced by the nuclear transfer formed sectors commonly, and differed from the parental types in appearance. All the zoospores produced by the nuclear hybrids were of normal size, and one-fifth of them contained both MR and CnR genes. Since zoospores are mostly uninucleate, these results indicated the occurrence of chromosome re-assortment or mitotic crossing-over following the production of transitory tetraploids, followed by diploidization during zoosporogenesis, thus suggesting the completion of events leading to a parasexual cycle. Hyphal fragment cultures from a nuclear hybrid tested showed considerable variation in growth rate, mycelial morphology, and level of resistance to metalaxyl, indicating uneven distribution and continuous segregation of different types of nuclei in mycelia during vegetative growth.

Growth rate and colony morphology of progenies of zoospores and selfed oospores of Phytophthora parasitica.

Rates of linear extension of mycelia of zoospore progenies of two tested isolates of Phytophthora parasitica were relatively uniform and similar to their respective parent. There was a continuous quantitative variation, however, in growth rate of progenies of selfed oospores. The character of colony pattern also showed variation in oospore progenies but not zoospore progenies. Results of the study suggest that heterozygous polygenes were involved in determining growth rate and colony morphology of these fungi and support the hypothesis that species of Phytophthora are diploid during the vegetative phase.

Isolation and Partial Characterization of α Hormones Produced by Phytophthora parasitica

Summary: Millipore filters placed underneath agar block cultures of Phytophthora parasitica were able to adsorb hormones α1 and α2 produced by A1 and A2 mating types of the fungus, respectively. Both hormones were released from moistened Millipore filters by extraction with ether, which on evaporation resulted in aqueous hormone extracts. Isolated hormones α1 and α2 stimulated oospore formation by A2 and A1 mating types of P. parasitica, respectively, when they were adsorbed on small pieces of Millipore filter, but not when they were added directly to agar cultures. Hormones α1 and α2 were removed from aqueous solutions by both cation and anion exchange resins, and could be eluted from the resins by ether. Both hormones α1 and α2 were able to pass through membranes with a molecular weight cut-off of 1000 and 500, but not 100 and 50.

Mating-type distribution of Phytophthora colocasiae on the island of Hawaii.

Heterothallic species of Phytophthora require the presence of opposite mating types known as A1 and A2 of the same or different species for oospore formation (12). Recent evidence suggests the regulation of this type of sexual reproduction by sex hormones (8). Phytophthora cinnamomi Rands, an unspecialized root pathogen (6), is widespread on the island of Hawaii (7). The mating-type distribution of this fungus isolated from the island is one-sided. All 47 isolates obtained from soils and plant roots were of the A2 type (7). A further study showed that all 252 isolates of P. cinnamomi from roots of various plant species collected at 21 locations throughout the island were also A2 type (Ko, unpublished data). Two isolates from macadamia cankers were the only record of A1 type from this island (3). Phytophthora colocasiae Rac., the causal organism of leaf blight of taro [Colocasia esculenta (L.) Schott], is also heterothallic and common in taro fields on the island of Hawaii. The fungus is a specialized foliar pathogen (5, 14). Because of its great contrast to P. cinnailmomi, the mating-type distribution of P. colacasiae throughout the island of Hawaii was investigated. Pieces of taro leaves and petioles infected with P. colocasiae were placed on the selective medium developed by Masago et al. (10). After a 3-da incubation at 24 C, mycelia from the edges of P. colocasiae colonies were transferred to V-8-agar plates (10% V-8 juice, 0.02% CaCO3 and 2a% agar). The mating type of each isolate was determined by pairing with an A1 type (P991) and an A2 type (P731) of Phytophthora parasitica Dastur. Both isolates of P. parasitica were supplied by G. A. Zentmyer.

Factors affecting protoplast formation by Rhizoctonia solani.

Novozym 234 was the most frequently used enzyme for production of Rhizoctonia solani protoplasts. Since manufacture of this enzyme was discontinued in the late 1990s, a new procedure was developed by testing lytic enzymes from Sigma and by examining factors affecting protoplast formation. The combination of 20 mg/mL Driselase and 10mg/mL lysing enzyme was effective in releasing protoplasts from R. solani. The optimal condition for enzyme treatment of mycelium was incubation at 37 degrees C for 15 min followed by 34 degrees C for 105 min. The amount of protoplasts produced was positively correlated with growth rate and negatively correlated with mycelial density. Under favorable conditions, R. solani mycelia released 1.68 x 10(6) protoplasts/mL that is comparable with that produced with Novozym 234. Among various media tested, the best solid medium for protoplast regeneration was 1% V-8 juice agar, while the best liquid medium was 10% potato dextrose broth.

Hormonal regulation of sexual reproduction and mating type change in heterothallic Pythium splendens

When different mating types of Pythium splendens were paired on opposite sides of a polycarbonate membrane, numerous oospores were produced by (+) but not (−) isolates, indicating that sexual reproduction of this fungus is a result of selfing of the (+) isolate in response to the stimulation by a hormone produced by the (−) isolate. Hormonal regulation of sexual reproduction in P. splendens was greatly affected by culture age and different isolates also differed in ability to produce hormone and to form oospores in response to the hormone. Four single-conidium subcultures of (+) isolate 461 of Pythium splendens produced oospores in sectors on fresh V-8 agar after being stored in sterile distilled water for 48–93 months. Single-spore cultures obtained from conidia produced on oospore sectors consisted of 293 (+), 3 (−) and 3 (±) type. All the 36 single-propagule colonies obtained from a stored culture originated from spherical structures smaller than conidia produced on V-8 agar. The mating type distribution of these colonies were 33 (+), 1 (−) and 2 (±). Single-conidium cultures from the ± type behaved either as (+) or (−). Hormone production and reception of the (−) variant were characteristic of the (−) wild type. Results suggested that oospore formation in single cultures by aged P. splendens is due to the appearance of the opposite mating type during the storage period and the (±) type is a transitional state in the process of mating type change.