Aims To purify and characterize antimicrobial compounds from Bacillus sp. strain N associated with rhabditid entomopathogenic nematode (EPN). Methods and Results The cell‐free culture filtrate of a bacterium associated with an EPN, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain three diketopiperazines (DKPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, 1H NMR, 13C NMR, 1H‐1H COSY, 1H‐13C HMBC) and Marfey's method. The compounds were identified as cyclo(l‐Pro‐l‐Leu), cyclo(d‐Pro‐l‐Leu) and cyclo(d‐Pro‐l‐Tyr), respectively. Three DKPs were active against all the five fungi tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and are more effective than the standard fungicide bavistin. The highest activity of 4 μg ml−1 by cyclo(l‐Pro‐l‐Leu) and cyclo(d‐Pro‐l‐Tyr) was recorded against P. expansum, a plant pathogen responsible for causing postharvest decay of stored apples and oranges. Cyclo(d‐Pro‐l‐Leu) recorded good antibacterial activity against all the four bacteria tested (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa), and cyclo(l‐Pro‐l‐Leu) and cyclo(d‐Pro‐l‐Tyr) recorded good activity only against Gram‐positive bacteria. To our knowledge, this is the first report of antifungal activity of the DKPs against the plant pathogenic fungi F. oxysporum, R. solani and P. expansum. The production of cyclo(l‐Pro‐l‐Leu), cyclo(d‐Pro‐l‐Leu) and cyclo‐(d‐Pro‐l‐Tyr) by a bacterium associated with EPN is also reported here for the first time. Conclusions Isolated DKPs demonstrated high antimicrobial activity against bacteria and fungi, especially against plant pathogenic fungi. We conclude that the bacterium associated with EPN is a promising source of natural bioactive secondary metabolites which may receive great benefit in the field of agriculture. Significance and Impact of the Study This study is a significant contribution to the knowledge of compounds unique from EPN bacteria as potential sources of new drugs in the agricultural and pharmacological industry.

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Bacterial strains associated with entomopathogenic nematodes (EPNs) Rhabditis (Oscheius) spp. were isolated from infected cadavers of Galleria mellonella. The obtained 18 isolates were subdivided into nine phylogenetically different genera based on comparative sequence analysis of their 16S rRNA genes. The isolates were affiliated to three different class namely γ-proteobacteria (Enterobacter, Proteus, Providencia, Pseudomonas, Stenotrophomonas), β-proteobacteria (Alcaligenes) and Bacilli (Bacillus, Enterococcus, Lysinibacillus). It was observed that Gram-positive strains (Bacilli) were more frequently associated with the EPN, whereas Gram-negative isolates were affiliated to six different genera with more genotypic diversity. Subsequently, all bacterial isolates used in this study were analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Eight restriction endonucleases (CfoI, HinfI, RsaI, DdeI, Sau3AI, AluI, HaeIII, and MspI) were examined and a total of 15 different genotypes were obtained, forming two heterogenous main clusters after analysis by un-weighted pair-group method using arithmetic averages.

Aims:  The aim of the present study was to purify and characterize a natural antimicrobial compound from Bacillus sp. strain N associated with a novel rhabditid entomopathogenic nematode. Methods and Results:  The cell‐free culture filtrate of a bacterium associated with a novel entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by column chromatography, and two bioactive compounds were isolated and their chemical structures were established based on spectral analysis. The compounds were identified as 3,4′,5‐trihydroxystilbene (1) and 3,5‐dihydroxy‐4‐isopropylstilbene (2). The presence of 3,4′,5‐trihydroxystilbene (resveratrol) is reported for the first time in bacteria. Compound 1 showed antibacterial activity against all the four test bacteria, whereas compound 2 was effective against the Gram‐positive bacteria only. Compounds 1 and 2 were active against all the five fungi tested and are more effective than bavistin, the standard fungicide. The antifungal activity of the compounds against the plant pathogenic fungi, Rhizoctonia solani is reported for the first time. Conclusions:  Cell‐free extract of the bacterium and isolated stilbenes demonstrated high antibacterial activity against bacteria and fungi especially against plant pathogenic fungi. We conclude that the bacterium‐associated EPN are promising sources of natural bioactive secondary metabolites. Significance and Impact of the Study:  Stilbene compounds can be used for the control of fungi and bacteria.

The aim of the present study was to determine the synergistic effects of diketopiperazines [cyclo-(L-Pro-L-Leu) (1), cyclo-(D-Pro-L-Leu) (2), and cyclo-(D-Pro-L-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp. on the growth of bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic antibacterial activities of the combination of diketopiperazines against pathogenic bacteria were assessed using the checkerboard assay and time-kill methods. The results of the present study showed that the combination effects of diketopiperazines were predominately synergistic (FIC index <0.5). Furthermore, time-kill study showed that the growth of the tested bacteria was completely attenuated with 4-12 h of treatment with 50:50 ratios of diketopiperazines. These results suggest that the combination of diketopiperazines may be microbiologically beneficial. The three diketopiperazines are nontoxic to normal human cell line (L231 lung epithelial) up to 200 m μg/ml. The in vitro synergistic activity of cyclo-(L-Pro-L-Leu), cyclo-(D-Pro-L-Leu), and cyclo-(D-Pro-L-Tyr) against bacteria is reported here for the first time. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (diketopiperazines).