Oospore Formation Research Articles

Isolation and Partial Characterization of Hormones Produced by Phytophthora parasitica

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.

Effect of conidium formation on oospore formation in lesions of soybean downy mildew caused by Peronospora manshurica.

Effect of conidium formation on oospore formation in lesions of soybean downy mildew caused by Peronospora manshurica.

Reassessment of apparent sterol requirement for sexual reproduction in Phytophthora.

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 β -sitosterol at 20μg/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 β-sitosterol. Addition of β-sitosterol at the concentrations of 1, 5, 10 and 20μg/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 β-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.

The relationship of oogonium and oospore formation of soybean downy mildew fungus, Peronospora manshurica, to photosynthesis of infected soybean leaves.

Effect of light and darkness on oospore formation of downy mildew fungus, Peronospora manshurica, of soybean, Glycine max, was investigated. Oospores were more abundantly formed in lesions floated on water at a light intensity of 20, 000lux than those in darkness. The ratio of total number of oogonia and oospores under light to that in darkness was 2.9-4.0. The total number of oogonia and oospores increased from the 3rd day of incubation and reached a maximum the 4th day of incubation in darkness. Under light, the total number of oogonia and oospores rapidly increased from the 2nd to 7th day of incubation. The oogonium was differentiated on the 3rd day of incubation and the oospore was formed on the 5th day of incubation in darkness. In contrast, under light, the oogonium was differentiated on the 2nd day of incubation, one day earlier than in darkness, and the oospore was formed on the 3rd day of incubation, 2 days earlier than in darkness. The number of oospores increased with the increase of incubation days in the light and photoperiod. Oospore formation was reduced by the treatment of lesions with 3-(3, 4-Dichlorophenyl)-1, 1-dimethylurea (DCMU) at concentrations of 10-6-10-4M under light. DCMU, however, did not affect the oospore formation at concentrations of ranging between 10-7-10-4M in darkness. This phenomenon supports the idea that oospore formation in lesions was decreased by means of the inhibition of photosynthesis of downy mildew lesions. It was concluded from these results that the oospore formation of soybean downy mildew fungus closely depends on the photosynthesis of host soybean leaves.

Factors Affecting the Induction of Sexual Reproduction in Phytophthora parasitica by P. colocasiae

When Phytophthora colocasiae (A2) was used as a hormone producer and P. parasitica (A1) as a hormone receptor, no sex organs of P. parasitica were observed in matings lasting for 7 h, but the amount of hormone produced was sufficient to stimulate the production of 341 oospores cm−2 6 d later. The maximum induction of sex organs was reached in matings lasting 48 h. Hormone production was inhibited by light, but the effect of light on oospore development was small. Temperatures of 10 and 15°C inhibited the growth and hormone production by P. colocasiae, and prevented P. parasitica from forming new sex organs after stimulation by hormone. The effect of temperature on hormone production differed from that on oospore formation. Hormone production was poor at 30°C, but oospore development was good at this temperature.

Influence of water and humidity on oospore formation of several downy mildew fungi.

Influence of water and humidity on oospore formation of several downy mildew fungi.

Oospore formation of Japanese radish downy mildew fungus.

Oospore formation of Japanese radish downy mildew fungus.

Effect of Steric and Nuclear Changes in Steroids and Triterpenoids on Sexual Reproduction in Phytophthora cactorum

The comparative biological activity of 21 naturally occurring or synthetically derived steroids, 7 tetracyclic and pentacylic triterpenoids, and antheridiol incubated with cultures of Phytophthora cactorum has been examined. There was greater dependence on precise steric features of the sterol side chain than on the extent of nuclear unsaturation in inducing oospore formation. There was no significant effect on oospore formation by changing nuclear unsaturation in ring B from Delta(5) to Delta(7) or to Delta(5,7). Converting the unsaturated sterol to its corresponding stanol resulted in a significant reduction in the number of oospores produced. The effectiveness of sterols bearing different side chains in inducing oospores was found to be in the following relative order: 24alpha-ethyl = trans-Delta(22)-24alpha-ethyl > trans-Delta(22)-24beta-ethyl = 24alpha-E-ethylidene = 24alpha-methyl > 24beta-methyl = trans-Delta(22)-24beta-methyl = 26-methyl = saturated C(7) side chain and C-20 R (17-alphaH, 20-alphaH, right-handed conformer) = cis-Delta(22)-C(7) side chain and C-20 R > saturated C(7) side chain and C-20 S (17-alphaH, 20-betaH, right-handed conformer) > no sterol = 29-hydroxyporiferasterol = 20alpha-hydroxycholesterol = 24xi-hydroxy-24-vinylcholesterol. Of the sterols examined the most significant stereochemical criterion for the induction of oospore formation was absence of bulk on the front face of C-20. This follows from the observation that 20-isocholesterol and 20alpha-hydroxycholesterol, in which a methyl and hydroxy group, respectively, project to the front in the right handed conformation, were inactive in stimulating production of oospores. None of the triterpenoids studied induced oospore formation to any significant degree. Oospore formation was not induced by antheridiol nor 29-hydroxyporiferasterol in combination or added separately to growing cultures of P. cactorum in the concentration range 0.01 - 10.0 milligrams per liter.

Chemical Stimulation of Oospore Formation in Phytophthora capsici

Oospore distribution patterns in pairings between compatible isolates of Phytophthora capsici suggested mutual induction of oospore formation. Oogonia and oospores formed near the A2 colony were smaller than those developing near the A' colony, suggesting that the smaller bodies were A2 products and the larger bodies were formed by A'. This interpretation was confirmed in pairings physically separated by polycarbonate membranes. Significantly more oospores were produced in synthetic medium containing Difco Bacto agar as compared to Difco Noble agar, Difco purified agar or SeaKem agarose.

Hormonal Regulation of Sexual Reproduction in Phytophthora

Induction of oospore formation by the complementary strain occurred in both cases when A1 and A2 isolates of Phytophthora parasitica were separated by polycarbonate membranes and up to 3 mm of agarose. The number of oospores produced increased as separation distance decreased. The homothallic species P. heveae induced oospore formation in both the A1 and A2 isolates of P. parasitica. Phytophthora megasperma var. sojae stimulated oospore formation in the A2 but not the A1 isolate of P. parasitica, while P. cactorum and P. katsurae stimulated the A1 but not the A2 isolate. Sexual reproduction of homothallic species of Phytophthora may thus also be controlled by α hormones. Sixteen types of chemically regulated sexuality among the members of Phytophthora, differing in hormone production and responsiveness to hormones, are postulated.

Electron microscopic study of oospore maturation and germination in an emasculate isolate of Saprolegnia ferax. 4. Nuclear cytology

Changes in nuclear ultrastructure during the formation, maturation, and germination of oospores produced by an agamospermous (emasculate) isolate of Saprolegnia ferax are described. Oogonium nuclei undergo synchronous meiotic divisions which appear identical to those previously reported in antheridiate species. Fusion of two typical gametic nuclei result in the formation of a single, unusually large, restitution nucleus within delimited oospheres. During the activation phase of germination the nuclei undergo three or four successive, near synchronous, mitotic divisions which appear similar to those described in other oomycetes. There was no evidence to support the occurrence of zygotic meiosis. Some nuclei in germinating oospores contain unusual spheroidal and lamellar inclusions, whereas others have abnormally large, irregularly lobed, profiles. Nuclear autolysis was also a common event in germlings. These results are compared with those in other oomycetes and the evidence supporting automictic sexuality discussed.

Phytophthora Blight of Pigeon Pea in India

Isolations were made from blighted pigeon-pea (Cajanus cajan) plants from different locations in India. A species of Phytophthora was consistently obtained from these locations and was proved to be the causal organism involved in the disease. Based on the sporangium shape and size, oogonium and oospore formation, temperature requirements, and pathogenicity tests, we have classified these isolates as P. drechsleri f. sp. cajani. The use of formae speciales was considered appropriate because of the specificity of these isolates to pigeon pea and Atylosia spp., wild relatives of the pigeon pea.

Influence of Water and Temperature on Oogonium and Oospore Formation of Downy Mildew Fungus, Peronospora manshurica, in Soybean Lesions

Oospores of downy mildew fungus, Peronospora manshurica, of soybean, Glycine max, were well formed when the lesions were immersed in water or under humid condition. Oospores were not formed when the lesions were submerged in water or kept under dry condition. In the immersion treatment at 5 C neither oogonium nor oospore was formed and at 25C only a few oogonia were formed. At 10 C oogonia and oospores were formed, but in small number. At 15 and 20 C abundant oogonia and oospores were formed. At 15 C oogonia were abundantly formed after 3 day-immersion and reached the maximum number after 4 day-immersion. Oospores developed after 2 days following oogonium formation. At 20 C oogonia were formed after 3 day-immersion and reached the maximum number after 4 day-immersion, as well at 15 C. Oospore development at 20 C was, however, 1 day earlier than that at 15 C. Size of oogonium thus obtained was 36.0×40.2 μm, and that of oospore was 27.6 μm in diameter on the average.

Control of homothallic oospore formation in Phytophthora parasitica by culture manipulations

Control of homothallic oospore formation in Phytophthora parasitica by culture manipulations

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.

Effect of water and temperature on the oogonium and oospore formation of Peronospora manshurica in lesions on Glycine soja.

Effect of water and temperature on the oogonium and oospore formation of Peronospora manshurica in lesions on Glycine soja.

Timing of events during oospore genesis in Saprolegnia diclina

The sequence and duration of events leading to oospore formation in Saprolegnia diclina were investigated using combined visual observation and time lapse photography of living oogonia and antheridia. Antheridia were always delimited before oogonia. Subsequent development of antheridia involved progressive vacuolation and eventual formation of in-growths into oogonia from some antheridia. The interval between formation of the septum delimiting an oogonium and formation of discrete oospheres from oosphere initials varied by not more than ± 5% of the mean value amongst the oogonia examined, but the timing of stages in oospore maturation was much more variable, within, as well as between oogonia.

Stimulation of oospore formation in Phytophthora by antagonistic species of Trichoderma and its ecological implications

Stimulation of oospore formation in Phytophthora by antagonistic species of Trichoderma and its ecological implications

Electron microscopy of early gametangial interaction in Phytophthora megasperma var. sojae

The ultrastructure of gametangial development and oospore formation was studied in Phytophthora megasperma var. sojae, a species with the paragynous antheridium. Except for the early gametangial interaction, the developmental stages of oogonial expansion, fertilization tube formation, oosphere and oospore formation were very similar to those reported for amphigynous species. The initial contact differs in the two types of gametangial interaction in that in amphigyny the oogonial hypha contacts and penetrates the antheridial initial from the side, whereas in paragyny the antheridial hypha touches and fuses with the oogonial initial from the side without penetrating it. In both cases the end result seems to be the same: the establishment of a firm adhesion zone between the gametangia. Dictyosome-derived vesicles were active in the formation of the contact zone, during oogonial expansion and wall thickening, and during extension of the fertilization tube. An extensive conversion of cytoplasm occurred during differentiation within the oogonium from a largely 'functional' cytoplasm rich in mitochondria, ribosomes, rough endoplasmic reticulum, and dictyosomes to a largely 'storage' cytoplasm within the oospore consisting of an ooplast and surrounding lipid layer.

Genetical Studies with Interspecific Crosses Between Phytophthora cinnamomi and Phytophthora parasitica

BOCCAS, B., and G. A. ZENTMYER. 1976. Genetical studies with interspecific crosses between Phytophthora cinnamomi and Phytophthora parasitica. Phytopathology 66: 47744 Two interspecific crosses between Phytophthoraparasitica and Phytophthora cinnamomi resulted in the formation of oospores, but only 5% were well formed. About 5% of the normal spores germinated and gave single oospore progeny which were genetically studied. The first cross produced only parental type progeny, but the second cross yielded heterogeneous progeny including parental type cultures and cultures with new associations of phenotypic characters. In both crosses, parental type progeny were interpreted as Additional key word: ploidy. arising from the germination of self-fertilized oospores from the parents. Progeny with apparent recombinant phenotypes could have resulted either from interspecific hybridization or from self fertilization. Evidence of recombination was obtained in relation to mating type, pathogenicity to citrus and Persea indica, and temperature response. Presumed recombinant progeny exhibited a protein pattern similar to P. purmitica, indicating that these progeny may have resulted from self fertilization. .