Adsorption of Tolaasins, the Toxins Behind Mushroom Bacterial Blotch, by Microbacterium spp. is Insufficient for Its Detoxification.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.

Detoxification process of tolaasins, lipodepsipeptides, by Microbacterium sp. K3-5.

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.

Complete Genome Sequence of Rehmannia Mosaic Virus Infecting Rehmannia glutinosa in Japan.

The complete genome sequence of isolate Jiou of rehmannia mosaic virus (ReMV) infecting Rehmannia glutinosa in Japan was obtained via Sanger sequencing. Isolate Jiou shared high nucleotide sequence identity (>94%) with other known ReMV isolates.

Incidence of pests and viral disease on pepino (Solanum muricatum Ait.) in Kanagawa Prefecture, Japan.

BACKGROUND: The solanaceous fruit crop pepino (Solanum muricatum Ait.), originating in the Andes, is grown commercially in South American countries and New Zealand. In these areas, pests and diseases of pepino have been identified well; however, to date, these have seldom been investigated in detail in Japan. Herein, we attempt to reconstruct an agricultural production system for commercial pepino crops in Japan, and evaluate the incidence of pests and viral diseases on pepino. The findings of this study will facilitate in developing a better crop system for the commercial cultivation of healthy pepino fruits. NEW INFORMATION: A total of 11 species, comprising nine insects and two mites, were recognized as pests of pepino plants in our experimental fields in Kanagawa Prefecture, central Honshu, Japan. Of these pest species, the two-spotted spider mite Tetranychus urticae Koch, 1836 and the cotton aphid Aphis gossypii Glover, 1877, were remarkably abundant than the other pest species. Eventually, 13 species, including two previously recorded, are currently recognized as the pests of pepino in Japan. With regard to viruses, we tested two species Alfalfa mosaic virus (AMV) and Cucumber mosaic virus (CMV), as well as three genera Carlavirus, Potexvirus, and Potyvirus. No virus was detected in symptomatic pepino leaves collected in our experimental fields. This is a first report on the identification of pests on pepino plants in Kanagawa Prefecture, Japan and elucidates the relationship between currently occurring pests of pepino plants and potential viral pathogens that they can transmit.

Culturable bacterial communities on leaf sheaths and panicles of rice plants in Japan.

Culturable bacterial communities on rice plants were investigated from 2001 to 2003. In total, 1,394 bacterial isolates were obtained from the uppermost leaf sheaths at 1 month before heading time and from leaf sheaths and panicles at heading time. The average culturable bacterial population on the leaf sheaths was larger at heading time than at 1 month previously. Furthermore, the population was significantly larger on panicles than on leaf sheaths, suggesting that the bacterial population is influenced by the organs of rice plants. Larger proportions of bacteria were obtained from the macerates of leaf sheaths after washing with phosphate buffer, and most culturable bacteria were verified to inhabit the inside or inner surface, rather than the outer surface, of the tissues. Verification of the bacterial composition based on 16S rRNA gene sequences revealed that genera of Sphingomonas, Microbacterium, Methylobacterium, and Acidovorax tended to be dominant colonizers on leaf sheaths, whereas Pseudomonas and Pantoea were isolated mainly from the panicles, indicating that leaf sheaths and panicles harbor distinct communities. Furthermore, the richness of bacterial genera was less on both leaf sheaths and panicles at heading time compared with that observed 1 month before heading time. Phylogenetic analyses using bacterial isolates belonging to the four dominant genera inhabiting leaf sheaths at heading time revealed that particular bacterial groups in each genus colonized the leaf sheaths.

Culturable leaf-associated bacteria on tomato plants and their potential as biological control agents.

Culturable leaf-associated bacteria inhabiting a plant have been considered as promising biological control agent (BCA) candidates because they can survive on the plant. We investigated the relationship between bacterial groups of culturable leaf-associated bacteria on greenhouse- and field-grown tomato leaves and their antifungal activities against tomato diseases in vitro and in vivo. In addition, the isolated bacteria were analyzed for N-acyl-homoserine lactone (AHL) and indole-3-acetic acid (IAA) production, which have been reported to associate with bacterial colonization, and resistance to a tomato alkaloid (alpha-tomatine). Leaf washings and subsequent leaf macerates were used to estimate the population size of epiphytic and more internal bacteria. Bacterial population sizes on leaves at the same position increased as the leaves aged under both greenhouse and field conditions. Field-grown tomatoes had significantly larger population sizes than greenhouse-grown tomatoes. Analysis of 16S rRNA gene (rDNA) sequencing using 887 culturable leaf-associated bacteria revealed a predominance of the Bacillus and Pseudomonas culturable leaf-associated bacterial groups on greenhouse- and field-grown tomatoes, respectively. Curtobacterium and Sphingomonas were frequently recovered from both locations. From the 2138 bacterial strains tested, we selected several strains having in vitro antifungal activity against three fungal pathogens of tomato: Botrytis cinerea, Fulvia fulva, and Alternaria solani. Among bacterial strains with strong in vitro antifungal activities, Bacillus and Pantoea tended to show strong antifungal activities, whereas Curtobacterium and Sphingomonas were not effective. The results indicated the differences in antifungal activity among predominant bacterial groups. Analysis of alpha-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to alpha-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular alpha-tomatine resistance, and AHL and IAA production had the highest protective value (91.7) against gray mold. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability of BCAs on tomato plants.

Phylogenetic study and multiplex PCR-based detection of Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli using gyrB and rpoD sequences.

In order to develop a detection method for the rice pathogens Burkholderia plantarii, Burkholderia glumae and Burkholderia gladioli, the phylogeny of six plant-pathogenic Burkholderia species was analysed using the combined nucleotide sequences of gyrB and rpoD. B. plantarii, B. glumae and B. gladioli formed tight monophyletic branches supported by high bootstrap probabilities. The high sequence similarity revealed a close phylogenetic relationship between B. glumae and B. plantarii. B. plantarii strains were divided into three subclusters comprising rice strains, whereas the single Vanda strain occupied a unique position in the phylogenetic tree. The gyrB and rpoD sequences of all B. glumae strains examined were highly conserved. In contrast, B. gladioli strains demonstrated a far greater sequence diversity, but this diversity did not correlate with pathovar, host plant or geographical origin of the strains. A multiplex-PCR protocol using specific primers from the gyrB sequences was designed that allowed the specific detection and identification of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these pathogenic species.

Production of quorum-sensing-related signal molecules by epiphytic bacteria inhabiting wheat heads.

The production of quorum-sensing-related signal molecules (QSRMs) among culturable bacteria comprising the community on wheat heads was investigated. The taxonomic position of 186 bacterial isolates obtained from ten heads was inferred based on 16S rRNA gene sequences, and their QSRM production was determined using two bioreporter strains of N-acylhomoserine lactones. Approximately 33% of isolates produced QSRMs, though the proportion of QSRM-producing isolates on a wheat head was significantly negatively correlated with population size. Most of the producing isolates were Pantoea species, most commonly Pantoea ananatis. Furthermore, the proportion of Pantoea ananatis that produced QSRMs was significantly negatively correlated with the number of bacterial genera (community richness) on each head. Finally, community richness was positively correlated with population size. Qualitative analysis using thin-layer-chromatography revealed that the QSRMs of Pantoea isolates were composed of at least two compounds. This is the first report indicating that Pantoea ananatis isolates inhabiting wheat heads are capable of producing QSRMs. QSRM production by Pantoea spp. may contribute to the predominance of this genus on wheat heads, particularly at relatively low population densities and community diversity.

Cloning and characterization of a gene rpg1 encoding polygalacturonase of Rhizopus oryzae.

The polygalacturonase (PG)-encoding gene (rpg1) of Rhizopus oryzae, the causal pathogen of rhizopus rot of mulberry, was cloned and sequenced. PGs were partially purified from incubation mixture of 2% pectin medium and their N-terminal amino acid sequences were determined by a gas-phase protein sequencer. RT-PCR was performed using degenerate primers designed from the amino acid sequences, which resulted in part of a PG-encoding gene being obtained. By 3'-RACE and TAIL-PCR analyses, the entire region of the PG-encoding gene was cloned and sequenced. The structural gene comprised 1199 bp coding for 383 amino acids with a putative signal peptide of 26 amino acids, and the open reading frame was interrupted by single intron of 47 bp. Phylogenetic analysis using the deduced amino acid sequence revealed that R. oryzae RPG1 belonged to a clade consisting of exo-PGs of ascomycete fungi.