In 2011, 25 isolates of Mycovellosiella nattrassii causing leaf mold on eggplant were collected from eggplant fields in Kochi Prefecture and tested for sensitivity to the succinate dehydrogenase inhibitor (SDHI) boscalid. In a test to determine the effective fungicide concentration that inhibits mycelia growth 50% relative to the control (EC50), 6 of 25 isolates were less sensitive to boscalid (EC50 > 16 ppm). They were resistant to boscalid but not to pentiopyrad in an inoculation test. A common nucleotide change was found in their SdhB gene encoding a target protein of boscalid, and the mutation was detected by PCR-RFLP.
For the stable production of Russell prairie gentian (Eustoma grandiflorum) flowers in hydroponic culture, we tested various applications of a granular mixture of azoxystrobin and metalaxyl M. The results indicated that application of 0.25 or 0.5 g/seedling of the mixture to the top of each peat block in the cell tray soon after transplantation of seedlings suppressed root rot caused by Pythium spinosum. This concentration of fungicidal ingredients in the nutrient solution is lower than the regulatory limit imposed by the Japanese government. The tests were repeated over 2 years, and similar results were obtained. Based on these results, we can control of root rot in Russell prairie gentian with a granular mixture of azoxystrobin and metalaxyl M.
Typical symptoms of bacterial canker disease (e.g., cankers with red exudates, white ooze exuding from pruning cuts, browning and discoloration beneath bark) were observed on trunks, branches and twigs of Actinidia deliciosa ‘Hayward’ in Nagano Prefecture, Japan, in April 2015. Thirteen strains of the causal bacterium were isolated from the diseased twigs and demonstrated by inoculation and reisolation to be pathogenic on A. deliciosa. The strains were gram-negative, aerobic rods with one to two polar flagella, and formed opaque, pale yellowish circular colonies. On the basis of biochemical and physiological characterization, PCR assays targeting ITS, hopZ3 and hopO1-2, and a multilocus sequence analysis (MLSA) using concatenated sequences of seven housekeeping genes (acnB, cts, gapA, gyrB, pfk, pgi and rpoD), we identified the pathogen as Pseudomonas syringae pv. actinidiae (Psa). However, the pathogen proved to be different from the four existing biovars (biovars 1, 2, 3 and 5) of Psa based on phenotypic characterization using API 20NE, and additional PCR assays, which showed that the pathogen did not possess hopH1, hopH3 or hopZ5 genes. Also, the results of the MLSA revealed that they clustered separately from the other Psa biovars. In addition, bioassays confirmed that they produced both phaseolotoxin and coronatine, although no plant pathogenic bacteria have ever been identified as producing both toxins. The combined results of genotypic and phenotypic analyses therefore support the classification of these strains as a novel biovar of Psa, for which the name “biovar 6” is proposed. In addition to biovar 6, three other biovars (biovars 1, 3 and 5) of Psa were confirmed as bacterial canker pathogens of kiwifruit in Japan. Although the distribution of biovar 6 is extremely limited at present, it may play an important role in helping us to understand the origin and evolution of Psa. Our results also show that toxin-related genes, which could be transmissible among bacteria, are not adequate markers to discriminate Psa biovars, although phaseolotoxin and coronatine biosynthetic genes have been utilized as PCR targets to identify biovars 1 and 2, respectively.
Among Rhizobium species, six species include plant pathogens as a member: R. radiobacter species complex (including R. nepotum and R. pusense), R. rhizogenes, R. vitis, R. rubi, R. larrymoorei, and R. skierniewicense. Based on their pathogenic states, the members belonging to the six species can be divided into three types, namely, crown gall bacteria carrying Ti plasmid, hairy root bacteria carrying Ri plasmid, and nonpathogenic bacteria carrying neither pathogenic plasmids. In this study, we developed a plasmid-profiling method using a multiplex colony-direct PCR to identify any pathogenic plasmid present in the respective strains and thus determine their pathogenic state. For that purpose, a universal primer set for both pathogenic plasmids and a specific primer set for the Ri plasmid were designed based on the sequences of virC1 and rolC, respectively. The universal primer set for 16S rRNA gene (16S rDNA), chosen as an internal control, was also added to the PCR mix to readily judge the success of the reaction and exclude false-negative reactions. Reliability of the method as a plasmid-profiling tool was then validated using the Rhizobium species containing plant pathogens and their relatives of 383 strains in total. As a result, two DNA fragments, one from virC1-specific amplification (278 bp) and the other (780–784 bp) from 16S rDNA as the internal control, were stably obtained from all crown gall bacteria used. For hairy root bacteria, three fragments derived from virC1 (278 bp), rolC (438 bp) and 16S rDNA were always observed. On the other hand, only a fragment derived from 16S rDNA was amplified from the other nonpathogenic bacteria. Therefore, this method is useful for rapid plasmid profiling of the Rhizobium species containing plant pathogens, which will improve the efficiency in areas such as surveillance and diagnosis of crown gall and hairy root diseases and epidemiological and ecological studies of the pathogens.