Yoshinao Aoki

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Abstract Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.

Isolation and characterisation of Bacillus amyloliquefaciens S13-3 as a biological control agent for anthracnose caused by Colletotrichum gloeosporioides

Abstract Bacillus amyloliquefaciens S13-3 was isolated from soil as a biological control agent for anthracnose caused by Colletotrichum gloeosporioides. In vitro bioassay demonstrated that S13-3 suppressed C. gloeosporioides mycelial growth. The biocontrol activity of S13-3 toward grape ripe rot caused by C. gloeosporioides was confirmed in a vineyard. S13-3 also reduced the severity of strawberry anthracnose caused by C. gloeosporioides on detached strawberry leaves. The finding that S13-3 showed tolerance to some chemical pesticides and copper suggested that S13-3 could be applied before and/or after chemical pesticide or Bordeaux mixture treatment in the field. S13-3 possesses ituD and lpa-14 genes, both of which play a role in iturin A production, in its genome and expressed their transcripts during culture. Iturin A production by S13-3 was detected in the culture medium and its concentration was 0.064 mg/mL in the medium after culture for 6 days, suggesting that the antagonistic activity of S13-3 toward C. gloeosporioides may depend on iturin A production. Due to the inhibitory effects of S13-3 on anthracnose caused by C. gloeosporioides, S13-3 is expected to contribute to the further improvement of integrated pest management systems against C. gloeosporioides, and to potentially reduce the amounts of chemical fungicides used in the field.

Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

BACKGROUND The occurrence of carboxylic acid amide (CAA)-fungicide-resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS The authors have developed a method, which utilises PCR-RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine-to-serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA-fungicide-resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. CONCLUSION It is proposed that the PCR-RFLP method is a reliable tool for the rapid detection of CAA-fungicide-resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance.

Cyclic lipopeptide iturin A structure-dependently induces defense response in Arabidopsis plants by activating SA and JA signaling pathways

Iturin A is the most well studied antifungal cyclic lipopeptide produced by Bacillus species that are frequently utilized as biological control agents. Iturin A not only shows strong antifungal activity against phytopathogens but also induces defense response in plants, thereby reducing plant disease severity. Here we report the defense signaling pathways triggered by iturin A in Arabidopsis salicylic acid (SA) or jasmonic acid (JA)-insensitive mutants. Iturin A activated the transcription of defense genes PR1 and PDF1.2 through the SA and JA signaling pathways, respectively. The role of iturin A as an elicitor was dependent on the cyclization of the seven amino acids and/or the β-hydroxy fatty acid chain. The iturin A derivative peptide, NH2-(L-Asn)-(D-Tyr)-(D-Asn)-(L-Gln)-(L-Pro)-(D-Asn)-(L-Ser)-COOH, completely suppressed PR1 and PDF1.2 gene expression in wild Arabidopsis plants. The identification of target molecules binding to iturin A and its derivative peptide is expected to shed new light on defense response in plants through the SA and JA signaling pathways.

Development of a multiplex allele-specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations

BACKGROUND DNA-based diagnosis has become a common tool for the evaluation of fungicide resistance in obligate phytopathogenic fungus Plasmopara viticola. RESULTS A multiplex allele-specific primer PCR assay has been developed for the rapid detection of fungicide resistance in P. viticola populations. With this assay, a glycine-to-alanine substitution at codon 143 of the P. viticola cytochrome b gene, which conferred QoI fungicide resistance, and a glycine-to-serine substitution at codon 1105 of the P. viticola cellulose synthase gene PvCesA3, which conferred CAA fungicide resistance, were detected simultaneously. CONCLUSION It is suggested that the present assay is a reliable tool for the rapid and simultaneous detection of QoI and CAA fungicide resistance alleles in P. viticola populations. The assay required only 2 h from the sampling of symptoms to the detection of resistance alleles to both fungicides.

Impact of Bacillus cereus NRKT on grape ripe rot disease through resveratrol synthesis in berry skin

BACKGROUND Vine growers are faced with the difficult problem of how to control grape ripe rot disease in vineyards because of fear of accumulation of pesticide residues on grape berries near harvest. Biological control is an alternative non-hazardous technique to control the diseases. RESULTS Application of resveratrol-synthesis-promoting bacterium, Bacillus cereus strain NRKT, reduced the incidence of grape ripe rot disease caused by Colletotrichum gloeosporioides in a vineyard. The application of NRKT to berry bunches upregulated the gene expression of stilbene synthase, a key enzyme for resveratrol synthesis in berry skins, thereby promoting resveratrol synthesis in berry skins. CONCLUSION The potential use of NRKT in vineyards is expected to contribute to the increase in resveratrol content in berry skins, thereby protecting grape berries against fungal diseases.

Peptidoglycan from fermentation by-product triggers defense responses in grapevine.

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses.

Detection and analysis of genetic variations in GOB locus of Plasmopara viticola by DNA sequence analysis

The population genetic structure of Plasmopara viticola in Japan was analyzed using grapevine downy mildew specimens collected from two islands, Honshu and Hokkaido. By simple sequence repeat analysis with the GOB microsatellite marker and DNA sequencing, an accurate copy number of the (CT)n(CTAT)n repeat was determined. Consequently, we found a large number of genetic variations in (CT)n and (CTAT)n repeats. Also, a single nucleotide polymorphism in the (CTAT)n repeat of the GOB locus was detected. These genetic variations may serve as a valuable tool to understand the population structure of P. viticola.

Antidiabetic effects of novel cell culture established from grapevine, Vitis vinifera cv. Koshu

Vitis vinifera cv. Koshu is an indigenous cultivar in Japan and has several characteristics that distinguish it from European V. vinifera. In Japan, Koshu is the most popular cultivar for wine making. We report herein a cell culture established from Koshu for use as a system for the production of resveratrol and its derivatives. Grape cell culture YU-1 was developed from the apex tissues of Koshu. YU-1 growth was favorably compared with BY-2 growth, a standard cell line in plant cell biology. Stilbene production and stilbene synthesis gene expression in YU-1 were upregulated by UV-C irradiation. YU-1 irradiated with UV-C decreased hemolymph sugar levels in model animals. Taken together, this study suggests that YU-1 may be used as a source of valuable medicinal components in plant cell bioreactor systems.

Characterisation of heteroplasmic status at codon 143 of the Botrytis cinerea cytochrome b gene in a semi‐quantitative AS‐PCR assay

BACKGROUND An in-depth understanding of quinone outside inhibitor (QoI)-fungicide-resistant Botrytis cinerea isolates in a vineyard is expected to contribute to the development of an optimum disease management programme for the control of grape grey mould. RESULTS The resistance and structure of the cytochrome b gene in B. cinerea collected from a Japanese vineyard were characterised. The semi-quantitative allele-specific primer-polymerase chain reaction (AS-PCR) assay developed in the present study was able to distinguish heteroplasmic status from homoplasmic status at codon 143 of the cytochrome b gene in QoI-fungicide-resistant B. cinerea from vineyards in Japan. With this assay it was demonstrated that the repeated introduction of QoI fungicide selection pressure increased the ratio of G143A-mutated cytochrome b genes in B. cinerea isolates. CONCLUSION It is proposed that the semi-quantitative AS-PCR assay is a reliable tool for the detection of QoI fungicide resistance and the evaluation of homoplasmic/heteroplasmic status at codon 143 of the cytochrome b gene in B. cinerea isolates.