Sam-Yeol Seo

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata.

ABSTRACT The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A2 (PLA2), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA2 inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA2-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA2 inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA2. X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA2 compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA2 inhibitors, which would be applicable for developing novel insecticides.

Two entomopathogenic bacteria, Xenorhabdus nematophila K1 and Photorhabdus temperata subsp. temperata ANU101 secrete factors enhancing Bt pathogenicity against the diamondback moth, Plutella xylostella.

Two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata, are known to be potent against the diamondback moth, Plutella xylostella, when the bacteria are injected into the hemocoel. This study investigated any pathogenic effect of their culture broth on P. xylostella by oral administration. Only culture broth of both bacterial species did not give enough pathogenic effects by the oral administration. However, when the culture broth was orally treated together with Bacillus thuringiensis (Bt), both cell-free culture broth significantly enhanced Bt pathogenicity against the 3rd instar larvae of P. xylostella. The culture broth was then fractionated into hexane, ethyl acetate, and aqueous extracts. Most synergistic effect on Bt pathogenicity was found in ethyl acetate extracts of both bacterial species. Thin layer chromatography of these extracts clearly showed that ethyl acetate extracts of both bacterial culture broths possessed metabolites that were different to those of hexane and aqueous extracts. These results suggest that the both entomopathogenic bacteria produce and secrete different factors to give significant synergistic effect on Bt pathogenicity.

Comparative Analysis of Immunosuppressive Metabolites Synthesized by an Entomopathogenic Bacterium, Photorhabdus temperata ssp. temperata, to Select Economic Bacterial Culture Media

An entomopathogenic bacterium, Photorhabdus temperata ssp. temperata (Ptt), suppresses insect immune responses and facilitates its symbiotic nematode development in target insects. The immunosuppressive activity of Ptt enhances pathogenicity of various microbial pesticides including Bacillus thuringiensis (Bt). This study was performed to select a cheap and efficient bacterial culture medium for large scale culturing of the bacteria. Relatively cheap industrial bacterial culture media (MY and M2) were compared to two research media, Luria-Bertani (LB) and tryptic soy broth (TSB). In all tested media, a constant initial population of Ptt multiplied and reached a stationary phase at 48 h. However, more bacterial colony densities were detected in LB and TSB at the stationary phase compared to two industrial media. All bacterial culture broth gave significant synergism to Bt pathogenicity against third instars of the diamondback moth, Plutella xylostella. Production of bacterial metabolites extracted by either hexane or ethyl acetate did not show any significant difference in total mass among four culture media. Reverse phase HPLC separated the four bacterial metabolites, which were not much different in quantities among four bacterial culture broths. This study suggests that two industrial bacterial culture media can be used to economically culture Ptt in a large scale.

Study on Development of Novel Biopesticides Using Entomopathogenic Bacterial Culture Broth of Xenorhabdus and Photorhabdus

Two groups of entomopathogenic bacteria, Xenorhabdus and Photorhabdus, are known to suppress insect immune responses by inhibiting eicosanoid biosynthesis. This study used these bacterial culture broths to develop novel biochemical insecticides against the diamondback moth, Plutella xylostella. Though the bacterial culture broths alone showed little insecticidal activity, they significantly enhanced pathogenicity of Bacillus thuringiensis against the fourth instar larvae of P. xylostella. Sterilization of the bacterial culture broth by autoclaving or membrane filtering did not influence the synergistic effect on the pathogenicity of B. thuringiensis. Three metablites identified in the culture broth of X. nematophila also showed similar synergistic effects. In field test, both entomopathogenic bacterial culture broth also enhanced the control efficacy of B. thuringiensis against P. xylostella.

Structure-activity Analysis of Benzylideneacetone for Effective Control of Plant Pests

Benzylideneacetone (BZA) is a compound derived from culture broth of an entomopathogenic bacterium, Xenorhabdus nematophila (Xn). Its immunosuppressive activity is caused by its inhibitory activity against eicosanoid biosynthesis. This BZA is being developed as an additive to enhance control efficacy of other commercial microbial insecticides. This study was focused on the enhancement of the immunosuppressive activity of BZA by generating its chemical derivatives toward decrease of its hydrophobicity. Two hydroxylated BZA and one sugar-conjugated BZA were chemically synthesized. All derivatives had the inhibitory activities of BZA against phospholipase () and phenoloxidase (PO) of the diamondback moth, Plutella xylostella, but BZA was the most potent. Mixtures of any BZA derivative with Bacillus thuringiensis (Bt) significantly increased pathogenicity of Bt. BZA also inhibited colony growth of four plant pathogenic fungi. However, BZA derivatives (especially the sugar-conjugated BZA) lost the antifungal activity. These results indicated that BZA and its derivatives inhibited catalytic activities of two immune-associated enzymes ( and PO) of P. xylostella and enhanced Bt pathogenicity. We suggest its use to control plant pathogenic fungi.

Biochemical Analysis of Physiological Stress Induced by High Frequency Sound Treatment in the Beet Armyworm, Spodoptera exigua

High frequency sounds disrupt physiological processes, such as feeding behavior, development and immune responses of Spodoptera exigua. We analyzed high frequency sounds with respect to biochemical changes in S. exigua. High frequency sound (5,000 Hz, 95 dB) suppressed protein synthesis and secretion of midgut epithelium. It also significantly inhibited a digestive enzyme activity of phospholipase . The gene expression of three different heat shock proteins and apolipophorin III was altered, particularly in midgut tissue in response to high frequency sound treatments. High frequency sound treatments significantly increased sugar and lipid levels in hemolymph plasma. These results suggest that high frequency sounds are a physiological stress that induces biochemical changes in S. exigua.

Enhanced Pathogenicity of Bacillus thuringiensis Mixed with a Culture Broth of an Entomopathogenic Bacterium, Xenorhabdus sp.

The entomopathogenic bacterium, sp., was isolated from an entomopathogenic nematode, . When these bacteria were injected into the hemocoel of the diamondback moth, , they caused significant mortality. However, the bacterium was not pathogenic when it was administered orally. This study showed that sp. significantly enhanced oral pathogenicity of (Bt) against the last instar larvae of . Different ratios of culture broth of sp. and Bt showed significantly different pathogenicities against . In field tests, the optimal bacterial mixture significantly enhanced control efficacy against compared to Bt treatment alone. These results demonstrated that sp. culture broth can be developed as a potent biopesticide by enhancing the insecticidal efficacy of Bt.

Comparative Analysis of Benzylideneacetone-derived Compounds on Insect Immunosuppressive and Antimicrobial Activities

Benzylinedeneacetone (BZA) is a bacterial metabolite which is synthesized by at least two entomopathogenic bacteria, namely Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata. It has been shown to possess inhibitory effects on insect cellular and humoral immune responses as well as antimicrobial activities against various species of bacteria and fungi. However, its relatively high phytotoxicity, and nonsystematic effect have thus far prevented its development into an optimal pesticide. This study screened five different BZA derivatives in order to select an optimal compound, which would have relatively high solubility and low phytotoxicity while retaining sufficient degrees of the immunosuppressive and antimicrobial activities associated with BZA. Hydroxylation of the benzene ring of BZA was found to significantly suppress its immunosuppressive and antimicrobial activities. Transformation of the ketone of BZA by carboxylation also suppressed the inhibitory activities. However, a shortening of the aliphatic chain of BZA into acetate form (4-hydroxyphenylacetic acid: HPA) did not decrease the inhibitory activity. HPA also showed much less phytotoxicity against the hot pepper plant Capsicum annuum, when compared to BZA. This study identified an optimal BZA derivative, which exhibited relatively little phytotoxicity, but retained a high degree of inhibitory activity to suppress insect immune responses and antimicrobial activities against plant pathogens.

Toxicity Evaluation of ‘Bt-Plus’ on Parasitoid and Predatory Natural Enemies

Effect of a new crop protectant `Bt-Plus` on natural enemies was analyzed in this study. Tested natural enemies included two parasitic species of and , and four predatory species of , , , and . `Bt-Plus` was formulated by combination of three entomopathogenic bacteria ( (Xn), subsp. (Ptt), (Bt)) and bacterial metabolite (BM). All three types of `Bt-Plus` showed significantly higher toxicities against fourth instar larvae than Bt single treatment. Two types of bacterial mixtures (`Xn+Bt` and `Ptt+Bt`) showed little toxicity to all natural enemies in both contact and oral feeding assays. However, `BM+Bt` showed significant toxicities especially to two predatory mites of and . The acaricidal effects of different bacterial metabolites were evaluated against two spotted spider mite, . All six BM chemicals showed significant acaricidal effects. The BM mixture used to prepare `Bt-Plus` showed a high acaricidal activity with a median lethal concentration at 218.7 ppm (95% confidence interval: 163.2 - 262.3). These toxic effects of bacterial metabolites were also proved by cytotoxicity test against Sf9 cells. Especially, benzylideneacetone, which was used as a main ingredient of `BM+Bt`, showed high cytotoxicity at its low micromolar concentration.

Application of a Remote-sensing Pheromone Trap for Analysis of Overwintering Population of the Oriental Fruit Moth, Grapholita molesta

A remote sensing pheromone trap called IT pheromone trap (Korean patent: 10-0982563) was applied to monitor overwintering population changes of the oriental fruit moth, Grapholita molesta, for three successive years in apple orchards. Males of the overwintering populations were attracted during April and May. However, the occurrence peak was delayed and extended to early June in 2010, at which the average spring temperature was significantly lower than the previous years. These overwintering populations could be monitored by the remote-sensing pheromone trap. When the remote-sensing pheromone traps were deployed to apple orchards of different provinces in Korea in 2010, the maximal overwintering populations of G. molesta were monitored at May in all areas. However, the population sizes monitored were significantly different among different localities. This study suggests a practical application of IT pheromone trap to monitor G. molesta in field conditions.