Genetic toolbox for Photorhabdus and Xenorhabdus: pSEVA based heterologous expression systems and CRISPR/Cpf1 based genome editing for rapid natural product profiling.

BACKGROUND: Bacteria of the genus Photorhabdus and Xenorhabdus are motile, Gram-negative bacteria that live in symbiosis with entomopathogenic nematodes. Due to their complex life cycle, they produce a large number of specialized metabolites (natural products) encoded in biosynthetic gene clusters (BGC). Genetic tools for Photorhabdus and Xenorhabdus have been rare and applicable to only a few strains. In the past, several tools have been developed for the activation of BGCs and the deletion of individual genes. However, these often have limited efficiency or are time consuming. Among the limitations, it is essential to have versatile expression systems and genome editing tools that could facilitate the practical work. RESULTS: In the present study, we developed several expression vectors and a CRISPR-Cpf1 genome editing vector for genetic manipulations in Photorhabdus and Xenorhabdus using SEVA plasmids. The SEVA collection is based on modular vectors that allow exchangeability of different elements (e.g. origin of replication and antibiotic selection markers with the ability to insert desired sequences for different end applications). Initially, we tested different SEVA vectors containing the broad host range origins and three different resistance genes for kanamycin, gentamycin and chloramphenicol, respectively. We demonstrated that these vectors are replicative not only in well-known representatives, e.g. Photorhabdus laumondii TTO1, but also in other rarely described strains like Xenorhabdus sp. TS4. For our CRISPR/Cpf1-based system, we used the pSEVA231 backbone to delete not only small genes but also large parts of BGCs. Furthermore, we were able to activate and refactor BGCs to obtain high production titers of high value compounds such as safracin B, a semisynthetic precursor for the anti-cancer drug ET-743. CONCLUSIONS: The results of this study provide new inducible expression vectors and a CRISPR/CPf1 encoding vector all based on the SEVA (Standard European Vector Architecture) collection, which can improve genetic manipulation and genome editing processes in Photorhabdus and Xenorhabdus.

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

STEINERNEMA ADAMSI N. SP. (RHABDITIDA: STEINERNEMATIDAE), A NEW ENTOMOPATHOGENIC NEMATODE FROM THAILAND.

A new species of entomopathogenic nematode, Steinernema adamsi n. sp., was recovered from the soil of a longan tree (Dimocarpus sp.) in Mueang Lamphun District, Thailand, using baiting techniques. Upon analysis of the nematode's morphological traits, we found it to be a new species of Steinernema and a member of the Longicaudatum clade. Molecular analyses of the ITS rDNA and D2D3 of 28S rDNA sequences further confirmed that S. adamsi n. sp. is a new species of the Longicaudatum clade, which is closely related to Steinernema guangdongense and Steinernema longicaudam. Using morphometric analysis, the infective juveniles measure between 774.69 and 956.96 µm, males have a size range of 905.44 to 1,281.98 µm, and females are within the range of 1,628.21 to 2,803.64 µm. We also identified the symbiotic bacteria associated with the nematode based on 16S sequences as Xenorhabdus spp. closely related toXenorhabdus griffiniae. Furthermore, we have successfully assessed a cryopreservation method for the long-term preservation of S. adamsi n. sp. Successful cryopreservation of this new species will allow for the longer preservation of its traits and will be valuable for its future use. The discovery of this new species has significant implications for the development of effective biological control agents in Thailand, and our work contributes to our understanding of the diversity and evolution of entomopathogenic nematodes.

Discovery of an antitumor compound from xenorhabdus stockiae HN_xs01.

Xenorhabdus, known for its symbiotic relationship with Entomopathogenic nematodes (EPNs), belongs to the Enterobacteriaceae family. This dual-host symbiotic nematode exhibits pathogenic traits, rendering it a promising biocontrol agent against insects. Our prior investigations revealed that Xenorhabdus stockiae HN_xs01, isolated in our laboratory, demonstrates exceptional potential in halting bacterial growth and displaying anti-tumor activity. Subsequently, we separated and purified the supernatant of the HN_xs01 strain and obtained a new compound with significant inhibitory activity on tumor cells, which we named XNAE. Through LC-MS analysis, the mass-to-nucleus ratio of XNAE was determined to be 254.24. Our findings indicated that XNAE exerts a time- and dose-dependent inhibition on B16 and HeLa cells. After 24 h, its IC50 for B16 and HeLa cells was 30.178 µg/mL and 33.015 µg/mL, respectively. Electron microscopy revealed conspicuous damage to subcellular structures, notably mitochondria and the cytoskeleton, resulting in a notable reduction in cell numbers among treated tumor cells. Interestingly, while XNAE exerted a more pronounced inhibitory effect on B16 cells compared to HeLa cells, it showed no discernible impact on HUVEC cells. Treatment of B16 cells with XNAE induced early apoptosis and led to cell cycle arrest in the G2 phase, as evidenced by flow cytometry analysis. The impressive capability of X. stockiae HN_xs01 in synthesizing bioactive secondary metabolites promises to significantly expand the reservoir of natural products. Further exploration to identify the bioactivity of these compounds holds the potential to shed light on their roles in bacteria-host interaction. Overall, these outcomes underscore the promising potential of XNAE as a bioactive compound for tumor treatment.

The effect of Xenorhabdus bacteria metabolites on Colorado potato beetle (Leptinotarsa decemlineata) adult feeding and larval survival.

Colorado Potato Beetle (CPB) is one of the most destructive potato pests that can quickly develop resistance to insecticides. Therefore, new safe and effective control strategies that are less susceptible to the development of resistance by CPB are urgently needed. Due to their complex mode of action, the likelihood of resistance development by target pests is generally low with antifeedants. In the present study, we assessed the effect of secondary metabolites of various Xenorhabdus bacteria species and strains on CPB adult feeding and on larval development. The metabolites were applied in the form of cell free supernatants (CFSs) from Xenorhabdus cultures. In bioassay 1, leaves treated with ten Xenorhabdus cultures were fed to CPB adults, and their feeding was assessed daily for one week. In bioassay 2, CPB egg masses were placed on the leaves treated with five bacterial cultures, and larval development to pupae was monitored. Out of the ten Xenorhabdus cultures tested, two strains exhibited a significant reduction in the feeding behavior of Colorado Potato Beetle adults, with reductions of up to 70% compared to the control. The effect of CFSs on larval development was variable, and when treated with X. khoisanae SGI 197, over 90% of larvae died in the first few days before reaching the 2nd instar, and complete mortality was achieved on the 8th day of the experiment. Our study is the first study to demonstrate the antifeedant effect of Xenorhabdus cultures towards herbivorous beetles, and the metabolites of these bacteria may have potential for CPB control. Clearly, the metabolites produced by X. khoisanae SGI-197 may be a promising tool for CPB larvae control with the potential to significantly decrease damage to potato plants.

The use of Phasmarhabditis nematodes and metabolites of Xenorhabdus bacteria in slug control.

Many species of slugs are considered serious pests in agriculture and horticulture around the world. In Europe, slugs of the genera Arion and Deroceras are the most harmful pests in agriculture. Therefore, the main goal of this study was to evaluate the effect of the whole-cell metabolites of 10 strains of five Xenorhabdus and three slug-parasitic nematodes (Phasmarhabditis hermaphrodita, Phasmarhabditis bohemica, and Phasmarhabditis apuliae) on the feeding behaviour and repellent effect on target slugs and evaluate a new possible means of biocontrol of these pests. The repellent and anti-feedant effects of nematode-killed insects, metabolites, slug-parasitic nematodes and a combination of metabolites and nematodes were studied through experimental designs: sand-filled plastic boxes divided into two parts in several modifications: with dead Galleria mellonella killed by nematodes, lettuce treated with bacterial metabolites and lettuce placed on the treated sand. We found that slugs avoid eating G. mellonella killed by nematodes, while they eat freeze-killed G. mellonella. Similarly, they avoid the consumption of lettuce in areas treated with bacterial metabolites (the most effective strains being Xenorhabus bovienii NFUST, Xenorhabdus kozodoii SLOV and JEGOR) with zero feeding in the treated side. All three Phasmarhabditis species also provided a significant anti-feedant/repellent effect. Our study is the first to show the repellent and anti-feedant effects of metabolites of Xenorhabdus bacteria against Arion vulgaris, and the results suggest that these substances have great potential for biocontrol. Our study is also the first to demonstrate the repellent effect of P. apuliae and P. bohemica. KEY POINTS: • Slugs avoid eating G. mellonella killed by entomopathogenic nematodes. • Bacterial metabolites have a strong repellent and antifeedant effect on slugs. • Presence of slug parasitic nematodes increases the repellent effect of metabolites.

The crystal structure of insecticidal protein Txp40 from Xenorhabdus nematophila reveals a two-domain unique binary toxin with homology to the toxin-antitoxin (TA) system.

Txp40 is a ubiquitous, conserved, and novel toxin from Xenorhabdus and Photorhabdus bacteria, toxic to a wide range of insect pests. However, the three-dimensional structure and toxicity mechanism for Txp40 or any of its sequence homologs are not yet known. Here, we are reporting the crystal structure of the insecticidal protein Txp40 from Xenorhabdus nematophila at 2.08 Å resolution. The Txp40 was structurally distinct from currently known insecticidal proteins. Txp40 consists of two structurally different domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), primarily joined by a 33-residue long linker peptide. Txp40 displayed proteolytic propensity. Txp40 gets proteolyzed, removing the linker peptide, which is essential for proper crystal packing. NTD adopts a novel fold composed of nine amphipathic helices and has no shared sequence or structural homology to any known proteins. CTD has structural homology with RNases of type II toxin-antitoxin (TA) complex belonging to the RelE/ParE toxin domain superfamily. NTD and CTD were individually toxic to Galleria mellonella larvae. However, maximal toxicity was observed when both domains were present. Our results suggested that the Txp40 acts as a two-domain binary toxin, which is unique and different from any known binary toxins and insecticidal proteins. Txp40 is also unique because it belongs to the prokaryotic RelE/ParE toxin family with a toxic effect on eukaryotic organisms, in contrast to other members of the same family. Broad insect specificity and unique binary toxin complex formation make Txp40 a viable candidate to overcome the development of resistance in insect pests.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity.

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Biosynthesis of selenium nanoparticles using cell-free extract of Xenorhabdus cabanillasii GU480990 and their potential mosquito larvicidal properties against yellow fever mosquito Aedes aegypti.

Nanomaterials are successful due to their numerous applications in various domains such as cancer treatment, environmental applications, drug and gene delivery. Selenium is a metalloid element with broad biological activities and low toxicity especially at the nanoscale. Several studies have shown that nanoparticles synthesized from microbial and plant extracts are effective against important pests and pathogens. This study describes the bio fabrication of selenium nanoparticles using cell free extract of Xenorhabdus cabanillasii (XC-SeNPs) and assessed their mosquito larvicidal properties. Crystallographic structure and size of XC-SeNPs were determined with UV-a spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), Energy-dispersive X-ray spectroscopy (EDAX), Zeta potential and Transmission electron microscopy (TEM). The significant surface plasmon resonance at 275 nm indicated the synthesis of XC-SeNPs from the pure cell-free extract of X. cabanillasii. The XRD result exhibits the crystalline nature of XC-SeNPs. The Zeta potential analysis confirmed that the surface charge of XC-SeNPs was -24.17 mV. TEM analysis revealed that synthesized XC-SeNPs were monodispersed, spherically shaped, and sized about 80-200 nm range. In addition, the larvicidal potentials of the bio-fabricated XC-SeNPs were assessed against the 4th-instar Ae. aegypti. XC-SeNPs displayed a dose-dependent larvicidal effect; the larval mortality was 13.3 % at the minimum evaluated concentration and increased to 72 % at higher dose treatments. The LC50 and LC90 concentration of XC-SeNPs against mosquito larvae were 79.4 and 722.4 ppm, respectively.

Development of an Efficient and Seamless Genetic Manipulation Method for Xenorhabdus and Its Application for Enhancing the Production of Fabclavines.

Xenorhabdus can produce numerous natural products, but their development has been hampered by the lack of a seamless genetic manipulation method. In this study, we compared several lethal genes and determined the sacB gene as the most effective counter-selection marker and then established a dual selection/counter-selection system by integrating neo and sacB genes into one cassette. This provides an efficient and seamless genetic manipulation method for Xenorhabdus. Using this method, DNA fragments ranging from 205 to 47,788 bp in length were seamlessly knocked out or replaced with impressively high positive rates of 80 to 100% in Xenorhabdus budapestensis XBD8. In addition, the method was successfully applied with good efficiency (45-100%) in Xenorhabdus nematophila CB6. To further validate the method, different constitutive promoters were used to replace the native fclC promoter in a batch experiment. The positivity rate remained consistently high, at 46.3%. In comparison to WT XBD8, the recombinant strain MX14 demonstrated a significant increase in the production of fabclavine 7 and fabclavine 8 by 4.97-fold and 3.22-fold, respectively, while the overall production of fabclavines was enhanced by 3.52-fold.

Identification of fabclavine derivatives, Fcl-7 and Fcl-8, from Xenorhabdus budapestensis as major antifungal natural products against Rhizoctonia solani.

AIMS: Black scurf disease, caused by Rhizoctonia solani, is a severe soil-borne and tuber-borne disease, which occurs and spreads in potato growing areas worldwide and poses a serious threat to potato production. New biofungicide is highly desirable for addressing the issue, and natural products (NPs) from Xenorhabdus spp. provide prolific resources for biofungicide development. In this study, we aim to identify antifungal NPs from Xenorhabdus spp. for the management of this disease. METHODS AND RESULTS: Out of the 22 Xenorhabdus strains investigated, Xenorhabdus budapestensis 8 (XBD8) was determined to be the most promising candidate with the measured IC50 value of its cell-free supernatant against R. solani as low as 0.19 ml l-1. The major antifungal compound in XBD8 started to be synthesized in the middle logarithmic phase and reached a stable level at stationary phase. Core gene deletion coupled with high-resolution mass spectrometry analysis determined the major antifungal NPs as fabclavine derivatives, Fcl-7 and 8, which showed broad-spectrum bioactivity against important pathogenic fungi. Impressively, the identified fabclavine derivatives effectively controlled black scurf disease in both greenhouse and field experiments, significantly improving tuber quality and increasing with marketable tuber yield from 29 300 to 35 494 kg ha-1, comparable with chemical fungicide fludioxonil. CONCLUSIONS: The fabclavine derivatives Fcl-7 and 8 were determined as the major antifungal NPs in XBD8, which demonstrated a bright prospect for the management of black scurf disease.

Xenorhabdus aichiensis sp. nov., Xenorhabdus anantnagensis sp. nov., and Xenorhabdus yunnanensis sp. nov., Isolated from Steinernema Entomopathogenic Nematodes.

Three bacterial strains, XENO-2T, XENO-7T, and XENO-10T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel Xenorhabdus species. In this study, we describe these new species by whole-genome and whole-proteome phylogenomic reconstructions, by calculating sequence identity scores using core genome sequences, and by phenotypic characterization. Phylogenomic reconstructions using ribosomal and house-keeping genes, and whole-genome and whole-proteome sequences show that XENO-2T and XENO-10T are closely related to Xenorhabdus japonica DSM 16522T and that XENO-7T is closely related to Xenorhabdus bovienii subsp. africana XENO-1T and to X. bovienii subsp. bovienii T228T. The dDDH values between XENO-2T and XENO-10T and between XENO-2T and X. japonica DSM 16522T are 56.4 and 51.8%, respectively. The dDDH value between XENO-10T and X. japonica DSM 16522T is 53.4%. The dDDH values between XENO-7T and X. bovienii subsp. africana XENO-1T and between XENO-7T and X. bovienii subsp. bovienii T228T are 63.6 and 69.4%, respectively. These dDDH values are below the 70% divergence threshold for prokaryotic species delineation. The newly described species are highly pathogenic to G. mellonella larvae, grow at pH between 5 and 9 (optimum 5-7), at salt concentrations of 1-3% (optimum 1-2%), and temperatures between 20 and 37 °C (optimum 28-30 °C). Biochemical tests such as lysine decarboxylase, ornithine decarboxylase, urease, gelatinase, citrate utilization, indole and acetoin production, and cytochrome oxidase tests allow to differentiate the novel species from their more closely related species. Considering these genetic and phenotypic divergencies, we propose the following new species: Xenorhabdus aichiensis sp. nov. with XENO-7T (= CCM 9233T = CCOS 2024T) as the type strain, Xenorhabdus anantnagensis sp. nov., with XENO-2T (= CCM 9237T = CCOS 2023T) as the type strain, and Xenorhabdus yunnanensis sp. nov., with XENO-10T (= CCM 9322T = CCOS 2071T) as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Enhancing the Production of Xenocoumacin 1 in Xenorhabdus nematophila CB6 by a Combinatorial Engineering Strategy.

Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici. However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus.

Host Association and Spatial Proximity Shape but Do Not Constrain Population Structure in the Mutualistic Symbiont Xenorhabdus bovienii.

To what extent are generalist species cohesive evolutionary units rather than a compilation of recently diverged lineages? We examine this question in the context of host specificity and geographic structure in the insect pathogen and nematode mutualist Xenorhabdus bovienii. This bacterial species partners with multiple nematode species across two clades in the genus Steinernema. We sequenced the genomes of 42 X. bovienii strains isolated from four different nematode species and three field sites within a 240-km2 region and compared them to globally available reference genomes. We hypothesized that X. bovienii would comprise several host-specific lineages, such that bacterial and nematode phylogenies would be largely congruent. Alternatively, we hypothesized that spatial proximity might be a dominant signal, as increasing geographic distance might lower shared selective pressures and opportunities for gene flow. We found partial support for both hypotheses. Isolates clustered largely by nematode host species but did not strictly match the nematode phylogeny, indicating that shifts in symbiont associations across nematode species and clades have occurred. Furthermore, both genetic similarity and gene flow decreased with geographic distance across nematode species, suggesting differentiation and constraints on gene flow across both factors, although no absolute barriers to gene flow were observed across the regional isolates. Several genes associated with biotic interactions were found to be undergoing selective sweeps within this regional population. The interactions included several insect toxins and genes implicated in microbial competition. Thus, gene flow maintains cohesiveness across host associations in this symbiont and may facilitate adaptive responses to a multipartite selective environment. IMPORTANCE Microbial populations and species are notoriously hard to delineate. We used a population genomics approach to examine the population structure and the spatial scale of gene flow in Xenorhabdus bovienii, an intriguing species that is both a specialized mutualistic symbiont of nematodes and a broadly virulent insect pathogen. We found a strong signature of nematode host association, as well as evidence for gene flow connecting isolates associated with different nematode host species and collected from distinct study sites. Furthermore, we saw signatures of selective sweeps for genes involved with nematode host associations, insect pathogenicity, and microbial competition. Thus, X. bovienii exemplifies the growing consensus that recombination not only maintains cohesion but can also allow the spread of niche-beneficial alleles.

Xenorhabdus bovienii subsp. africana subsp. nov., isolated from Steinernema africanum entomopathogenic nematodes.

Four Gram-negative bacterial strains isolated from Steinernema africanum entomopathogenic nematodes were biochemically and molecularly characterized to determine their taxonomic position. Results of 16S rRNA gene sequencing indicated that they belong to the class Gammaproteobacteria, family Morganellaceae, genus Xenorhabdus, and that they are conspecific. The average 16S rRNA gene sequence similarity between the newly isolated strains and the type strain of its more closely related species, Xenorhabdus bovienii T228T, is 99.4 %. We therefore selected only one of them, XENO-1T, for further molecular characterization using whole genome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions show that XENO-1T is closely related to the type strain of X. bovienii, T228T, and to several other strains that are thought to belong to this species. To clarify their taxonomic identities, we calculated average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values. We observed that the ANI and dDDH values between XENO-1T and X. bovienii T228T are 96.3 and 71.2 %, respectively, suggesting that XENO-1T represents a novel subspecies within the X. bovienii species. Noteworthy, the dDDH values between XENO-1T and several other X. bovienii strains are between 68.7 and 70.9 % and ANI values are between 95.8 and 96.4 %, which could be interpreted, in some instances, as that XENO-1T represents a new species. Considering that for taxonomic description the genomic sequences of the type strains are compared, and to avoid future taxonomic conflicts, we therefore propose to assign XENO-1T to a new subspecies within X. bovienii. ANI and dDDH values between XENO-1T and any other of the species with validly published names of the genus are lower than 96 and 70 %, respectively, supporting its novel status. Biochemical tests and in silico genomic comparisons show that XENO-1T exhibit a unique physiological profile that differs from all the Xenorhabdus species with validly published names and from their more closely related taxa. Based on this, we propose that strain XENO-1T represents a new subspecies within the X. bovienii species, for which we propose the name X. bovienii subsp. africana subsp. nov, with XENO-1T (=CCM 9244T=CCOS 2015T) as the type strain.

Type 6 secretion system components hcp and vgrG support mutualistic partnership between Xenorhabdus bovienii symbiont and Steinernema jollieti host.

Xenorhabdus, like other Gram-negative bacteria, possesses a Type 6 Secretion System (T6SS) which acts as a contact-dependent molecular syringe, delivering diverse proteins (effectors) directly into other cells. The number of T6SS loci encoded in Xenorhabdus genomes are variable both at the inter and intraspecific level. Some environmental isolates of Xenorhabdus bovienii, encode at least one T6SS locus while others possess two loci. Previous work conducted by our team demonstrated that X. bovienii [Jollieti strain SS-2004], which has two T6SSs (T6SS-1 and T6SS-2), hcp genes are required for biofilm formation. Additionally, while T6SS-1 hcp gene plays a role in the antibacterial competition, T6SS-2 hcp does not. In this study, we tested the hypothesis that vgrG genes are also involved in mutualistic and pathogenic interactions. For this purpose, targeted mutagenesis together with wet lab experiments including colonization, competition, biofilm, and virulence experiments, were carried out to assess the role of vgrG in the mutualistic and antagonistic interactions in the life cycle of XBJ. Our results revealed that vgrG genes are not required for biofilm formation but play a role in outcompeting other Xenorhabdus bacteria. Additionally, both vgrG and hcp genes are required to fully colonize the nematode host. We also demonstrated that hcp and vgrG genes in both T6SS clusters are needed to support the reproductive fitness of the nematodes. Overall, results from this study revealed that in X. bovieni jollieti strain, the twoT6SS clusters play an important role in the fitness of the nematodes in relation to colonization and reproduction. These results lay a foundation for further investigations on the functional significance of T6SSs in the mutualistic and pathogenic lifecycle of Xenorhabdus spp.

The deterrent ability of Xenorhabdus nematophila and Photorhabdus laumondii compounds as a potential novel tool for Lobesia botrana (Lepidoptera: Tortricidae) management.

The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had âˆ¼ 55% and âˆ¼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in âˆ¼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.

Disease caused by Neofusicoccum parvum in pruning wounds of grapevine shoots and its control by Trichoderma spp. and Xenorhabdus szentirmaii.

Neofusicoccum parvum, is a fungal pathogen and one of the etiological agents of dieback disease in grapevines. The fungus causes deterioration of vines due to vascular colonization and/or production of toxins. We report herein the inhibitory effects of Trichoderma spp. isolates and the antifungal effects of cell-free supernatants (CFS) from Xenorhabdus and Photorhabdus bacteria against N. parvum in agar plates. We also evaluated the effects of the most effective fungi and bacteria against the pathogen in pruning wounds of vine shoots. All isolates of Trichoderma exhibited antifungal activity ranging between 82 and 97.5% at 14 days of post-treatment. All Xenorhabdus and Photorhabdus CFS at 10 and 33% concentrations inhibited mycelial growth with X. szentirmaii PAM 11 and PAM 25 causing the highest inhibition (>74%). In the shoot experiments, T. asperellum IB 01/13 and T. asperellum Quality®, X. szentirmaii PAM 11 (undiluted growth culture and CFS) suppressed the fungus by ≥ 93%. Our study highlights the potential of Trichoderma and X. szentirmaii PAM 11 for use as biofungicides in the management of N. parvum in grapevines. Further studies should be conducted to develop formulations of Trichoderma and Xenorhabdus that enhance stability in shelf-life and increase the efficacy of N. parvum control in grapevines under field conditions.

The modulation effect of the Steinernema carpocapsae - Xenorhabdus nematophila complex on immune-related genes in Drosophila suzukii larvae.

Larvae of the invasive pest Drosophila suzukii are susceptible to the Steinernema carpocapsae - Xenorhabdus nematophila complex and an assessment of the immune-regulatory system activation in this insect was performed to understand the response to the nematode infection. The expressions of 14 immune-related genes of different pathways (Imd, Toll, Jak-STAT, ProPO, JNK, TGF-ß) were analyzed using qRT-PCR to determine variations after nematode penetration (90 min and 4 h) and after bacterial release (14 h). Before the bacteria were present, the nematodes were not recognized by the immune system of the larvae and practically none of the analyzed pathways presented variations when compared with the non-infected larvae. However, after the X. nematophila were released, PGRP-LC was activated leading to the gene upregulation of antimicrobial peptides of both the Toll and Imd pathways. Interestingly, the cellular response was inactive during the infection course as Jak/STAT and pro-phenoloxidase genes remained unresponsive to the presence of both pathogens. These results illustrate how D. suzukii immune pathways responded differently to the nematode and bacteria along the infection course.

The role of Photorhabdus-induced bioluminescence and red cadaver coloration on the deterrence of insect scavengers from entomopathogenic nematode-infected cadavers.

Photorhabdus spp. and Xenorhabdus spp. bacteria produce a variety of molecules that inhibit bacterial and fungal contamination as well as deter scavenging invertebrates and some vertebrates in soil. Certain Heterorhabditis/Photorhabdus-infected insect cadavers can be bioluminescent in the dark and/or turn red from the production of anthraquinone pigments. The role of these traits remains unresolved. The aim of the present study was to evaluate the role of red color (anthraquinone) and bioluminescence on the deterrence of insect scavengers. Our data shows that scavenger deterrent factor (SDF) is not related to red cadaver coloration or bioluminescence activity as crickets and ants did not consume Galleria mellonella cadavers infected by P. laumondii strain 48-02 and X. bovienii. Both bacteria exhibit SDF activity but do not produce anthraquinone. Also, the insects were not affected by anthraquinone in agar plugs prepared with supernatant from induced P. laumondii Δpptase Pcep-KM-antA (SVS-275) mutant strain, which overproduces anthraquinone. Since bioluminescence and anthraquinone are not responsible for SDF activity against insect scavengers, more studies are needed to elucidate the SDF compound from Xenorhabdus and Photorhabdus bacteria.