Distribution of photo-induced and non-photo-induced sporulator physiotypes ofBipolaris oryzae in Japan

The distribution of three sporulator physiotypes ofBipolaris oryzae, namely, photo-induced, and non-photo-induced (I) and (II), was investigated. Of 407 isolates, 99% belonged to the photo-induced type, in which conidial development was under photo-control of the antagonistic action of blue/UV-A and near-UV radiation mediated through the ‘mycochrome’ system at conidiophore induction and conidiophore maturation stages. Of the remainder, 1 isolate belonged to the non-photo-induced (I) type, and 4 isolates belonged to the non-photo-induced (II) type. Conidial development in the former of these was photo-controlled by the ‘mycochrome’ system at conidiophore maturation stage alone, while in the latter it was not affected by light conditions. No difference was found between the three physiotypes in restriction fragment length polymorphisms (RFLPs) of rDNA. However, random amplified polymorphic DNA (RAPD) revealed polymorphisms between photo-induced and non-photo-induced isolates and showed that non-photo-induced (I) and (II) strains were clustered in the same group, suggesting that they are genetically close. Photo-induced sporulators ofB. oryzae were confirmed to be widely distributed in paddy fields in Japan.     

Characterization of the BLR1 gene encoding a putative blue-light regulator in the phytopathogenic fungus Bipolaris oryzae

Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR1, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white-collar 1 (WC-1). The deduced amino acid sequence of BLR1 showed high degrees of similarity to other fungal blue-light regulator protein. Disruption of the BLR1 gene demonstrated that this gene is essential for conidial development after conidiophore formation and for near-UV radiation-enhanced photolyase gene expression.     

Identity of Pyrenophora isolates causing net-type and spot-type lesions on barley

Causative fungi of net-type and spot-type lesions on barley were isolated and identified as anamorphs of Pyrenophora teres and Pyrenophora japonica respectively. Comparison of morphological characteristics showed that isolates of the two species are separable on the basis of differences in colony texture, conidiophore shape, conidial arrangement and conidial dimensions. The two species could also be distinguished from each other on account of adult-plant reactions exhibited by Dampier barley after inoculation by various isolates from South Africa, one isolate from Germany and one isolate from Washington State in North America.     

Inheritance of photo-control of conidial development in the fungusBipolaris oryzae

The inheritance of light dependence for conidial development inBipolaris oryzae was analyzed using single-ascospore isolates. When a photo-induced strain was crossed with another photo-induced strain, only photo-induced progeny were produced. When a photo-induced strain was crossed with a non-photo-induced (I) strain, photo-induced and nonphoto-induced (I) progeny were produced in a ratio of 1∶1. However, when a photo-induced strain was crossed with a non-photo-induced (II) strain, a non-photo-induced (I) progeny were formed in addition to parental types. It was suggested that genes for photo-control of conidiophore induction and genes for photo-control of conidiophore maturation are located on the same chromosome.     

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.     

Colonization of roots of cultivated Solanum lycopersicum by dark septate and other ascomycetous endophytes

Tomato (Solanum lycopersicum L.) roots from four different crop sites in Colombia were surface sterilized and 51 fungal isolates were obtained and conserved for further analysis. Based on microscopical observations and growth characteristics, 20 fungal isolates corresponded to genus Fusarium, six presented asexual conidia different from Fusarium, eight were sterile mycelia, seven of which had dark septate hyphae and 17 did not continue to grow on plates after being recovered from conservation. Growth on different media, detailed morphological characterization and ITS region sequencing of the six sporulating and eight sterile isolates revealed that they belonged to different orders of Ascomycota and that the sterile dark septate endophytes did not correspond to the well known Phialocephala group. Interactions of nine isolates with tomato plantlets were assessed in vitro. No effect on shoot development was revealed, but three isolates caused brown spots in roots. Colonization patterns as analyzed by confocal microscopy differed among the isolates and ranged from epidermal to cortical penetration. Altogether 11 new isolates from root endophytic fungi were obtained, seven of which showed features of dark septate endophytes. Four known morphotypes were represented by five isolates, while six isolates belonged to five morphotypes of putative new unknown species.     

可可西里低温适生拮抗芽孢杆菌的筛选鉴定及脂肽化合物分析

极端环境特殊微生物资源研究和开发具有广阔的应用前景和研究意义.对分离筛选自青海可可西里境内植被根围的8株在4和10 ℃条件下生长良好的低温适生芽孢杆菌进行鉴定分析.结果表明: 通过生理生化特征分析、rep-PCR指纹图谱分析、16S rDNA及gyrB基因序列分析鉴定,8株供试菌株分别为莫哈韦芽孢杆菌(Bacillus mojavensis)3株,解淀粉芽孢杆菌(B. amyloliquefaciens)1株和简单芽孢杆菌(B. simplex)4株.采用平板对峙试验从中筛选到4株对油菜菌核病原真菌(Sclerotinia sclerotiorum)及水稻白叶枯病原细菌(Xanthomonas oryzae pv. oryzae)均具有显著拮抗活性的生防菌株;采用MALDI-TOF-MS质谱分析生防菌株的拮抗活性物质,结果显示菌株KKD-1 (B. mojavensis)产生脂肽类化合物泛革素和表面活性素,菌株KKD-2(B. amyloliquefaciens)产生脂肽类化合物伊枯草菌素A、泛革素和表面活性素,推断生防菌株的拮抗活性可能与脂肽化合物的合成及分泌有关.该研究为低温适生性芽孢杆菌生物肥料和生物农药的研发提供了菌株资源.     

Light regulation of asexual development in the rice blast fungus, Magnaporthe oryzae

Light is a major environmental factor that influences many biological processes. We characterized the roles of light in asexual development (including the formation of aerial hyphae and conidiophore) in Magnaporthe oryzae, which is the causal agent of rice blast disease. Our data revealed a complex nature of light regulation in the asexual developments of M. oryzae. Asexual development of M. oryzae is suppressed by blue light in a light/dark cycling environment and asexual spore release is controlled by both blue and red light. We demonstrated that even very dim light, about 10 micromol m(-2), is sufficient to suppress spore-release behavior in M. oryzae. We also generated knockout strains of a blue light receptor, mgwc-1, the M. oryzae homolog of white collar-1 in Neurospora crassa, and demonstrated blue-light-specific regulation in the asexual development and spore release in M. oryzae. Our findings in this agriculturally important pathogen, M. oryzae, broaden our understanding of the roles of light in fungal development.     

Light-Dependent Induction of Sekiguchi Lesion Formation by Bipolaris oryzae in Rice cv. Sekiguchi-asahi

The effect of light irradiation on Sekiguchi lesion formation induced by infection with Bipolaris oryzae on rice sl mutants was investigated. Although the Sekiguchi lesions formed exclusively under daylight fluorescent lamps, brown spots and necrotic spot lesions formed in the dark. The Sekiguchi lesions were larger than the brown spot lesions. When leaf blades inoculated with B. oryzae were irradiated through band-pass filters of BPB-45, BPB-50, BPB-55, and BPB-60, Sekiguchi lesions were formed regardless of the BPB filters' wavelengths. Under black-light lamps, near-infrared fluorescent lamps, or in the dark, however, brown spot lesions were induced by B. oryzae . The effective wavelength region for light-dependent Sekiguchi lesion formation was 400–700 nm. Furthermore, there was a direct correlation between the length of the radiation wavelengths and the size of the Sekiguchi lesions.     

The cell cycle gene MoCDC15 regulates hyphal growth, asexual development and plant infection in the rice blast pathogen Magnaporthe oryzae

Rice blast, caused by the pathogen Magnaporthe oryzae, is a serious hindrance to rice production and has emerged as an important model for the characterization of molecular mechanisms relevant to pathogenic development in plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M.oryzae infection and spread over a large area. However, relatively little is known regarding the molecular mechanisms that underlie conidiation in M. oryzae. To better characterize these mechanisms, we identified a conidiation-defective mutant, ATMT0225B6 (MoCDC15(T-DNA)), in which a T-DNA insertion disrupted a gene that encodes a homolog of fission yeast cdc15, and generated a second strain containing a disruption in the same allele (ΔMoCDC15(T-DNA)). The cdc15 gene has been shown to act as a coordinator of the cell cycle in yeast. Functional analysis of the MoCDC15(T-DNA) and ΔMoCDC15(T-DNA) mutants revealed that MoCDC15 is required for conidiation, preinfection development and pathogenicity in M. oryzae. Conidia from these mutants were viable, but failed to adhere to hydrophobic surface, a crucial step required for subsequent pathogenic development. All phenotypic defects observed in mutants were rescued in a strain complemented with wild type MoCDC15. Together, these data indicate that MoCDC15 functions as a coordinator of several biological processes important for pathogenic development in M. oryzae.     

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

The Supernatant of a Conidial Suspension of Magnaporthe oryzae Contains a Factor that Promotes the Infection of Rice Plants

Many factors produced by the pathogen Magnaporthe oryzae enhance its ability to infect rice. We found a novel infection-promoting activity in the supernatant of a conidia suspension (SCS) of M . oryzae . The addition of SCS promoted the invasion of excised rice leaf sheaths by infectious hyphae. The activity was heat-stable and was found in SCSs from five virulent and three avirulent isolates of M . oryzae on the rice cv. Nipponbare ( Pia ). The effect was exclusively detected in compatible interactions. The infection of rice plants by non-rice blast fungi was not enhanced by SCS. These results suggest that SCS includes a heat-stable factor(s) that promotes M . oryzae infection during compatible interactions.     

Plasmid and pathogenicity in Xanthomonas oryzae pathovar oryzae, the bacterial blight pathogen of Oryza sativa

Xanthomonas oryzae pv. oryzae , the causative agent for bacterial leaf blight of rice, comprises diverse groups of strains differing in biochemical and pathological characteristics. A collection of X.o . pv. oryzae strains differing in geographical origin was screened for the presence of plasmids. Out of 17 isolates of X.o. pv. oryzae , 14 harboured plasmids of which two isolates (XoP5, XoC26) had two plasmids each and one isolate (XoR20) had three. The remaining isolates contained a single plasmid of identical mobility. Finger print analysis of plasmids was carried out using Eco RI for 10 isolates. The restriction fragment pattern was distinct for each isolate. They were classified under three groups based on cluster analysis using the unweighted pair group method with averages (UPGMA). Of the 18 plasmids, the plasmid pMA36 ( X.o. pv. oryzae XoC36) was further characterized. This plasmid was cured by acridine orange at the frequency rate of 10%. The cured strain was transformed with pMA36 at a frequency of 2.3 times 10² transformants μg^-1 of plasmid DNA. The plasmid-cured strain was virulent on rice but symptom development was delayed when compared to wild and transformed strains. The wild type strain ( X.o. pv. oryzae XoC36) was resistant to ampicillin, carbenicillin and rifampicin whereas the cured strain was resistant to carbenicillin and rifampicin but sensitive to ampicillin. The transformant was resistant to the three antibiotics indicating that the plasmid pMA36 codes for ampicillin resistance. The plasmid influenced the pathogenicity of X.o. pv. oryzae.     

Action spectra for the blue and near ultraviolet reversible photoreaction in the induction of fungal conidiation

Induction of conidiation in Alternaria tomato (Cke.) Weber and Helminthosporium oryzae V. Breda de Haan was repeatedly controlled by ulternating doses of near ultraviolet light and blue light. These fungi did not form conidia in total darkness. However, when dark-grown colonies were exposed to near altraviolet light, conidiophore formation was induced, and conidia were then produced upon return to darkness. Conidiation was inhibited by blue light applied immediately after near ultraviolet light. Furthermore, the final response depended on the last kind of light received. The action spectrum for induction of conidiation showed a peak near 300 nm with a shoulder between 310 nm and 330 nm, as is common in photoconidiation of various fungi. The action spectrum for the reversion of induction of conidiation showed a peak near 447 nm with a shoulder between 400 nm and 440 nm, one minor peak around 478 nm and another one near 385 nm. This is very similar to the spectrum for the ubiquitous blue light receptor (cryptochrome).     

不同寄主来源的串珠镰孢生物学性状及其在无性后代的遗传与变异

自安徽、山东、湖北、江苏等地多点采集棉花红腐病、水稻恶苗病、玉米穗腐病的病组织,经分离、鉴定和纯化,获得107个串珠镰孢（Fusarium monilifoFine）菌株。对上述来源于棉花、水稻、玉米的串珠镰孢的菌落形态、生长速率和产孢量等生物学性状及其在无性后代的遗传与变异进行了研究。结果表明,不同寄主来源的串珠镰孢菌株的菌丝生长温度范围和最适温度大致相同,但在菌落形态特别是色素方面存在明显差异,生长速率和产孢量也存在显著差异。棉花菌株的平均生长速率最大,玉米菌株生长速率最小,水稻菌株生长速率居中,相同群体的不同菌株间生长速率有极显著差异;玉米菌株产孢量最大,棉花菌株产孢量最小,水稻菌株产孢量居中。方差分析显示,不同寄主菌株群体间产孢量存在显著差异,而同一寄主群体的不同菌株间产孢量均无显著差异,说明菌株产孢量大小主要与其寄主种类有关,而与地区来源关系不大。遗传测定结果表明,分离自棉花、玉米和水稻的串珠镰孢的菌落形态和生长速率在单分生孢子后代均可稳定遗传;产孢量性状遗传有两种情况：分离自棉花和水稻的串珠镰孢菌株Fm1和Fm31的产孢量性状在单分生孢子后代均可稳定遗传,而分离白玉米的串珠镰孢菌株Fm19的产孢量性状在单分生孢子第一代（CG1）发生变异。     

Candida krusei and Kloeckera apis inhibit the causal agent of pineapple fusariosis, Fusarium guttiforme

Studies based on microbial ecology and antagonistic interactions play an important role in the development of new alternative strategies in controlling plant pathogens and are relevant to further biotechnological applications. Antagonistic interactions between the yeasts Candida krusei and Kloeckera apis isolated from rotten pineapple fruits, and two isolates of the pathogenic filamentous fungus Fusarium guttiforme (Syn.: Fusarium subglutinans f. sp. ananas) resistant and susceptible to fungicide benzimidazole were studied in broth culture, and on plate assays. The yeasts significantly reduced Fusarium conidial germination after 24h of cocultivation in broth culture, and also mycelial growth on plate assays. Slide coculture appeared to show attachment of yeasts to the hyphal surface and also slight morphological abnormalities caused by C. krusei. Filtrates of cocultures of fungi and yeasts inhibited fungal growth, but filtrates of the yeast cultures alone did not, suggesting that the antagonistic action of the yeasts is inducible. The F. guttiforme isolate sensitive to benzimidazole was most affected by both yeasts in pineapple juice, reaching a maximum of 36.5?% germ tube inhibition. This isolate was also inhibited by yeasts in mycocinogenic plate assay. These results demonstrated that C. krusei and K. apis are effective in inhibiting F. guttiforme growth and that the mode of action is associated with hyperparasitism and mycocinogenic activity.     

青海柴达木极端干旱沙地分离芽孢杆菌的分子鉴定及拮抗活性分析

【目的】研究高原极地环境微生物资源。【方法】采用rep-PCR指纹图谱分析、gyrB基因及16S rDNA基因序列分析等多项分子鉴定技术对分离自青海柴达木极端干旱沙地的8株芽孢杆菌菌株进行分类鉴定;通过平板对峙及接种离体叶片试验检测分离菌株的拮抗活性及对病原菌侵染的防效;采用MALDI-TOF-MS质谱分析生防菌株的活性成分。【结果】8株分离菌株鉴定为Bacillus amyloliquefaciens(6株)、Bacillus axarquiensis(1株)和Bacillus atrophaeus(1株);各菌株对油菜菌核病原真菌(Sclerotinia sclerotiorum)均具有显著的拮抗活性;接种离体叶片试验表明菌株对油菜菌核病菌的侵染具有较好防效;MALDI-TOF-MS质谱分析结果显示菌株DGL1(B.amyloliquefaciens)产生脂肽化合物Fengycin,菌株DGL6(B.axarquiensis)产生脂肽化合物Surfactin、BacillomycinsD和Fengycin,菌株DCD1(B.atrophaeus)产生脂肽化合物Surfactin、Fengycin。【结论】为高原干旱沙地极端环境微生物资源研究及生防菌资源开发和应用提供了研究材料。     

Isolation of Bacillus strains from the rhizosphere of cereals and in vitro screening for antagonism against phytopathogenic, food-borne pathogenic and spoilage micro-organisms

Bacillus strains were isolated from the rhizosphere of cereals in order to be used as natural biocontrol agents (BCAs). They were screened for antagonism in vitro against various test micro-organisms. The isolates showing antagonism were identified to species level. A combination of techniques was employed for the isolation of Bacillus species. Using the direct method, only one of the 25 isolates screened showed antagonistic properties. This strain (IFS-01) was identified by means of API test strips and the ATB Plus computer programme. It proved to be Bacillus subtilis and consequently has been designated as Bacillus subtilis IFS-01. This strain produced either a broad spectrum antimicrobial compound or several compounds with different activities. The fungi and Gram-positive bacteria were more sensitive to the antagonistic isolate than the Gram-negative bacteria. A Bacillus strain producing BCAs which can be used as biopesticides or organic preservatives has been isolated and identified.