Bacterial Communities and Virulence Associated with Pine Wood Nematode Bursaphelenchus xylophilus from Different Pinus spp.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is a destructive threat to pine forests. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. This study investigated variation in bacterial communities and the virulence of surface-sterilized B. xylophilus from different Pinus spp. The predominant culturable bacteria of nematodes from different pines were Stenotrophomonas and Pseudomonas. Biolog EcoPlate analysis showed that metabolic diversity of bacteria in B. xylophilus from P. massoniana was the highest, followed by P. thunbergii and P. densiflora. High-throughput sequencing analysis indicated that bacterial diversity and community structure in nematodes from the different pine species varied, and the dominant bacteria were Stenotrophomonas and Elizabethkingia. The virulence determination of B. xylophilus showed that the nematodes from P. massoniana had the greatest virulence, followed by the nematodes from P. thunbergii and P. densiflora. After the nematodes were inoculated onto P. thunbergii, the relative abundance of the predominant bacteria changed greatly, and some new bacterial species emerged. Meanwhile, the virulence of all the nematode isolates increased after passage through P. thunbergii. These inferred that some bacteria associated with B. xylophilus isolated from different pine species might be helpful to adjust the PWN's parasitic adaptability.

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes.

During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode-bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the 'bicornutum' group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.

The infection of Harmonia axyridis by a parasitic nematode is mediated by entomopathogenic bacteria and triggers sex-specific host immune responses.

The harlequin ladybird Harmonia axyridis is native to Asia but has been introduced into many countries as a biological control agent. It is now considered an invasive pest, threatening the biodiversity of native ladybirds globally, in part because of its superior immune system. H. axyridis is infected and killed by the parasitic nematode Parasitylenchus bifurcatus, which could therefore be developed as a biological strategy to counter the spread of this insect pest. However, effective control requires an understanding of the tripartite relationship between H. axyridis, P. bifurcatus and their potential bacterial mutualists. Here we describe the isolation of two species of nematode-associated bacteria (Serratia marcescens and Providencia rettgeri) which were highly virulent against H. axyridis in survival experiments. In addition, contact between the nematodes and beetles led to the sex-specific modulation of multiple host immunity-related genes after 24 and 48 h, with many genes encoding antimicrobial peptides rapidly and stably repressed in females whereas the same genes were initially induced in males before suppression at the later time point. These data provide evidence that the female immune system responds much more strongly to the nematodes and provokes, in turn, a more robust invasion strategy involving the bacterial mutualists.

Ascarosides Promote the Prevalence of Ophiostomatoid Fungi and an Invasive Pathogenic Nematode, Bursaphelenchus xylophilus.

Understanding the coevolution of pathogens and their associated mycoflora depend upon a proper elucidation of the basis of their chemical communication. In the case of pine wilt disease, the mutual interactions between cerambycid beetles, invasive pathogenic nematodes, (Bursaphelenchus xylophilus) and their symbiotic ophiostomatoid fungi provide a unique opportunity to understand the role of small molecules in mediating their chemical communication. Nematodes produce ascarosides, a highly conserved family of small molecules that serve essential functions in nematode biology and ecology. Here we demonstrated that the associated fungi, one of the key natural food resources of pine wood nematodes, can detect and respond to these ascarosides. We found that ascarosides significantly increase the growth of L. pini-densiflorae and Sporothrix sp. 1, which are native fungal species in China that form a symbiotic relationship with pinewood nematodes. Hyphal mass of L. pini-densiflorae increased when treated with asc-C5 compared to other ophiostomatoid species. Field results demonstrated that in forests where higher numbers of PWN were isolated from beetle galleries, L. pini-densiflorae had been prevalent; the same results were confirmed in laboratory studies. Furthermore, when treated with asc-C5, L. pini-densiflorae responded by increasing its production of spores, which leads to a higher likelihood of dispersal by insect vectors, hence explaining the dominance of L. pini-densiflorae over S. sp. 1 in the Tianwang and Nanlu Mountains within the Northern Forestry Centre of China. These findings provide an emphatic representation of coevolution of pine wood nematode and its associated fungi. Our results lay a broader foundation for a better understanding of inter-kingdom mutualisms and the chemical signals that mediate their establishment.

Molecular diversity of Photorhabdus and Xenorhabdus bacteria, symbionts of Heterorhabditis and Steinernema nematodes retrieved from soil in Benin.

The diversity of 43 bacterial strains isolated from Beninese entomopathogenic nematodes was investigated molecularly by analyzing the 16S rRNA, recA, and gyrB genes. Based on 16S rRNA sequence analysis, 15 bacterial strains were identified as Xenorhabdus sp., 27 strains as Photorhabdus sp., and one as Serratia sp. The Xenorhabdus strains were isolated from Steinernema nematodes and identified as Xenorhabdus indica based on 16S rRNA gene and concatenated recA and gyrB sequence analysis. However, analysis of 16S rRNA and concatenated recA and gyrB gene sequences of the Photorhabdus strains, all isolated from Heterorhabditis nematodes, resulted in two separate sub-clusters (A) and (B) within the Photorhabdus luminescens group, distinct from the existing subspecies. They share low sequence similarities with nearest phylogenetic neighbors Photorhabdus luminescens subsp. luminescens HbT, Photorhabdus luminescens subsp. caribbeanensis HG29T, and Photorhabdus luminescens subsp. noenieputensis AM7T.

The insect pathogenic bacterium Xenorhabdus innexi has attenuated virulence in multiple insect model hosts yet encodes a potent mosquitocidal toxin.

BACKGROUND: Xenorhabdus innexi is a bacterial symbiont of Steinernema scapterisci nematodes, which is a cricket-specialist parasite and together the nematode and bacteria infect and kill crickets. Curiously, X. innexi expresses a potent extracellular mosquitocidal toxin activity in culture supernatants. We sequenced a draft genome of X. innexi and compared it to the genomes of related pathogens to elucidate the nature of specialization. RESULTS: Using green fluorescent protein-expressing X. innexi we confirm previous reports using culture-dependent techniques that X. innexi colonizes its nematode host at low levels (~3-8 cells per nematode), relative to other Xenorhabdus-Steinernema associations. We found that compared to the well-characterized entomopathogenic nematode symbiont X. nematophila, X. innexi fails to suppress the insect phenoloxidase immune pathway and is attenuated for virulence and reproduction in the Lepidoptera Galleria mellonella and Manduca sexta, as well as the dipteran Drosophila melanogaster. To assess if, compared to other Xenorhabdus spp., X. innexi has a reduced capacity to synthesize virulence determinants, we obtained and analyzed a draft genome sequence. We found no evidence for several hallmarks of Xenorhabdus spp. toxicity, including Tc and Mcf toxins. Similar to other Xenorhabdus genomes, we found numerous loci predicted to encode non-ribosomal peptide/polyketide synthetases. Anti-SMASH predictions of these loci revealed one, related to the fcl locus that encodes fabclavines and zmn locus that encodes zeamines, as a likely candidate to encode the X. innexi mosquitocidal toxin biosynthetic machinery, which we designated Xlt. In support of this hypothesis, two mutants each with an insertion in an Xlt biosynthesis gene cluster lacked the mosquitocidal compound based on HPLC/MS analysis and neither produced toxin to the levels of the wild type parent. CONCLUSIONS: The X. innexi genome will be a valuable resource in identifying loci encoding new metabolites of interest, but also in future comparative studies of nematode-bacterial symbiosis and niche partitioning among bacterial pathogens.

Isolation and identification of Xenorhabdus and Photorhabdus bacteria associated with entomopathogenic nematodes and their larvicidal activity against Aedes aegypti.

BACKGROUND: Aedes aegypti is a potential vector of West Nile, Japanese encephalitis, chikungunya, dengue and Zika viruses. Alternative control measurements of the vector are needed to overcome the problems of environmental contamination and chemical resistance. Xenorhabdus and Photorhabdus are symbionts in the intestine of entomopathogenic nematodes (EPNs) Steinernema spp. and Heterorhabditis spp. These bacteria are able to produce a broad range of bioactive compounds including antimicrobial, antiparasitic, cytotoxic and insecticidal compounds. The objectives of this study were to identify Xenorhabdus and Photorhabdus isolated from EPNs in upper northern Thailand and to study their larvicidal activity against Ae. aegypti larvae. RESULTS: A total of 60 isolates of symbiotic bacteria isolated from EPNs consisted of Xenorhabdus (32 isolates) and Photorhabdus (28 isolates). Based on recA gene sequencing, BLASTN and phylogenetic analysis, 27 isolates of Xenorhabdus were identical and closely related to X. stockiae, 4 isolates were identical to X. miraniensis, and one isolate was identical to X. ehlersii. Twenty-seven isolates of Photorhabdus were closely related to P. luminescens akhurstii and P. luminescens hainanensis, and only one isolate was identical and closely related to P. luminescens laumondii. Xenorhabdus and Photorhabdus were lethal to Ae aegypti larvae. Xenorhabdus ehlersii bMH9.2_TH showed 100% efficiency for killing larvae of both fed and unfed conditions, the highest for control of Ae. aegypti larvae and X. stockiae (bLPA18.4_TH) was likely to be effective in killing Ae. aegypti larvae given the mortality rates above 60% at 72 h and 96 h. CONCLUSIONS: The common species in the study area are X. stockiae, P. luminescens akhurstii, and P. luminescens hainanensis. Three symbiotic associations identified included P. luminescens akhurstii-H. gerrardi, P. luminescens hainanensis-H. gerrardi and X. ehlersii-S. Scarabaei which are new observations of importance to our knowledge of the biodiversity of, and relationships between, EPNs and their symbiotic bacteria. Based on the biological assay, X. ehlersii bMH9.2_TH begins to kill Ae. aegypti larvae within 48 h and has the most potential as a pathogen to the larvae. These data indicate that X. ehlersii may be an alternative biological control agent for Ae. aegypti and other mosquitoes.

Three Novel Xenorhabdus-Steinernema Associations and Evidence of Strains of X. khoisanae Switching Between Different Clades.

Xenorhabdus species are normally closely associated with entomopathogenic nematodes of the family Steinernematidae. Strain F2, isolated from Steinernema nguyeni, was identified as Xenorhabdus bovienii and strains J194 and SB10, isolated from Steinernema jeffreyense and Steinernema sacchari as Xenorhabdus khoisanae, based on phenotypic characteristics and sequencing of 16S rRNA and housekeeping genes dnaN, gltX, gyrB, infB and recA. All three strains produced antimicrobial compounds that inhibited the growth of Gram-positive and Gram-negative bacteria. This is the first report of associations between strains of the symbiotic bacteria X. bovienii with S. nguyeni, and X. khoisanae with S. jeffreyense and S. sacchari. This provides evidence that strains of Xenorhabdus spp. may switch between nematode species within the same clade and between different clades.

Understanding pine wilt disease: roles of the pine endophytic bacteria and of the bacteria carried by the disease-causing pinewood nematode.

Pine wilt disease (PWD) is one of the most destructive diseases in trees of the genus Pinus and is responsible for environmental and economic losses around the world. The only known causal agent of the disease is the pinewood nematode (PWN) Bursaphelenchus xylophilus. Despite that, bacteria belonging to several different genera have been found associated with PWN and their roles in the development of PWD have been suggested. Molecular methodologies and the new era of genomics have revealed different perspectives to the problem, recognizing the manifold interactions between different organisms involved in the disease. Here, we reviewed the possible roles of nematode-carried bacteria in PWD, what could be the definition of this group of microorganisms and questioned their origin as possible endophytes, discussing their relation within the endophytic community of pine trees. The diversity of the nematode-carried bacteria and the diversity of pine tree endophytes, reported until now, is revised in detail in this review. What could signify a synergetic effect with PWN harming the plant, or what could equip bacteria with functions to control the presence of nematodes inside the tree, is outlined as two possible roles of the microbial community in the etiology of this disease. An emphasis is put on the potential revealed by the genomic data of isolated organisms in their potential activities as effective tools in PWD management.

Versatile glycoside hydrolase family 18 chitinases for fungi ingestion and reproduction in the pinewood nematode Bursaphelenchus xylophilus.

The glycoside hydrolase family 18 (GH18) of chitinases is a gene family widely expressed in archaes, prokaryotes and eukaryotes, and hydrolyzes the ß-1,4-linkages in chitin. The pinewood nematode Bursaphelenchus xylophilus is one of the organisms that produces GH18 chitinases. Notably, B. xylophilus has a higher number of GH18 chitinases compared with the obligate plant-parasitic nematodes Meloidogyne incognita and Meloidogyne hapla. In this study, seven GH18 chitinases were identified and cloned from B. xylophilus based on genomic analyses. The deduced amino acid sequences of all these genes contained an N-terminal signal peptide and a GH18 catalytic domain. Phylogenetic analysis showed that the origin of B. xylophilus GH18 chitinases was independent of those from fungi and bacteria. Real-time quantitative reverse transcription PCR analysis indicated that GH18 chitinase genes had discrete expression patterns, representing almost all the life stages of B. xylophilus. In situ hybridisation showed that the mRNA of GH18 chitinase genes of B. xylophilus were detected mainly in the spermatheca, esophageal gland cells, seminal vesicle and eggs. RNA interference (RNAi) results revealed different roles of GH18 chitinase genes in B. xylophilus. Bx-chi-1, Bx-chi-2 and Bx-chi-7 were associated with reproduction, fungal cell-wall degradation and egg hatching, respectively. Bx-chi-5 and Bx-chi-6 may be involved in sperm metabolism. In conclusion, this study demonstrates that GH18 chitinases have multiple functions in the life cycle of B. xylophilus.

Evidence for an Opportunistic and Endophytic Lifestyle of the Bursaphelenchus xylophilus-Associated Bacteria Serratia marcescens PWN146 Isolated from Wilting Pinus pinaster.

Pine wilt disease (PWD) results from the interaction of three elements: the pathogenic nematode, Bursaphelenchus xylophilus; the insect-vector, Monochamus sp.; and the host tree, mostly Pinus species. Bacteria isolated from B. xylophilus may be a fourth element in this complex disease. However, the precise role of bacteria in this interaction is unclear as both plant-beneficial and as plant-pathogenic bacteria may be associated with PWD. Using whole genome sequencing and phenotypic characterization, we were able to investigate in more detail the genetic repertoire of Serratia marcescens PWN146, a bacterium associated with B. xylophilus. We show clear evidence that S. marcescens PWN146 is able to withstand and colonize the plant environment, without having any deleterious effects towards a susceptible host (Pinus thunbergii), B. xylophilus nor to the nematode model C. elegans. This bacterium is able to tolerate growth in presence of xenobiotic/organic compounds, and use phenylacetic acid as carbon source. Furthermore, we present a detailed list of S. marcescens PWN146 potentials to interfere with plant metabolism via hormonal pathways and/or nutritional acquisition, and to be competitive against other bacteria and/or fungi in terms of resource acquisition or production of antimicrobial compounds. Further investigation is required to understand the role of bacteria in PWD. We have now reinforced the theory that B. xylophilus-associated bacteria may have a plant origin.

Effects of Endobacterium (Stenotrophomonas maltophilia) on Pathogenesis-Related Gene Expression of Pine Wood Nematode (Bursaphelenchus xylophilus) and Pine Wilt Disease.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is responsible for devastating epidemics in pine trees in Asia and Europe. Recent studies showed that bacteria carried by the PWN might be involved in PWD. However, the molecular mechanism of the interaction between bacteria and the PWN remained unclear. Now that the whole genome of B. xylophilus (Bursaphelenchus xylophilus) is published, transcriptome analysis is a unique method to study the role played by bacteria in PWN. In this study, the transcriptome of aseptic B. xylophilus, B. xylophilus treated with endobacterium (Stenotrophomonas maltophilia NSPmBx03) and fungus B. xylophilus were sequenced. We found that 61 genes were up-regulated and 830 were down-regulated in B. xylophilus after treatment with the endobacterium; 178 genes were up-regulated and 1122 were down-regulated in fungus B. xylophilus compared with aseptic B. xylophilus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to study the significantly changed biological functions and pathways for these differentially expressed genes. Many pathogenesis-related genes, including glutathinone S-transferase, pectate lyase, ATP-binding cassette transporter and cytochrome P450, were up-regulated after B. xylophilus were treated with the endobacterium. In addition, we found that bacteria enhanced the virulence of PWN. These findings indicate that endobacteria might play an important role in the development and virulence of PWN and will improve our understanding of the regulatory mechanisms involved in the interaction between bacteria and the PWN.

The genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus.

BACKGROUND: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. RESULTS: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. CONCLUSIONS: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment.

Sequencing and Analysis of the Pseudomonas fluorescens GcM5-1A Genome: A Pathogen Living in the Surface Coat of Bursaphelenchus xylophilus.

It is known that several bacteria are adherent to the surface coat of pine wood nematode (Bursaphelenchus xylophilus), but their function and role in the pathogenesis of pine wilt disease remains debatable. The Pseudomonas fluorescens GcM5-1A is a bacterium isolated from the surface coat of pine wood nematodes. In previous studies, GcM5-1A was evident in connection with the pathogenicity of pine wilt disease. In this study, we report the de novo sequencing of the GcM5-1A genome. A 600-Mb collection of high-quality reads was obtained and assembled into sequence contigs spanning a 6.01-Mb length. Sequence annotation predicted 5,413 open reading frames, of which 2,988 were homologous to genes in the other four sequenced P. fluorescens isolates (SBW25, WH6, Pf0-1 and Pf-5) and 1,137 were unique to GcM5-1A. Phylogenetic studies and genome comparison revealed that GcM5-1A is more closely related to SBW25 and WH6 isolates than to Pf0-1 and Pf-5 isolates. Towards study of pathogenesis, we identified 79 candidate virulence factors in the genome of GcM5-1A, including the Alg, Fl, Waa gene families, and genes coding the major pathogenic protein fliC. In addition, genes for a complete T3SS system were identified in the genome of GcM5-1A. Such systems have proved to play a critical role in subverting and colonizing the host organisms of many gram-negative pathogenic bacteria. Although the functions of the candidate virulence factors need yet to be deciphered experimentally, the availability of this genome provides a basic platform to obtain informative clues to be addressed in future studies by the pine wilt disease research community.

Bacterial Diversity and Community Structure in the Pine Wood Nematode Bursaphelenchus xylophilus and B. mucronatus with Different Virulence by High-Throughput Sequencing of the 16S rDNA.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.

A staining method for assessing the viability of Esteya vermicola conidia.

The viability of conidia of Esteya vermicola, a potentially important biocontrol agent against the pinewood nematode Bursaphelenchus xylophilus, is usually determined by cultivation for 18-48 h in culture medium. As an alternative to this labor-intensive method, we have developed a rapid, simple, and low-cost staining method for assessing E vermicola conidia survival rates. A mixture of neutral red and methylene blue was found to be the most optimal among several stains that also included safranin O and Janus green B. This mixture stained nonviable conidia blue, in contrast to viable conidia, which were stained red in the cytoplasm and blue in the cell wall. This method may be particularly useful for traditional research laboratories, as it provides rapid results using common, relatively inexpensive laboratory equipment.

Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus.

BACKGROUND: Pine wilt disease (PWD) caused by the pinewood nematode Bursaphelenchus xylophilus is one of the most serious forest diseases in the world. The role of B. xylophilus-associated bacteria in PWD and their interaction with the nematode, have recently been under substantial investigation. Several studies report a potential contribution of the bacteria for the PWD development, either as a helper to enhance the pathogenicity of the nematode or as a pathogenic agent expressing interesting traits related to lifestyle host-adaptation. RESULTS: We investigated the nematode-bacteria interaction under a severe oxidative stress (OS) condition using a pro-oxidant hydrogen peroxide and explored the adhesion ability of these bacteria to the cuticle surface of the nematodes. Our results clearly demonstrated a beneficial effect of the Serratia spp. (isolates LCN-4, LCN-16 and PWN-146) to B. xylophilus under the OS condition. Serratia spp. was found to be extremely OS-resistant, and promote survival of B. xylophilus and down-regulate two B. xylophilus catalase genes (Bxy-ctl-1 and Bxy-ctl-2). In addition, we show that the virulent isolate (Ka4) of B. xylophilus survives better than the avirulent (C14-5) isolate under the OS condition. The bacterial effect was transverse for both B. xylophilus isolates. We could not observe a strong and specific adhesion of these bacteria on the B. xylophilus cuticle surface. CONCLUSIONS: We report, for the first time, that B. xylophilus associated bacteria may assist the nematode opportunistically in the disease, and that a virulent B. xylophilus isolate displayed a higher tolerance towards the OS conditions than an avirulent isolate.

Host deception: predaceous fungus, Esteya vermicola, entices pine wood nematode by mimicking the scent of pine tree for nutrient.

BACKGROUND: A nematophagous fungus, Esteya vermicola, is recorded as the first endoparasitic fungus of pine wood nematode (PWN), Bursaphelenchus xylophilus, in last century. E. vermicola exhibited high infectivity toward PWN in the laboratory conditions and conidia spraying of this fungus on Japanese red pine, Pinus densiflora, seedlings in the field protected the pine trees from pine wilt disease to some extent, indicating that it is a potential bio-control agent against PWN. Previous research had demonstrated that the living fungal mycelia of E. vermicola continuously produced certain volatile organic compounds (VOCs), which were responsible for the PWN attraction. However, identity of these VOCs remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report the identification of α-pinene, ß-pinene, and camphor produced by living mycelia of E. vermicola, the same volatile compounds emitted from PWN host pine tree, as the major VOCs for PWN attraction using gas chromatography-mass spectrometry (GC-MS). In addition, we also confirmed the host deception behavior of E. vermicola to PWN by using synthetic VOCs in a straightforward laboratory bioassay. CONCLUSIONS/SIGNIFICANCE: This research result has demonstrated that the endoparasitic nematophagous fungus, E. vermicola, mimics the scent of PWN host pine tree to entice PWN for the nutrient. The identification of the attractive VOCs emitted from the fungus E. vermicola is of significance in better understanding parasitic mechanism of the fungus and the co-evolution in the two organisms and will aid management of the pine wilt disease.

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus.

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.

Phylogenetic analysis of Deladenus nematodes parasitizing northeastern North American Sirex species.

The parasitic nematode Deladenus siricidicola is a biological control agent of the invasive woodwasp, Sirex noctilio. Since the discovery of S. noctilio in pine forests of northeastern North America in 2005, a biological control program involving the Kamona strain of D. siricidicola has been under consideration. However, North American pine forests have indigenous Sirex spp. and likely harbor a unique assemblage of associated nematodes. We assessed phylogenetic relationships among native Deladenus spp. in the northeastern United States and the Kamona strain of D. siricidicola. We sequenced three genes (mtCO1, LSU, and ITS) from nematodes extracted from parasitized Sirex spp. collected inside and outside of the range of S. noctilio. Our analyses suggest cospeciation between four North American Sirex spp. and their associated nematode parasites. Within two S. noctilio individuals we found nematodes that we hypothesize are normally associated with Sirex nigricornis. One individual of the native S. nigricornis contained Deladenus normally associated with S. noctilio. We discuss nematode-host fidelity in this system and the potential for non-target impacts of a biological control program using D. siricidicola against S. noctilio.