Shigenobu Yoshida

Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.

Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2

Bacillus amyloliquefaciens strain RC-2 produced seven antifungal compounds (1-7) secreted into the culture filtrate. These compounds inhibited the development of mulberry anthracnose caused by the fungus, Colletotrichum dematium. Chemical structural analyses by NMR and FAB-MS revealed that all these compounds were iturins (cyclic peptides with the following sequence: L-Asn --> D-Tyr --> D-Asn --> L-Gln --> L-Pro --> D-Asn --> L-Ser --> D-beta-amino acid -->) and compounds 1-6 are identical to iturins A-2-A-7, respectively. Compound 7 (iturin A-8) is a new iturin, which has a -(CH(2))(10)CH(CH(3))CH(2)CH(3) group as a side chain in the beta-amino acid in the molecule.

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 (α-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 α-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to α-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular α-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.

Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol

The mycotoxin deoxynivalenol (DON) causes serious problems worldwide in the production of crops such as wheat and barley because of its toxicity toward humans and livestock. A bacterial culture capable of degrading DON was obtained from soil samples collected in wheat fields using an enrichment culture procedure. The isolated bacterium, designated strain WSN05-2, completely removed 1,000xa0μg/mL of DON from the culture medium after incubation for 10xa0days. On the basis of phylogenetic studies, WSN05-2 was classified as a bacterium belonging to the genus Nocardioides. WSN05-2 showed significant growth in culture medium with DON as the sole carbon source. High-performance liquid chromatography analysis indicated the presence of a major initial metabolite of DON in the culture supernatant. The metabolite was identified as 3-epi-deoxynivalenol (3-epi-DON) by mass spectrometry and 1H and 13C nuclear magnetic resonance analysis. The amount of DON on wheat grain was reduced by about 90% at 7xa0days after inoculation with WSN05-2. This is the first report of a Nocardioides sp. strain able to degrade DON and of the yet unknown 3-epi-DON as an intermediate in the degradation of DON by a microorganism.

Biodegradable plastic-degrading enzyme from Pseudozyma antarctica: cloning, sequencing, and characterization

Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61–68xa0%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8u2009±u20096.3xa0U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(ε-caprolactone), and poly(lactic acid).

Phyllosphere yeasts rapidly break down biodegradable plastics

The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands.

Thirteen novel deoxynivalenol‐degrading bacteria are classified within two genera with distinct degradation mechanisms

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.

Biological and phylogenetic characterization of Fusarium oxysporum complex, which causes yellows on Brassica spp., and proposal of F. oxysporum f. sp. rapae, a novel forma specialis pathogenic on B. rapa in Japan.

Although the causal agent of yellows of Brassica rapa (turnip, pak choi, and narinosa) in Japan was reported in 1996 to be Fusarium oxysporum f. sp. conglutinans, this classification has remained inconclusive because of a lack of detailed genetic and pathogenic studies. Therefore, we analyzed the taxonomic position of this organism using Japanese isolates of F. oxysporum complex obtained from diseased individuals of various B. rapa subspecies. Phylogenetic analyses using partial sequences of the rDNA intergenic spacer region and the mating-type gene (MAT1-1-1alpha-box) showed that B. rapa and cabbage isolates belong to different monophyletic clades that separated at early evolutionary stages. Additionally, correlations were observed between the molecular phylogeny and the vegetative compatibility groups. Isolates from turnip, komatsuna, and narinosa (B. rapa group) did not show pathogenicity against cabbage or broccoli (B. oleracea group), although they caused severe symptoms on their original host species. In contrast, cabbage isolates had significantly higher (P = 0.05) virulence on B. oleracea than on B. rapa crops. Our results indicate that F. oxysporum complex isolates from B. rapa and B. oleracea are not only phylogenetically distinct but also differ in host specificity. Therefore, we propose a novel forma specialis, F. oxysporum f. sp. rapae, which causes yellows on B. rapa, including turnip, komatsuna, pak choi, and narinosa.

Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants

To improve the biodegradation of biodegradable plastic (BP) mulch films, 1227 fungal strains were isolated from plant surface (phylloplane) and evaluated for BP-degrading ability. Among them, B47-9 a strain isolated from the leaf surface of barley showed the strongest ability to degrade poly-(butylene succinate-co-butylene adipate) (PBSA) and poly-(butylene succinate) (PBS) films. The strain grew on the surface of soil-mounted BP films, produced breaks along the direction of hyphal growth indicated that it secreted a BP-degrading enzyme, and has directly contributing to accelerating the degradation of film. Treatment with the culture filtrate decomposed 91.2u2009wt%, 23.7u2009wt%, and 14.6u2009wt% of PBSA, PBS, and commercially available BP polymer blended mulch film, respectively, on unsterlized soil within 6u2009days. The PCR-DGGE analysis of the transition of soil microbial community during film degradation revealed that the process was accompanied with drastic changes in the population of soil fungi and Acantamoeba spp., as well as the growth of inoculated strain B47-9. It has a potential for application in the development of an effective method for accelerating degradation of used plastics under actual field conditions.

Colletotrichum dematium produces phytotoxins in anthracnose lesions of mulberry leaves.

ABSTRACT Colletotrichum dematium, the causal agent of mulberry anthracnose, was examined to produce phytotoxins in vitro and in planta. Raw and autoclaved mulberry leaves infected with the fungus, as well as the fungus incubated with several solid or liquid media, were extracted with acetone. Extracts obtained from the fungus grown on raw and autoclaved mulberry leaves caused brown necrotic lesions on susceptible mulberry leaves when they were placed (10 mul) on the wounded adaxial surface. Whereas, no extracts obtained from media, except inoculated medium containing homogenized mulberry leaves, induced the necrosis, suggesting that the fungus produced phytotoxins in planta and that some components in mulberry leaves may be indispensable substrates for producing the toxins. The phytotoxins obtained from the diseased leaves induced necrosis on nonhost plants leaves as well as on mulberry leaves. The toxins were present in the border of anthracnose lesions on the leaves, and the sensitivity to the toxin correlated with that to the fungus infection in each susceptible or resistant mulberry cultivar. These results suggest that the phytotoxins are host nonspecific and play a role in fungal pathogenesis in the development of the lesions. Four toxic compounds were isolated and purified from anthracnose lesions. However, due to the low yield, the chemical structure of the compounds could not be identified.