Identification of an entomopathogenic bacterium, Xenorhabdus ehlersii KSY, from Steinernema longicaudum GNUS101 and its immunosuppressive activity against insect host by inhibiting eicosanoid biosynthesis.

Steinernema longicaudum GNUS101, an entomopathogenic nematode, was isolated from soils in Korea. Its internal transcribed space sequence was highly similar to the known S. longicaudum species. Infective juveniles (IJs) of S. longicaudum were highly virulent to lepidopteran and coleopteran insects. Two different bacteria were isolated from the hemolymph of lepidopteran larvae infected with S. longicaudum. They exhibited blue and red colonies on nutrient bromothymol blue agar. The red-colored bacterium was identified as Enterococcus mundtii KHY while the blue-colored bacterium was identified as Xenorhabdus ehlersii KSY based on 16S rRNA sequencing and biochemical characters. The bacterial species showed different growth rates, with X. ehlersii KSY growing more slowly than E. mundtii KHY. Both bacteria were entomopathogenic, but showed differences in suppressing host immune responses. X. ehlersii KSY, but not E. mundtii KHY, showed inhibitory activity against cellular immune responses of Spodoptera exigua larvae including hemocyte-spreading behavior and nodule formation in bacteria-cultured broth. Its immunosuppressive activity was reversed by adding arachidonic acid, an eicosanoid biosynthesis precursor. Furthermore, organic extracts of X. ehlersii KSY using hexane or ethyl acetate showed inhibitory activity against cellular immune responses of S. exigua larvae. Arachidonic acid addition to S. exigua larvae infected with X. ehlersii significantly rescued the survival rate of target insect. Of the two bacteria isolated from S. longicaudum GNUS101, only X. ehlersii induced immunosuppression of target insect by inhibiting eicosanoid biosynthesis.

Kushenol A and 8-prenylkaempferol, tyrosinase inhibitors, derived from Sophora flavescens.

Tyrosinase is known for an enzyme that plays a key role in producing the initial precursor of melanin biosynthesis. Inhibition of the catalytic reaction of this enzyme led to some advantage such as skin-whitening and anti-insect agents. To find a natural compound with inhibitory activity towards tyrosinase, the five flavonoids of kushenol A (1), 8-prenylkaempferol (2), kushenol C (3), formononetin (4) and 8-prenylnaringenin (5) were isolated by column chromatography from a 95% methanol extract of Sophora flavescens. The ability of these flavonoids to block the conversion of L-tyrosine to L-DOPA by tyrosinase was tested in vitro. Compounds 1 and 2 exhibited potent inhibitory activity, with IC50 values less than 10 µM. Furthermore, enzyme kinetics and molecular docking analysis revealed the formation of a binary encounter complex between compounds 1-4 and the enzyme. Also, all of the isolated compounds (1-5) were confirmed to possess antioxidant activity.

Benzaldehyde Synergizes the Response of Female Xyleborinus saxesenii (Coleoptera: Curculionidae, Scolytinae) to Ethanol.

The ambrosia beetle, Xyleborinus saxesenii Ratzeburg (Coleoptera: Curculionidae, Scolytinae), infests physiologically stressed apple and peach trees in Korea. Dispersing females utilize the degradation product ethanol and host-related volatiles to locate and colonize new host trees. We examined the extent to which 12 chemicals emitted from fruit trees act synergistically with ethanol to attract X. saxesenii. The addition of benzaldehyde to ethanol significantly increased beetle attraction, although benzaldehyde was not attractive by itself. The addition of (-)-α-pinene, ethyl butyrate, ethyl isovalerate, (R)-(+)-limonene, 3-methyl-1-butanol, ethyl tiglate, (+)-aromadendrene, vanillin, 2-butanol, styrene, or ethyl 3,3-dimethylacrylate to ethanol had no effect on beetle attraction. In a dose-response test, the addition of 5-50% benzaldehyde doses synergistically increased the number of beetle captures; however, trap catches did not increase as the benzaldehyde dosage increased. The synergistic influence of benzaldehyde on beetle response to ethanol was lower in early spring than in late summer to early fall, probably because synthetic benzaldehyde emissions from field lures were overwhelmed by background levels of natural benzaldehyde emitted from peach twigs in the flowering stage.

Cutaneous Microflora from Geographically Isolated Groups of Bradysia agrestis, an Insect Vector of Diverse Plant Pathogens.

Larvae of Bradysia agrestis, an insect vector that transports plant pathogens, were sampled from geographically isolated regions in Korea to identify their cutaneous fungal and bacterial flora. Sampled areas were chosen within the distribution range of B. agrestis; each site was more than 91 km apart to ensure geographical segregation. We isolated 76 microbial (fungi and bacteria) strains (site 1, 29; site 2, 29; site 3, 18 strains) that were identified on the basis of morphological differences. Species identification was molecularly confirmed by determination of universal fungal internal transcribed spacer and bacterial 16S rRNA gene sequences in comparison to sequences in the EzTaxon database and the NCBI GenBank database, and their phylogenetic relationships were determined. The fungal isolates belonged to 2 phyla, 5 classes, and 7 genera; bacterial species belonged to 23 genera and 32 species. Microbial diversity differed significantly among the geographical groups with respect to Margalef's richness (3.9, 3.6, and 4.5), Menhinick's index (2.65, 2.46, and 3.30), Simpson's index (0.06, 0.12, and 0.01), and Shannon's index (2.50, 2.17, and 2.58). Although the microbial genera distribution or diversity values clearly varied among geographical groups, common genera were identified in all groups, including the fungal genus Cladosporium, and the bacterial genera Bacillus and Rhodococcus. According to classic principles of co-evolutionary relationship, these genera might have a closer association with their host insect vector B. agrestis than other genera identified. Some cutaneous bacterial genera (e.g., Pseudomonas) displaying weak interdependency with insect vectors may be hazardous to agricultural environments via mechanical transmission via B. agrestis. This study provides comprehensive information regarding the cutaneous microflora of B. agrestis, which can help in the control of such pests for crop management.

Identification and bacterial characteristics of Xenorhabdus hominickii ANU101 from an entomopathogenic nematode, Steinernema monticolum.

An entomopathogenic nematode, Steinernema monticolum, was collected in Korea. Its identity was confirmed by morphological and molecular characters. Its symbiotic bacterium, Xenorhabdus hominickii ANU101, was isolated and assessed in terms of bacterial characteristics. Sixty-eight different carbon sources were utilized by X. hominickii ANU101 out of 95 different sources from a Biolog assay. Compared to other Xenorhabdus species, X. hominickii ANU101 was relatively susceptible to high temperatures and did not grow above 34°C. Furthermore, its growth rate was much slower than other Xenorhabdus species. X. hominickii exhibited insecticidal activities against coleopteran, dipteran, and lepidopteran insect pests. The bacterial virulence was not correlated with its host nematode virulence with respect to relative insecticidal activity against target insects. X. hominickii ANU101 exhibited antibiotics tolerance. The bacterium possesses four different plasmids (Xh-P1 (104,132bp), Xh-P2 (95,975bp), Xh-P3 (88,536bp), and Xh-P4 (11,403bp)) and encodes 332 open reading frames. Subsequent predicted genes include toxin/antitoxins comprising a multidrug export ATP-binding/permease. This study reports bacterial characters of X. hominickii and its entomopathogenicity.

First Report of Myrothecium roridum Causing Leaf and Stem Rot Disease on Peperomia quadrangularis in Korea.

In 2010, symptoms of leaf and stem rot were observed on potted plants (Peperomia quadrangularis) in a greenhouse in Yongin, Korea. The causative pathogen was identified as Myrothecium roridum based on morphological data, internal transcribed spacer sequence analysis, and pathogenicity test. To our knowledge, this is the first report of M. roridum causing leaf and stem rot disease on P. quadrangularis in Korea and elsewhere worldwide.

Development of a high efficient "Dual Bt-Plus" insecticide using a primary form of an entomopathogenic bacterium, Xenorhabdus nematophila.

A phase variation has been reported in an entomopathogenic bacterium, Xenorhabdus nematophila. Compared with a wild-type primary form, a secondary form usually loses several physiological and biochemical characters. This study showed that the phase variation of X. nematophila caused a significant alteration in its immunosuppressive activity and subsequent entomopathogenicity. A secondary form of X. nematophila was detected in laboratory colonies and exhibited significant differences in dye absorption and entomopathogenicity. In addition, the secondary form was different in its production of eicosanoid-biosynthesis inhibitors (EBIs) compared with the primary form of X. nematophila. Production of oxindole and phydroxypropionic acid was significantly reduced in the culture broth of the secondary form of X. nematophila. The reduced EBI production resulted in significant suppression in the inhibitory effects on cellular nodule formation and phenoloxidase activity. Culture broth of the primary form of X. nematophila enhanced the pathogenicity of Bacillus thuringiensis ( Bt) significantly more than the culture broth of the secondary form. Furthermore, this study developed a highly efficient "Dual Bt-Plus" to control both lepidopteran insect pests Plutella xylostella and Spodoptera exigua, by mixing two effective Bt strains along with the addition of potent bacterial metabolites or 100-fold concentrated X. nematophila culture broth.