Serological detection of red rot disease initiation stages of microbial pathogen, Pythium porphyrae (Oomycota) on Porphyra yezoensis

Pythium porphyrae (Oomycota), a pathogen causing red rot diseasein Porphyra spp., can at present only be detected when colonizationof the host thallus has already occurred and so it is often too late to takeappropriate disease control measures. The paper presents an account of an effective methdology for early detection of the disease. Since Py.porphyrae zoospores are the primary means of pathogen dispersal,polyclonal antibodies (Pabs) were raised against the surface components ofzoospores and encysted zoospores. Using these Pabs the disease initiationstages of the Pythium porphyrae were detected on the surface of Porphyra thalli by immunofluorescence assay. The specificity of theseantibodies and the efficacy of immunofluorescence assay in the detectionof red rot disease are discussed.     

Rapid detection of Pythium porphyrae in commercial samples of dried Porphyra yezoensis sheets by polymerase chain reaction

The fungal parasite Pythium porphyrae is the causative organism of red rot disease in Porphyra cultivation farms. The detection of P. porphyrae from dried Porphyra yezoensis sheets was achieved using the species-specific primers PP-1 (5′-TGTGTTCTGTGCT-CCTCTCG-3′) and PP-2 (5′-CCCAAATTGGTGTTGCCTCC-3′) with the polymerase chain reaction (PCR). The DNA sequence (707 bp) of PCR product was found to be identical to that amplified from ITS rDNA extracted from a type species of P. porphyrae (IFO 30800, The Institute of Fermentation, Osaka, Japan). Quantities of the product amplified varied with the time when samples were harvested after the occurrence of red rot disease in Porphyra farms. This simple, rapid, and inexpensive method should have great applications in furthering quality control and determination of quality ranking in the Porphyra processing industry.     

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L⁻¹), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L⁻¹)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L⁻¹), and thallus disintegration (in more than 4000 zoospores L⁻¹). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.     

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta)

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.     

紫菜腐霉激发子基因家族特征及其在感染过程中的作用

[背景]激发子(elicitin)是卵菌(Oomycetes)疫霉和腐霉分泌的可诱发宿主产生免疫反应的小分子化合物.[目的]鉴定紫菜腐霉激发子基因家族,分析其结构特征和在感染宿主过程中可能的作用机制.[方法]运用同源比对法筛查紫菜腐霉NBRC33253基因组中激发子基因家族成员,利用生物信息学工具分析激发子家族的理化性...     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Isolation and evaluation of a strain of Pyropia yezoensis (Bangiales,Rhodophyta) resistant to red rot disease

Red rot disease (Pythium porphyrae) resistant strains of Pyropia yezoensis, AP1 and AP2, were isolated from live cells taken from lesions on infected P. yezoensis blades. The degree of resistance of these strains to red rot disease was evaluated over a range of environmental conditions including temperature (10, 15, and 20 °C), salinity (20, 26, and 32 ppt), and pH (7.5, 8.0, and 8.5). These conditions are within the range that red rot disease naturally occurs on Pyropia blades. P. yezoensis and Pyropia suborbiculata with low and high partial resistance to red rot disease, respectively, were used as controls. Infection with red rot disease occurred under all environmental conditions. The incidence, severity, and expansion of the disease increased with increasing temperature and decreasing salinity and pH. The resistance of the strains P. yezoensis-AP1 and P. yezoensis-AP2 was higher than that of P. yezoensis, but lower than that of P. suborbiculata. The degree of resistance was not significantly different between the AP1 and AP2 strains. These strains can therefore be considered to exhibit stable partial resistance to P. porphyrae, and as a good starting point for the development of more resistant strains that will prevent or reduce the incidence of red rot disease on Pyropia farms.     

New species of unicellular obligate parasite, <Emphasis Type="Italic">Olpidiopsis pyropiae</Emphasis> sp. nov., that plagues <Emphasis Type="Italic">Pyropia</Emphasis> sea farms in Korea

Pyropia (Porphyra) sea farms are plagued with many diseases, similar to a land crop field. Olpidiopsis disease has been one of the most serious diseases causing multimillion dollars of economic loss every year. From 3 years of epidemiological studies in Pyropia sea farms, we found that the pathogen of Olpidiopsis disease in Korea is different from the oomycete, Olpidiopsis porphyrae, which is known to infect the commercially cultivated Pyropia yezoensis in Japan. The Korean species showed a clearly different small subunit (SSU) 18S rRNA sequence (90.4 % identity) and lacked four intron-like insertions, which are present in O. porphyrae. We therefore established a new species named Olpidiopsis pyropiae. The infection process and asexual life history of O. pyropiae were similar to O. porphyrae. Infection started when zoospores attached to the surface of Pyropia blade, lost flagella, and produced thin germ tubes that penetrated the cell walls of the host. Spherical multinucleate thalli developed within the cytoplasm of its algal host, which grew into mature sporangia within the next 2 days. The shape and size of sporangium was similar to that of the Japanese species, except for longer discharge tubes in O. pyropiae. Transmission electron microscopy revealed the presence of K-body-like organelles with tubular inclusions located close to the nucleus, which is one of the key characters of the genus. However, phylogenetic analysis based on SSU ribosomal RNA (rRNA) gene data showed a loose affinity of the Korean species with the other marine Olpidiopsis spp.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.     

Effect of organic soil amendments on the incidence of stalk rot of maize

Five organic materials, Calopogonium sp. leaves, rice straw, mixed wood saw dust of Terminalia sp. Triplochyton sp. and Kaya sp., fresh guinea grass and poultry manure were added to soil with a recent history of high stalk rot of maize caused by Macrophomina phaseolina and Fusarium moniliforme. The two stalk rot organisms reacted differently to the organic amendments. All the amendments produced significantly less Fusarium stalk rot disease than the unamended control. Amendment of soil with fresh Calopogonium leaves was almost ineffective in controlling the incidence of Macrophomina phaseolina. Organic amendment with poultry manure increased the disease casued by M. phaseolina. The other amendments appeared to be equally effective in checking the disease. Increased microbial population due to amendment may play some role in disease suppression.     

Root rot of onion caused byPythium irregulare andPythium coloratum

Pythium irregulare andPythium coloratum were isolated consistently from roots of onion plants exhibiting root rot and associated foliar symptoms in fields located in Organe County, NY.P. coloratum predominated following extremely wet weather in 1984 and 1986, whileP. irregulare was prevalent following moderately wet weather in 1985. Both species produced root rot symptoms similar to field symptoms when 12 week old onion plants (cv Downing Yellow Globe) were inoculated and incubated in a growth chamber at 14°C for 10 days. This is the first report ofP. irregulare andP. coloratum as causes of root rot of onion.     

Recent trends in nori products and markets in Asia

Since 1960s, modern techniques for nori (Porhyra) cultivation, artificial seeding of conchospores, floatingnet cultivation and freeze storage of nets, have been introduced into Japan, Korea and China. Owing to recent developments of these techniques, mechanization of processing and adaptation of fast-growing varieties of Porphyra, annual yield of nori have increased rapidly. Individual problems have, however, arisen in each country. In Japan, as the result of overproduction of nori, the farmers' profit level has, in general, decreased. As a consequence, the number of nori farmers, especially among those who would enter the industry, has declined markedly, and this is currently a serious problem. In Korea, there is a pressing need to improve the quality of nori, which is more important than increasing production. In China, the technical level of producing high quality Porphyra through cultivation and processing high quality nori is relatively undeveloped. There is thus a critical requirement to develop basic techniques in both cultivation and processing of nori in China.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Grape marc compost: microbial studies and suppression of soil-borne mycosis in vegetable seedlings

Compost suppression of soil-borne diseases in horticultural crops has been attributed to the activities of antagonistic microorganisms. A great diversity of microorganisms, capable of suppressing pathogens naturally colonize compost. A large number of microbes appeared in microbiological analyses of grape marc compost. Most microorganisms were bacteria. Average percentages were 31% mesophilic and 28% thermophylic bacteria, 16% mesophilic actinomycetes and 20% thermophylic actinomycetes. Only a few mould and yeast morphologies were obtained, 4% and 1% respectively. Antagonist in vitro assays were performed with 432 microbial morphologies isolated from grape marc compost. The microbes isolated were extremely effective antagonists in in vitro assays against all the fungal pathogens tested. Seven microorganisms were selected for further bioassay with Rhizoctonia solani in radish, Fusarium oxysporum f. sp. radicis-cucumerinum in melon, and Phytophthora parasitica in tomato and two microorganisms with Pythium aphanidermatum in cucumber. Those experiments indicate that grape marc compost reduces the severity of Pythium damping-off in cucumber, but does not reduce the severity of Phytophthora root rot in tomato, Fusarium oxysporum f. sp. radicis-cucumerinum in melon and Rhizoctonia solani in radish. Better suppressive effects were not demonstrated by either compost or vermiculite amended with microbes selected from grape marc compost.     

Fusarium root rot of coneflower seedlings and integrated control using <Emphasis Type="Italic">Trichoderma</Emphasis> and fungicides

Fusarium root rot (Fusarium spp.) is one of the most important seedling diseases of coneflower (Echinacea spp.) in Alberta greenhouses. Effects of microbial antagonists (Trichoderma spp.) and fungicides, including difenoconazole, fludioxonil, and a mixture of fludioxonil, metalaxyl and difenoconazole, on the management of this disease, were investigated in Alberta. Twenty Trichoderma isolates demonstrated antagonistic activity to Fusarium in agar plate bioassays, with inhibition rates ranging from 44 to 65%. Some Trichoderma isolates significantly ( p < 0.05) reduced disease incidence and severity on seedlings in greenhouse experiments. An in vitro bioassay indicated that difenoconazole and the mixture equally inhibited the growth of both Fusarium and Trichoderma, but, while fludioxonil strongly inhibited the growth of Fusarium, it had little effect on Trichoderma, according to the dose--response models developed ( p < 0.01, R²= 0.902-0.998). Two Trichoderma isolates, T1 and T13 were applied singly or in combination with a low rate of fludioxonil in greenhouse evaluations. The results suggested that fludioxonil and Trichoderma could be integrated into a disease management program for fusarium root rot in coneflower.     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Pathway,inhibition and regulation of methyl <Emphasis Type="Italic">tertiary</Emphasis> butyl ether oxidation in a filamentous fungus, <Emphasis Type="Italic">Graphium</Emphasis> sp.

The filamentous fungus Graphium sp. (ATCC 58400) co-metabolically oxidizes the gasoline oxygenate methyl tertiary butyl ether (MTBE) after growth on gaseous n-alkanes. In this study, the enzymology and regulation of MTBE oxidation by propane-grown mycelia of Graphium sp. were further investigated and defined. The trends observed during MTBE oxidation closely resembled those described for propane-grown cells of the bacterium Mycobacterium vaccae JOB5. Propane-grown mycelia initially oxidized the majority (∼95%) of MTBE to tertiary butyl formate (TBF), and this ester was biotically hydrolyzed to tertiary butyl alcohol (TBA). However, unlike M. vaccae JOB5, our results collectively suggest that propane-grown mycelia only have a limited capacity to degrade TBA. None of the products of MTBE exerted a physiologically relevant regulatory effect on the rate of MTBE or propane oxidation, and no significant effect of TBA was observed on the rate of TBF hydrolysis. Together, these results suggest that the regulatory effects of MTBE oxidation intermediates proposed for MTBE-degrading organisms such as Mycobacterium austroafricanum are not universally relevant mechanisms for MTBE-degrading organisms. The results of this study are discussed in terms of their impact on our understanding of the diversity of aerobic MTBE-degrading organisms and pathways and enzymes involved in these processes.     

一株高效拮抗向日葵菌核菌的细菌菌株YC16的筛选及其作用效果研究

[目的]筛选高效拮抗向日葵菌核菌的细菌菌株,为开发防治菌核菌病害、提高向日葵产量的生物菌剂提供菌种资源。[方法]以羧甲基纤维素钠(CMC)、小麦秸秆纤维素为唯一碳源的无机盐培养基,分离高效降解纤维素的细菌菌株;采用纤维素降解菌与菌核菌的平板对峙方法,进一步筛选拮抗菌核菌的菌株;利用16S rDNA序列鉴定菌株、PDYA平板对峙实验检验上述所选拮抗菌株的抑菌谱;采用离体向日葵新鲜叶片、草炭土基质盆栽实验,观察拮抗菌菌株抑制菌核菌生长的能力;温室盆栽和田间试验条件下,研究其防治向日葵菌核菌病害、促进生长和提高产量的效果。[结果]筛选了一株高效抑制菌核菌的细菌YC16,经过16S rDNA序列分析,鉴定为解淀粉芽孢杆菌。YC16菌株能够抑制8种病原真菌生长,包括齐整小核菌、腐皮镰孢菌、尖孢镰刀菌、稻梨孢、辣椒疫霉、镰刀菌、尖镰孢黄瓜专化型和向日葵菌核菌;抑制菌核菌感染叶片,抑制率达到了80.42%;抑制盆栽基质中菌核菌的菌丝生长,基质表面菌丝密度比对照减少了50%以上。盆栽接种YC16的向日葵生物量比对照提高54.9%,田间向日葵接种YC16菌剂对菌核菌引发的盘腐病防治效果达39%-100%,产量提高24.4%-30.2%。[结论]YC16生物菌剂施用于土壤,能够有效防治向日葵的茎腐病和盘腐病,展现了防治向日葵菌核病和提高产量的双重效果,是一株具有良好应用前景的高效菌种资源。     

Three new <Emphasis Type="Italic">Ophiostoma</Emphasis> species isolated from Japanese red pine

Three new species of Ophiostoma found on Japanese red pine are described as Ophiostoma pusillum sp. nov., O. botuliforme sp. nov., and O. nigrogranum sp. nov. Ophiostoma pusillum is characterized by oblong ascospores and a Hyalorhinocladiella anamorph. Ophiostoma botuliforme has ostioles covered with a hyaline gelatinous cap, allantoid ascospores, and a Pesotum anamorph with hyaline to pale brown stipes. Ophiostoma nigrogranum has hyaline ostiolar hyphae with rounded tips, allantoid ascospores, and sclerotium-like structures.Contribution no. 172, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba