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

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) 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 PLA(2). 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 PLA(2) 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 PLA(2) inhibitors, which would be applicable for developing novel insecticides.     

Effects of entomopathogenic bacterium Photorhabdus temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis (Lepidoptera: Crambidae)

Photorhabdus temperata is an entomopathogenic bacterium that is associated with nematodes of the Heterorhabditidae family in a symbiotic relationship. This study investigated the effects of P. temperata infection on the intestinal microbiota of the sugarcane stalk borer Diatraea saccharalis. Histopathology of the infection was also investigated using scanning electron microscopy. Groups of 20 larvae were infected by injection of approximately 50 bacterial cells directly into the hemocoel. After different periods of infection, larvae were dissected and different tissues were used for bacterial cell quantification. P. temperata was highly virulent with an LD₅₀ of 16.2 bacterial cells at 48 h post-infection. Infected larvae started dying as soon as 30 h post-infection with a LT₅₀ value of 33.8 h (confidence limits 32.2–35.6) and an LT₉₀ value of 44.8 h (CL 40.8–51.4). Following death of the larvae, bacteria from the midgut did not invade the hemocoel. In the midgut epithelium, P. temperata occupied the space underneath the basal lamina. The cultivable intestinal bacterial populations decreased as soon as 1 h post-infection and at 48 h post-infection, 90% of the gut microbiota had died. The role of P. temperata in control of the midgut microbiota was discussed.     

The effects of the piroplasm Babesia bigemina on the survival and reproduction of the blue tick,Boophilus decoloratus

Seven strains of the insect pathogenic fungus Verticillium lecanii have been examined in the Calliphora erythrocephala bioassay for the production, in surface culture on Czapek Dox medium, of insecticidal secondary metabolites. One strain which had lost its pathogenicity on storage yielded no active compounds. The remainder yielded dipicolinic acid (pyridine-2,6-dicarboxylic acid) which was responsible for the insecticidal activity of acidic extracts. Neutral extracts from two strains contained novel insecticidal C₂₅ compounds.     

Involvement of oxidative stress and growth at high cell density in the viable but nonculturable state of Photorhabdus temperata ssp. temperata strain K122

Photorhabdus temperata ssp. temperata strain K122 represents a promising source of bioinsecticide. When cultured in an optimized medium, P. temperata exhibited restricted survival in terms of colony-forming ability on solid medium, which remained lower than the total cell counts. Membrane integrity assessment by flow cytometry showed that almost 100% of P. temperata cells were viable indicating that this bacterium enters in the viable but nonculturable state (VBNC). According to the double staining results, hydrogen peroxide was demonstrated to be responsible of P. temperata VBNC state. Addition of catalase or sodium pyruvate upon the inoculation of P. temperata on agar plates promoted the recovery of nonculturable cells up to 24 h incubation. Further, growth at high cell density enhanced the VBNC state of this bacterium. This should evidenced extracellular signals accumulation involved in quorum sensing mechanism. Elucidation of this state is interesting for both toxicity study and production of P. temperata useful as bioinsecticide.     

Dexamethasone,a Specific PLA2 Inhibitor,Allows Nonpathogenic Pseudomonas fluorescens to Induce Virulence Against Spodoptera exigua

Phospholipase A₂ (PLA₂) catalyzes phospholipids at sn-2 position to release arachidonic acid (20:4n-6). The arachidonic acid is further oxidized to form different eicosanoids, which play biological mediators to express cellular or humoral immune reactions in response to pathogen infection. Xeno-rahbdus and Photorhabdus, the symbiotic bacteria of entomopathogenic nematodes, have been known to inhibit PLA₂ to express their pathogenicity. This research aimed to test a hypothesis that other entomopathogenic bacteria also inhibit PLA₂ to express their pathogenicity in Spodopera exigua. Two bacterial species of Enterococcus faecalis and Pseudomonas fluorescens presumably different in ento-mopathogenicity were analyzed in their PLA₂ inhibitory activities. A pathogenic E. faecalis induced significantly immunodepression of S. exigua by inhibiting PLA₂ activity because the bacteria-infected S. exigua recovered immune reactions after the addition of arachidonic acid. However, the nonpathogenic P. fluorescens did not induce immunodepression because the addition of arachidonic acid to P. fluorescens-infected S. exigua did not further increase immune capacities while dexamethasone, a PLA₂ inhibitor, could decrease the immune activities. Injection of E. faecalis along with 10 μg of dexamethasone significantly increased pathogenicity in comparison with the bacteria alone. Moreover, the addition of dexamethasone transformed nonpathogenic P. fluorescens into pathogenic bacterium. This study suggests an evidence that PLA₂ is an inhibitory target even for entomopathogenic bacteria not related with entomopathogenic nematodes, and that the inhibition of PLA₂ determines the bacterial virulence in S. exigua.     

Laboratory assessment of entomopathogenic nematode symbiotic bacteria to control maize pest,Ostrinia furnacalis,and fungi diseases,Bipolaris maydis and Curvularia lunata

The application of entomopathogenic nematodes (EPN) and their symbiotic bacteria as biological control approaches depend on their lethal parasites to pest and antifungal activities against plant pathogenic fungi. We have collected 23 symbiotic bacterial strains from 23 EPN isolates gathered from different regions of China. In the present study, the insecticidal and antifungal activities of all these bacterial isolates were evaluated in the laboratory. Bioassay results showed that the broth and crude extract of all these 23 EPN symbiotic bacteria strains have, to a certain extent, oral insecticidal activity and/or growth inhibition to the larvae of Ostrinia furnacalis and antifungal activity against Bipolaris maydis and Curvularia lunata. Among these strains, SY5 exhibited highest insecticidal and antifungal activities to O. furnacalis, B. maydis and C. lunata. The adversity resistance of strain SY5 showed that the antifungal activity of the broth was more stable than the insecticidal activity, and the stability of antifungal activity to B. maydis and C. lunata was different.     

Growth-dependent toxicity of Photorhabdus temperata in katydid Paratlanticus ussuriensis

The oral toxicity of the symbiotic bacteria Photorhabdus temperata was investigated in various developmental stages of Paratlanticus ussuriensis. Supernatants of Photorhabdus culture medium were mixed into an artificial diet, which was fed to various stages of immature nymphs and adults of P. ussuriensis. Mortality was highest in the first instar nymph but decreased in older stages of immature nymphs. Adult females were not killed upon oral ingestion of P. ussuriensis, but their fecundity was significantly inhibited to 29.3% of that of control. In addition, the effects of oral ingestion of the symbiont culture media on the expression rates of three heat shock protein 70 genes (hsp70a, hsp70b, and hsp70c) in third instar nymphs of P. ussuriensis were determined by quantitative real-time RT-PCR analysis. There were no significant changes in expression levels in comparison with control, which suggests that hsp genes may not be associated with the mechanism of Photorhabdus toxicity. Our results imply that Photorhabdus culture media is highly effective in killing younger immature nymphs and also suppressing adult reproduction of P. ussuriensis.     

Enteric bacteria of field-collected Colorado potato beetle larvae inhibit growth of the entomopathogens Photorhabdus temperata and Beauveria bassiana

The nematode Heterorhabditis marelatus fails to reproduce in the Colorado potato beetle, Leptinotarsa decemlineata, possibly due to interference from the enteric bacteria of the beetle. Specifically, the enteric bacteria inhibit the growth of Photorhabdus temperata, the enteric symbiont of the nematode, in vitro. However, previous work was based on a laboratory culture of L. decemlineata, and we wished to determine if similar bacteria were present in the field. Therefore, we cultured the enteric bacteria of fourth-instar larvae collected from the field at two locations in Maryland and Virginia. Representatives of the genera Pantoea, Enterobacter, Pseudomonas, Acinetobacter, Serratia, Stenotrophomonas, Curtobacterium, Bacillus, Lactococcus and Enterococcus were identified by sequencing of their 16S rDNA. Isolates belonging to the genera Pantoea, Enterobacter, Pseudomonas, Serratia and Bacillus inhibited the growth of P. temperata. A number of these isolates also inhibited the entomopathogenic fungus Beauveria bassiana in vitro.     

Mosquitocidal activity of anthraquinones isolated from symbiotic bacteria Photorhabdus of entomopathogenic nematode

Two anthraquinones were isolated from the symbiotic bacteria Photorhabdus temperata of entomopathogenic nematodes Heterorhabditis spp. by repeated column chromatography. They were abundantly present in the culture medium and identified as 1,3-dimethoxy-8-hydroxy-9,10-anthraquinone and 3-methoxychrysazine by spectral analysis. The isolated anthraquinones were highly lethal to larvae of Culex pipiens pallens. Our results suggest that anthraquinones might be useful as biopesticides for the biological control of mosquitoes.     

Stability and Activities of Antibiotics Produced during Infection of the Insect Galleria mellonella by Two Isolates of Xenorhabdus nematophilus

Xenorhabdus nematophilus subsp. dutki, an entomopathogenic bacterium, is vectored by steinernematid nematodes into insects, where it produces broad-spectrum antibiotics. The use of the nematode-bacterium complex against soil-dwelling pest insects could introduce antibiotics into the soil via the dead insect fragments during the emergence phase of the nematodes. Studies on the stability and activities of these antibiotics produced in the insect Galleria mellonella may contribute to assessing the possible impact of antibiotics on soil bacteria. Two isolates of X. nematophilus subsp. dutki (isolates GI and SFU) produced xenocoumacins 1 and 2 in cadavers of G. mellonella larvae in a 1:1 ratio. Total xenocoumacin 1 and 2 production was 800 ng/200 mg (wet weight) of insect tissue for the GI isolate. Antibiotic activity of water extracts from insects that had been infected with X. nematophilus was stable at 60°C for 1 h and after repeated freeze-thaw cycles. The antibiotic titer of extracts held at 27°C declined by day 10. The spectrum of bacterial species killed by antibiotics produced in insect cadavers varied with the isolate of X. nematophilus. Levels of antibiotic activity were greater in vivo than in tryptic soy broth, which may represent a nutrient effect. The bacterial isolate, culture condition, and presence of nematodes influenced the total antibiotic production in vivo. However, the levels of activity were not correlated with bacterial levels in the different growth environments. Insect cadavers with antibiotic activity transiently lowered the numbers of the bacteria in the soil, the extent of decline varying with the strain of X. nematophilus and the time of sampling.     

Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

For low-cost production of Photorhabdus temperata ssp. temperata strain K122 bioinsecticide, a cheap complex medium was optimized. Diluted seawater was used as the source of micronutrients, especially sodium chloride, involved in the improvement of cell density, culturability and oral toxicity of the bacterium P. temperata against Ephestia kuehniella larvae. Thus, the new formulated medium was composed only of 10 g/l of soya bean meal, used as the carbon and nitrogen main source, mixed in sevenfold diluted seawater. At such conditions, several limitations of P. temperata bioinsecticide productions were shown to be overcome. The appearance of variants small colony polymorphism was completely avoided. Thus, the strain K122 was maintained at the primary form even after prolonged incubation. Moreover, the viable but nonculturable state was partially overcome, since the ability of P. temperata cells to form colonies on the solid medium was prolonged until 78 h of incubation. In addition, when cultured in the complex medium, P. temperata cells were produced at high cell density of 12 × 10⁸ cells/ml and exhibited 81.48% improvement of oral toxicity compared to those produced in the optimized medium. With such medium, the large-scale bioinsecticides production into 3-l fully controlled fermenter improved the total cell counts, CFU counts and oral toxicity by 20, 5.81 and 16.73%, respectively. This should contribute to a significant reduction of production cost of highly potent P. temperata strain K122 cells, useful as a bioinsecticide.     

Pathogenicity of Moraxella osloensis, a Bacterium Associated with the Nematode Phasmarhabditis hermaphrodita, to the Slug Deroceras reticulatum

Moraxella osloensis, a gram-negative bacterium, is associated with Phasmarhabditis hermaphrodita, a nematode parasite of slugs. This bacterium-feeding nematode has potential for the biological control of slugs, especially the grey garden slug, Deroceras reticulatum. Infective juveniles of P. hermaphrodita invade the shell cavity of the slug, develop into self-fertilizing hermaphrodites, and produce progeny, resulting in host death. However, the role of the associated bacterium in the pathogenicity of the nematode to the slug is unknown. We discovered that M. osloensis alone is pathogenic to D. reticulatum after injection into the shell cavity or hemocoel of the slug. The bacteria from 60-h cultures were more pathogenic than the bacteria from 40-h cultures, as indicated by the higher and more rapid mortality of the slugs injected with the former. Coinjection of penicillin and streptomycin with the 60-h bacterial culture reduced its pathogenicity to the slug. Further work suggested that the reduction and loss of pathogenicity of the aged infective juveniles of P. hermaphrodita to D. reticulatum result from the loss of M. osloensis from the aged nematodes. Also, axenic J1/J2 nematodes were nonpathogenic after injection into the shell cavity. Therefore, we conclude that the bacterium is the sole killing agent of D. reticulatum in the nematode-bacterium complex and that P. hermaphrodita acts only as a vector to transport the bacterium into the shell cavity of the slug. The identification of the toxic metabolites produced by M. osloensis is being pursued.     

Gibberellins synthesized by the entomopathogenic bacterium,Photorhabdus temperata M1021 as one of the factors of rice plant growth promotion

In the present study, different types of gibberellins (GAs) in the culture filtrate (CF) of Photorhabdus temperata M1021 were quantified. The analysis of CF helped in profiling various bioactive GAs: GA₁, GA₃, GA₄, and GA₇. Several physiologically inactive GAs: GA₉, GA₁₂, and GA₂₀ were detected as well. Siderophore production was also investigated by growing P. temperata M1021 on chrome azurol-S blue agar plates. Furthermore, the strain was inoculated into ‘Waito-C’ (Oryza sativa L.) rice plants, which significantly (P < 0.05) increased plant growth attributes such as plant length, chlorophyll content, and fresh and dry biomass compared with those in controls. In a separate experiment, canola (Brassica napus L.) seeds treated with CF of M1021 were significantly (P < 0.05) accelerated germination rate as well as biomass production. Findings of the present study suggest that the strain M1021 contributes an important role in the plant growth by synthesizing a wide array of bioactive metabolites.     

Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae)

The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.     

Pathogenicity and repulsion for toxin‐producing bacteria of dominant bacteria on the surface of American pine wood nematodes

Bacteria were isolated from the surface of two samples of American pine wood nematodes to identify methods of controlling pine wilt disease. The dominant bacterial strains were identified, and their toxicity and pathogenicity, in addition to their competitiveness with other pathogenic bacteria, were measured to ascertain how bacteria on the surface of American pine wood nematodes might be used to prevent and control pine wilt disease. The bacterial isolates show that the dominant bacteria carried by the two samples of pine wood nematodes are US4, US5, Smal‐007 and Rrad‐006. Based on routine staining, morphological observation and 16S rDNA sequence analysis, the four strains were identified as Delftia lacustris, Pseudomonas putida, Stenotrophomonas maltophilia and Rhizobium nepotum. The incubation of four dominant bacterial strains and Chinese dominant bacterial strains on the surface of aseptic nematodes and in nutrient broth showed that Smal‐007 and Rrad‐006 have strong competitiveness on the surface of pine wood nematodes. Using a bacterial culture medium to measure the propensity of pine seedlings to wilt, all the American dominant bacterial strains were shown to be less toxic than the Chinese dominant strains. If pine seedlings are inoculated with both bacterial and aseptic pine wood nematodes, American dominant bacterial strains present less pathogenicity than the Chinese dominant bacterial strains. In particular, Smal‐007 and Rrad‐006 show the lowest pathogenicity. If pine seedlings are inoculated with both bacterial and wild pine wood nematodes, American dominant bacterial strains significantly reduce the pathogenicity of wild pine wood nematodes isolated from Zhejiang Province, China. The effects of Smal‐007 and Rrad‐006 are confirmed as the most prominent. The American dominant strains Smal‐007 and Rrad‐006 satisfy two main requirements: excellent repulsion performance and low pathogenicity. Therefore, they can be used as candidate strains for biocontrol bacteria.     

Heterologous Expression of Bacillus thuringiensis Vegetative Insecticidal Protein-Encoding Gene vip3LB in Photorhabdus temperata Strain K122 and Oral Toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities.     

Differences in the virulence of Heterorhabditis bacteriophora and Steinernema scarabaei to three white grub species: The relative contribution of the nematodes and their symbiotic bacteria

Because susceptibility of white grub species to entomopathogenic nematodes differs, we compared the virulence of Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbiotic bacteria of the nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, to the three white grub species, Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. Both bacteria were pathogenic to all three grub species even at 2 cells/grub. However, the median lethal dose at 48 h post injection and median lethal time at 20 cells/grub showed that P. temperata was more virulent than X. koppenhoeferi to C. borealis. Although H. bacteriophora is less pathogenic than S. scarabaei to R. majalis and P. japonica, their symbiotic bacteria did not differ in virulence against these two grub species, and they also showed similar growth patterns both in vitro and inside R. majalis larvae at 20 °C. We then tested the pathogenicity of oral- and intrahemocoel-introduced H. bacteriophora to R. majalis to determine whether nematodes are able to successfully vector the bacteria into the hemolymph. Hemocoel injected H. bacteriophora was pathogenic to R. majalis indicating successful bacterial release, but orally introduced H. bacteriophora were not. Dissection of grubs confirmed that the orally introduced H. bacteriophora were unable to penetrate into the hemolymph through the gut wall. We conclude that the low susceptibility of R. majalis to H. bacteriophora is not due to the symbiotic bacteria but rather to the nematode’s poor ability to penetrate through the gut wall and the cuticle to vector the bacteria into the hemolymph.     

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H₂O₂ was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10⁸, 1.15 × 10⁸ cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012