Formation Of Zoosporangia Research Articles

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins.

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co-immunoprecipitation (Co-IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co-IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co-localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH-sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR-1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase-mediated vesicular secretion of antimicrobial PR-1, PDF1.2 and possibly other defence-related compounds.

Lycosides, Unusual Carotenoid-Derived Terpenoid Glycosides from a Vegetable Juice, Inhibit Asexual Reproduction of the Plant Pathogen Phytophthora.

Vegetable juices, typical culture media for the plant pathogen Phytophthora, effectively induce its asexual reproduction (zoosporangia formation). However, some chromatographic fractions from a vegetable juice were found to inhibit asexual reproduction. Bioassay-guided chromatographic steps led to the isolation of four novel compounds, named lycosides A-D, 1-4, that could be metabolic products from a carotenoid. They showed 50% inhibitory activity against the asexual reproduction of P. capsici at 2.1-7.6 μM. The structure-activity relationship and the universality of the inhibitory activity within the Phytophthora genus were also investigated. In addition, the quantitative analysis of lycosides in fresh vegetables and vegetable juices revealed that tomato is the source of these active substances. These food-derived chemicals could help provide safe agents to control the outbreak of the agricultural pest Phytophthora in fields.

Antagonism of antifungal metabolites from Streptomyces griseus H7602 against Phytophthora capsici

In this study, evidences for antagonism were established by production of antifungal metabolites from Streptomyces griseus H7602, which were active to inhibit mycelial growth of Phytophthora capsici in the in vitro assays. Mycelial growth and zoosporangia formation of P. capsici was strongly inhibited in the medium containing the cell free culture filtrate of S. griseus H7602. Antifungal metabolites from the cell free culture filtrate of S. griseus H7602 showed substantial antagonistic effects on P. capsici. In addition, a purified antifungal compound was separated from the antifungal metabolites of S. griseus H7602 and identified to be 1H-pyrrole-2-carboxylic acid (PCA) by spectra analyses. PCA showed strong antifungal activity and was evaluated for the first time for its antagonism against P. capsici under in vitro conditions. Minimum inhibitory concentration (MIC) value of PCA was low (4 µg ml(-1)), and the mycelial growth of P. capsici was almost inhibited at concentration of 64 µg ml(-1). This study suggests that the PCA may be useful as biofungicides against P. capsici, and the prominent antagonism of antifungal metabolites from S. griseus H7602 highlights it as a candidate for biocontrol of P. capsici.

고추역병 생물적방제 근권세균의 선발 및 근권정착 능력 연구

하남성지역의 고추역병 발병포장 건전한 고추뿌리와 근권토양에서 세균 507균주를 분리하였는데, 그중에서 고추역병균에 길항효과가 우수한 G28-6, G28-5, I7-6과 H14-1균주를 선발하였다. In vitro에서 선발된 균주들은 고추역병균의 균사생장, 유주자낭과 유주자 형성, 그리고 피낭포자의 발아를 현저하게 억제하였고, Pot시험에서 선발된 균주 모두 70% 이상의 방제효과를 나타냈고, 포트시험에서 방제효과가 우수하였던 G28-6균주는 고추역병 자연발생 비닐하우스에서 79.4%의 방제가를 보여 공시된 화학농약 80% Mancozeb 보다 훨씬 높은 방제효과를 보였다. 뿐만 아니라 선발된 4균주 모두 in vitro에서 우수한 근권정착능력을 보였다. 고추역병 방제에 우수한 길항세균으로 선발된 G28-6균주의 16S rRNA gene 염기서열분석과 그의 형태적, 생리적 특성들을 Bergey's manual of Systematic Bacteriology를 기준으로 비교하여 Pseudomonas aurantiaca로 동정하였다. Four promising biocontrol agents against Phytophthora capsici were selected from 507 bacterial isolates collected from rhizosphere soils and roots of pepper plants. In vitro experiment, these four biocontrol agents inhibited mycelial growth, germination of cystospores, and formation of zoosporangia and zoospores of Phytophthora capsici. In the pot experiment, the four biocontrol agents showed control efficiency higher than 70%. In greenhouse experiment, the isolates G28-6 gave the control value of 79.4%. These four biocontrol agents successfully colonized in the population density beyond 105 cfu/g on roots of pepper in vitro. The isolates G28-6 was identified as Pseudomonas aurantiaca, based on its cultural, morphological, and biochemical characterization and 16S rRNA gene sequence analysis.

Characterization of a new fungal antagonist of Phytophthora capsici

The capacity of many microorganisms for antagonism towards pathogens is unknown and previously uncharacterized species may be potentially useful in this respect. This work presents the evaluation of the capacity of a previously uncharacterized Ascomycete, denominated here as UA to protect chili pepper ( Capsicum annuum) against Phytophthora capsici, and other soil-borne plant pathogens and establishes the role of UA as a biocontrol agent. Inoculation of UA 2 days before the inoculation of P. capsici led to 77.8% survival of pepper plants. Simultaneous confrontation of both microorganisms in vitro led to 53.1% growth inhibition of P. capsici, while the inoculation of P. capsici 3 days after the inoculation with UA improved growth inhibition up to 73%. Simultaneous confrontation in vitro of UA with Fusarium oxysporum and Fusarium solani led to 41.2 and 50% growth inhibition, respectively, but had no effect on Rhizoctonia solani or a binucleate Rhizoctonia isolate. Moreover, formation of zoosporangia and the germination of zoospores were completely inhibited by exposure to undiluted filter sterilized filtrate. UA produces septate mycelia, but could not be classified in detail due to a lack of spores or reproductive structures. However, sequencing of Internal Transcribed Spacer 1, 2 and the 5.8S genes indicated that this fungus is a member of the Ascomycetes.

A novel method for efficient and abundant production of Phytophthora brassicae zoospores on Brussels sprout leaf discs

BackgroundPhytophthora species are notorious oomycete pathogens that cause diseases on a wide range of plants. Our understanding how these pathogens are able to infect their host plants will benefit greatly from information obtained from model systems representative for plant-Phytophthora interactions. One attractive model system is the interaction between Arabidopsis and Phytophthora brassicae. Under laboratory conditions, Arabidopsis can be easily infected with mycelial plugs as inoculum. In the disease cycle, however, sporangia or zoospores are the infectious propagules. Since the current P. brassicae zoospore isolation methods are generally regarded as inefficient, we aimed at developing an alternative method for obtaining high concentrations of P. brassicae zoospores.ResultsP. brassicae isolates were tested for pathogenicity on Brussels sprout plants (Brassica oleracea var. gemmifera). Microscopic examination of leaves, stems and roots infected with a GFP-tagged transformant of P. brassicae clearly demonstrated the susceptibility of the various tissues. Leaf discs were cut from infected Brussels sprout leaves, transferred to microwell plates and submerged in small amounts of water. In the leaf discs the hyphae proliferated and abundant formation of zoosporangia was observed. Upon maturation the zoosporangia released zoospores in high amounts and zoospore production continued during a period of at least four weeks. The zoospores were shown to be infectious on Brussels sprouts and Arabidopsis.ConclusionThe in vitro leaf disc method established from P. brassicae infected Brussels sprout leaves facilitates convenient and high-throughput production of infectious zoospores and is thus suitable to drive small and large scale inoculation experiments. The system has the advantage that zoospores are produced continuously over a period of at least one month.

Characterization of Antibiotic Substance Produced by Serratia plymuthica A21-4 and the Biological Control Activity against Pepper Phytophthora Blight

The biocontrol agent, Serratia plymuthica A21-4, has been developed for controlling pepper Phytophthora blight. Serratia plymuthica A21-4 strongly inhibits the mycelial growth, zoospore formation, and cyst germination of Phytophthora capsici in vitro. The application of a cell suspension of strain A21-4 to pepper plants in pot experiments and in greenhouse successfully controlled the disease. The bacteria produced a potent antifungal substance which was a key factor in the suppression of Phytophthora capsici. The most active chemical com-pound was isolated and purified by antifungal activity-guided fractionation. The chemical structure was identified as a chlorinated macrolide by spectroscopic (UV, IR, MS, and NMR) data, and was named macrocyclic lactone A21-4. The active compound significantly inhibited the formation of zoosporangia and zoospore and germination of cyst of P. capsici at concentrations lower than . The effective concentrations of the macrocyclic lactone A21-4 for of mycelial growth inhibition were against P. capsici, Pythium ultimum, Sclerotinia sclerotiorum and Botrytis cinerea, respectively.

Effects of light quality on the reproduction and morphology of sporophytes of Laminaria japonica (Phaeophyceae)

Responses of the sporophytes of Laminaria japonica to blue, red and white lights were investigated using segments, discs and whole plants. The relative growth rate with regard to the blade's length and width showed no difference among the light conditions during 2 weeks of culturing. On the contrary, the elongation rates of the stipe and the holdfast were 1.7–2.5 times higher under red light than that under white light as a control. Callus formation in the segments was also promoted more under red light conditions than under white or blue light. Blue light irradiation resulted in a 48% and 12% decrease in the relative growth rate of the stipe and the holdfast, respectively, in comparison with the control. When sporophyte discs were cultured for 10 weeks, all discs formed zoosporangial sori 2–3 weeks earlier under blue light than under white light. However, red light inhibited the formation of zoosporangia. Indoleacetic acid (IAA) oxidase activity was about two-fold higher under blue light than under red light, suggesting that the difference in response between the blue and red light conditions is related to the internal IAA level of the tissues. The developmental, reproductive and morphological effects of blue and red lights will be useful for the artificial control of the life cycle of Laminaria plants and for the development of more efficient cultivation techniques.

Ultrastructural study of the brown alga Petroderma maculiforme (Phaeophyceae) in the free-living state and in lichen symbiosis with the intertidal marine fungus Verrucaria tavaresiae (Ascomycotina)

A comparative TEM study was carried out on the phaeophyte Petroderma maculiforme, collected from San Francisco Bay both as free-living thalli and as phycobiont of the lichen fungus Verrucaria tavaresiae. The free-living alga usually had thicker cell walls and substantially greater reserves in storage bodies than the lichenized form. Cell division patterns were altered in symbiosis, and formation of zoosporangia was not observed on the lichenized filaments. Both free-living and lichenized Petroderma showed an abundance of densely osmiophilic deposits associated with vesicles, which were interpreted as physodes; Golgi and ER appeared to be involved in the elaboration of their contents. Plastids of both free-living and lichenized forms possessed a large pyrenoid deeply penetrated by tubular invaginations of the plastidial membranes. This pyrenoid was often enfolded by lobes of the plastid as previously reported, but also commonly occurred as a broad-based protrusion from the surface of the plastid. Smaller, exserted pyrenoids with a very narrow base and no tubular invaginations were sometimes also observed on plastids of the lichenized alga, but were not seen on those of free-living thalli. The significance of this variation in pyrenoid type was not clear. The considerable variation in pyrenoid position and form in P. maculiforme suggests that the protruding, enfolded, and stellate pyrenoid positions known in brown algae are not profoundly different from each other developmentally.

Bacillus stearothermophilus YC4194에 의한 Pythium 모잘록병의 생물학적 방제

Bacillus stearothermophilus YC4l94는 in vitro 와 in vivo 에서 병원균 Pythium aphanidermatum에 대한 억제 효과를 보였다 in vitro 실험에서 P. aphanidermatum의 유주포자낭 형성과 피낭포자 발아를 억제하였을 뿐 아니라 식물체에 적접 길항균 제제 처리 시 50% 이상의 방제가를 보였다. 그리고 다른 화학 살균제와 비슷한 방제효과를 보여 미생물 제제로서의 개발 가능성을 보여주었다. In vitro and in vivo activities of a biocontrol agent, Bacillus stearothermophilus strain YC4194 was evaluated for the control of Pythium damping-off of cucumber. B. stearothermophilus YC4194 inhibited germination of cystospores and formation of zoosporangia of Pythium aphanidermatum in vitro. Incorporation of a bentonite and talc based formulation(10<TEX>$^{9}$</TEX> cfu/g) of B. stearothermophilus YC4194 to the nursery soils (10 g/ι soil) resulted In a significant (p=0.01) reduction in the disease severity of cucumber damping-off after inoculation with P. aphanidermatum. The control efficacy of B. stearothermophilus YC4194 formulation was not different from that of the fungicides, dimethomorph, metalaxyl, ethaboxam. When the cucumber plants were transplanted to the soil inoculated with P. aphanidermatum zoospores, the B. stearothermophilus YC4194 maintained the high population density in rhizosphere soil upto 10<TEX>$^{7}$</TEX> cfu/g until 15 days after treatment.

Vegetative survival, akinete and zoosporangium formation and germination in some selected algae as affected by nutrients, pH, metals, and pesticides.

Lack of nitrogen, phosphorus, or all nutrients, extremes of pH (< 4, > 11), presence of 'heavy' metals (Co, Cu, Zn, Hg, Pb; 0.5-10 ppm) or pesticides (carbofuran, 2,4-D, dithane, phorate, or bavistin; 1-50 ppm) decreased to various extent or completely inhibited the survival or vegetative cells in all studied algae. The formation of akinetes, their viability and germination in Anabaena iyengarii, Westiellopsis prolifica, Nostochopsis lobatus and Pithophora oedogonia and the formation of zoosporangia, their viability, and the germination of zoospores in Cladophora glomerata and Rhizoclonium hieroglyphicum was affected. The formation of viable akinetes or zoosporangia was shown to be directly linked with vegetative cell survival and growth; it could not be induced by any chemical stress imposed.

Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release, development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta)

Formation of zoosporangia and cleavage of zoosporangial cytoplasm to zoospores in Pythium porphyrae is absolutely dependent on extracellular calcium. Calcium ion could be substituted neither by monovalent nor divalent cations tested. Increased concentrations of extracellular calcium did not affect the release of zoospores from zoosporangia but inhibited the zoospore motility. Chelating calcium ion by EGTA has decreased the ability of encysted zoospores to germinate and form appressoria. The increased external calcium-ion concentration has decreased the infectivity of Porphyra yezoensis thalli in a linear fashion apparently indicating a role of calcium in the signaling mechanism.

Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release, development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta).

Formation of zoosporangia and cleavage of zoosporangial cytoplasm to zoospores in Pythium porphyrae is absolutely dependent on extracellular calcium. Calcium ion could be substituted neither by monovalent nor divalent cations tested. Increased concentrations of extracellular calcium did not affect the release of zoospores from zoosporangia but inhibited the zoospore motility. Chelating calcium ion by EGTA has decreased the ability of encysted zoospores to germinate and form appressoria. The increased external calcium-ion concentration has decreased the infectivity of Porphyra yezoensis thalli in a linear fashion apparently indicating a role of calcium in the signaling mechanism.

Low Concentrations of the Isoflavone Genistein Influence in vitro Asexual Reproduction and Growth ofPhytophthora sojae

Isolates of Phytophthora sojae were evaluated for their sensitivity to a series of low concentrations of genistein (0.01, 0.1, 1, 3, and 10 μg/ml) during various stages of growth and asexual reproduction. Genistein was applied during mycelial growth, formation of zoosporangia, release of zoospores, and zoospore germination. At 10 μg/ml, genistein inhibited radial growth of all isolates in solid culture. Changes in fungal morphology, such as increased branching and swelling of hyphae, were observed at all concentrations of genistein tested. Growth of mycelium in broth containing 10 μg of genistein per ml decreased colonies resulting from subsequently formed and germinated zoospores, even when genistein was removed during zoosporangia formation. Genistein also decreased zoosporangia production and release of zoospores. The strongest inhibition of P. sojae reproduction was obtained when genistein was applied during zoosporangia formation ; this occurred at concentrations as low as 0.01 to 1 μg/ml. Genistein in the medium containing zoospores caused a general reduction in asexual reproduction across isolates, with 10 μg/ml causing the most dramatic reduction. There was strong evidence of intraspecific variation in the responses of the isolates of P sojae in all processes tested.

Factors influencing zoosporangia formation by Phytophthora fragariae in vitro

Factors influencing asexual sporulation in vitro were studied for three isolates of Phytophthora fragariae. Maximum numbers of zoosporangia were obtained when an 11 or 15 mm diam disc, cut at the edge of a 15- or 20-d-old colony on a semi-defined medium, was incubated in 6 ml sterile glass-distilled water, in a 4·5 cm diam Petri dish, at 13 or 22°C, depending on the isolate. Isolates varied in their response to the addition of nutrients to the flooding medium. Although visible light had no effect on asexual sporulation near-uv radiation inhibited zoosporangia production by two of the isolates. The role of nutrients as promoters and/or inhibitors of zoosporangia formation is discussed.

Effect of fungicides on stages of the life cycle of Phytophthora cinnamomi

AbstractAn assessment was made of the effects of several fungicides on the formation of zoosporangia, the release of zoospores from zoosporangia, zoospore motility, germ tube formation and mycelial growth, by Phytophthora cinnamomi Rands. Compounds active against mycelial growth (such as metalaxyl and furalaxyl), as well as those active against zoospores (such as captafol and zineb), could be detected by a lupin (Lupinus angustifolius L.) assay which has been developed. This assay may be useful as a screening procedure to detect new fungicides.

病原性水生糸状菌の生理生態に関する研究-II

The resistance of S. parasitica aganist malachite green was variable according to its life-time; i. e. the growth of hyphae was restrained completely in pepton-solution with 0.02 ppm., and the formation of zoosporangia or gemmae, and the germination of resting spores were checked with 0.2 ppm. (Fig. 1, Table 3) The correlation diagram between the concentration of M. G. and the time need to kill the hyphae or sporangia on the eggs of Hypomesus oliaus was shown as a straight line when both factors were expressed in logarithum (Fig. 2). As the infection of the disease could not be checked unless the formation of zoospore ceased, it may be safely said that the disease is carried chiefly through zoospores (Table 3). From these experiments, it is suggested that in practice controling the disease, it may be enough to treat the trout eggs for about 20 minutes in 5 ppm. of M. G., and in the case of the eel it may be necessary to use more concentrated M. G. solution than 0.2 ppm.

Studies on the downy mildew of rice plant.

1. In the present paper the writers deal with the results of a study on the oospore germination of Sclerophthora macrospora (Sacc.) Thirum., Shaw et Naras., the causal fungus of downy mildew of rice plant, and inoculation tests by oospores. 2. When the infected leaves were kept on soil flooded with water in autumn, the oospores became germinable at the end of January, and remained viable at least until May. The percentage of germination ranged from 4.08 to 0.14 during January to May. However, oospores of the infected leaves left on dry soil showed no germination. If the infected leaves were burried 2cm deep in the soil, slight germination of oosporores was obtained irrespective of the water condition in soil. 3. Germinability of the oospores was noticed after about 2 months when the infected leaves were kept on soil with flooded water in early March, May, and at the end of August, respectively. The mature oospores in the rotted leaves can germinate within 48 hours in distilled water. 4. Crushing of rotted leaves is not a necessary measure for the germination of the oospores with high germinability. 5. Distilled water may be a favorable medium for oospore germination. Soil extract did not promote germination. Tap water used seems to check the germination. Addition of 5×10-2-10-3 percent peptone in distilled water inhibited the germination. 2% sucrose solution rather accelerated the germination, but it retarded slightly the formation of zoosporangia at the tip of germ tubes. 6. The optimum temperature for the oospore germination seemed to lie between 18-20°C, the highest 25-26°C, and the minimum somewhat lower than 10-12°C. 7. In inoculation experiments to rice seedlings using oospores, a consideraby high perce-ntage of the disease incidence may sometimes be obtained. 8. Owing to the present results, the writers consider the oospores to be fairly important infection source to wild grasses in autumn and to the rice seedlings in the nursery stage.