Photorhabdus-Derived Secondary Metabolites Reduce Root Infection by Meloidogyne incognita in Cowpea.

Root-knot nematodes (RKNs) cause significant economic damage to crop plants, spurring demand for safe, affordable, and sustainable nematicides. A previous study by our research team showed that the combination of two nematicidal secondary metabolites (SMs) derived from Photorhabdus bacteria, trans-cinnamic acid (t-CA), and (4E)-5-phenylpent-4-enoic acid (PPA) have a synergistic effect against RKNs in vitro. In this study, we considered in planta assays to assess the effects of this SM mixture on the virulence and reproductive fitness of the RKN Meloidogyne incognita in a cowpea. Factorial combinations of five t-CA + PPA concentrations (0, 9.0, 22.9, 57.8, and 91.0 µg/ml) and two nematode inoculation conditions (presence or absence) were evaluated in 6-week growth chamber experiments. Results from this study showed that a single root application of the t-CA + PPA mixture significantly reduced the penetration of M. incognita infective juveniles (J2s) into the cowpea roots. The potential toxicity of t-CA + PPA on RKN-susceptible cowpea seedlings was also investigated. The effect of t-CA + PPA × nematode inoculation interactions and the t-CA + PPA mixture did not show significant phytotoxic effects, nor did it adversely affect plant growth parameters or alter leaf chlorophyll content. Total leaf chlorophyll and chlorophyll b content were significantly reduced (by 15 and 22%, respectively) only by the nematode inoculum and not by any of the SM treatments. Our results suggest that a single root application of a mixture of t-CA and PPA reduces M. incognita J2's ability to infect the roots without impairing plant growth or chlorophyll content.

Transcriptomic Analysis of Steinernema Nematodes Highlights Metabolic Costs Associated to Xenorhabdus Endosymbiont Association and Rearing Conditions.

Entomopathogenic nematodes of the genus Steinernema have a mutualistic relationship with bacteria of the genus Xenorhabdus and together they form an antagonist partnership against their insect hosts. The nematodes (third-stage infective juveniles, or IJs) protect the bacteria from the external environmental stressors and vector them from one insect host to another. Xenorhabdus produce secondary metabolites and antimicrobial compounds inside the insect that protect the cadaver from soil saprobes and scavengers. The bacteria also become the nematodes' food, allowing them to grow and reproduce. Despite these benefits, it is yet unclear what the potential metabolic costs for Steinernema IJs are relative to the maintenance and vectoring of Xenorhabdus. In this study, we performed a comparative dual RNA-seq analysis of IJs of two nematode-bacteria partnerships: Steinernema carpocapsae-Xenorhabdus nematophila and Steinernema. puntauvense-Xenorhbdus bovienii. For each association, three conditions were studied: (1) IJs reared in the insect (in vivo colonized), (2) colonized IJs reared on liver-kidney agar (in vitro colonized), and (3) IJs depleted by the bacteria reared on liver-kidney agar (in vitro aposymbiotic). Our study revealed the downregulation of numerous genes involved in metabolism pathways, such as carbohydrate, amino acid, and lipid metabolism when IJs were reared in vitro, both colonized and without the symbiont. This downregulation appears to impact the longevity pathway, with the involvement of glycogen and trehalose metabolism, as well as arginine metabolism. Additionally, a differential expression of the venom protein known to be secreted by the nematodes was observed when both Steinernema species were depleted of their symbiotic partners. These results suggest Steinernema IJs may have a mechanism to adapt their virulence in absence of their symbionts.

Identification of novel prophage regions in Xenorhabdus nematophila genome and gene expression analysis during phage-like particle induction.

BACKGROUND: Entomopathogenic Xenorhabdus bacteria are endosymbionts of Steinernema nematodes and together they form an insecticidal mutualistic association that infects a wide range of insect species. Xenorhabdus produce an arsenal of toxins and secondary metabolites that kill the insect host. In addition, they can induce the production of diverse phage particles. A few studies have focused on one integrated phage responsible for producing a phage tail-like bacteriocin, associated with an antimicrobial activity against other Xenorhabdus species. However, very little is known about the diversity of prophage regions in Xenorhabdus species. METHODS: In the present study, we identified several prophage regions in the genome of Xenorhabdus nematophila AN6/1. We performed a preliminary study on the relative expression of genes in these prophage regions. We also investigated some genes (not contained in prophage region) known to be involved in SOS bacterial response (recA and lexA) associated with mitomycin C and UV exposure. RESULTS: We described two integrated prophage regions (designated Xnp3 and Xnp4) not previously described in the genome of Xenorhabdus nematophila AN6/1. The Xnp3 prophage region appears very similar to complete Mu-like bacteriophage. These prophages regions are not unique to X. nematophila species, although they appear less conserved among Xenorhabdus species when compared to the previously described p1 prophage region. Our results showed that mitomycin C exposure induced an up-regulation of recA and lexA suggesting activation of SOS response. In addition, mitomycin C and UV exposure seems to lead to up-regulation of genes in three of the four integrated prophages regions.

Selective Toxicity of Secondary Metabolites from the Entomopathogenic Bacterium Photorhabdus luminescens sonorensis against Selected Plant Parasitic Nematodes of the Tylenchina Suborder.

Entomopathogenic Photorhabdus bacteria (Enterobacteriaceae: Gamma-proteobacteria), the natural symbionts of Heterorhabditis nematodes, are a rich source for the discovery of biologically active secondary metabolites (SMs). This study describes the isolation of three nematicidal SMs from in vitro culture supernatants of the Arizona-native Photorhabdus luminescens sonorensis strain Caborca by bioactivity-guided fractionation. Nuclear magnetic resonance spectroscopy and comparison to authentic synthetic standards identified these bioactive metabolites as trans-cinnamic acid (t-CA), (4E)-5-phenylpent-4-enoic acid (PPA), and indole. PPA and t-CA displayed potent, concentration-dependent nematicidal activities against the root-knot nematode (Meloidogyne incognita) and the citrus nematode (Tylenchulus semipenetrans), two economically and globally important plant parasitic nematodes (PPNs) that are ubiquitous in the United States. Southwest. Indole showed potent, concentration-dependent nematistatic activity by inducing the temporary rigid paralysis of the same targeted nematodes. While paralysis was persistent in the presence of indole, the nematodes recovered upon removal of the compound. All three SMs were found to be selective against the tested PPNs, exerting little effects on non-target species such as the bacteria-feeding nematode Caenorhabditis elegans or the entomopathogenic nematodes Steinernema carpocapsae, Heterorhabditis bacteriophora, and Hymenocallis sonorensis. Moreover, none of these SMs showed cytotoxicity against normal or neoplastic human cells. The combination of t-CA + PPA + indole had a synergistic nematicidal effect on both targeted PPNs. Two-component mixtures prepared from these SMs revealed complex, compound-, and nematode species-dependent interactions. These results justify further investigations into the chemical ecology of Photorhabdus SMs, and recommend t-CA, PPA and indole, alone or in combinations, as lead compounds for the development of selective and environmentally benign nematicides against the tested PPNs. IMPORTANCE Two phenylpropanoid and one alkaloid secondary metabolites were isolated and identified from culture filtrates of Photorhabdus l. sonorensis strain Caborca. The three identified metabolites showed selective nematicidal and/or nematistatic activities against two important plant parasitic nematodes, the root-knot nematode (Meloidogyne incognita) and the citrus nematode (Tylenchulus semipenetrans). The mixture of all three metabolites had a synergistic nematicidal effect on both targeted nematodes, while other combinations showed compound- and nematode-dependent interactions.

Bacterial transfer from Pristionchus entomophagus nematodes to the invasive ant Myrmica rubra and the potential for colony mortality in coastal Maine.

The necromenic nematode Pristionchus entomophagus has been frequently found in nests of the invasive European ant Myrmica rubra in coastal Maine, United States, and may contribute to ant mortality and collapse of colonies by transferring environmental bacteria. Paenibacillus and several other bacterial species were found in the digestive tracts of nematodes harvested from collapsed ant colonies. Serratia marcescens, Serratia nematodiphila, and Pseudomonas fluorescens were collected from the hemolymph of nematode-infected wax moth (Galleria mellonella) larvae. Virulence against waxworms varied by the site of origin of the nematodes. In adult nematodes, bacteria were highly concentrated in the digestive tract with none observed on the cuticle. In contrast, juveniles had more on the cuticle than in the digestive tract. Host species was the primary factor affecting bacterial community profiles, but Spiroplasma sp. and Serratia marcescens sequences were shared across ants, nematodes, and nematode-exposed G. mellonella larvae.

R-type bacteriocins of Xenorhabdus bovienii determine the outcome of interspecies competition in a natural host environment.

Xenorhabdus species are bacterial symbionts of Steinernema nematodes and pathogens of susceptible insects. Different species of Steinernema nematodes carrying specific species of Xenorhabdus can invade the same insect, thereby setting up competition for nutrients within the insect environment. While Xenorhabdus species produce both diverse antibiotic compounds and prophage-derived R-type bacteriocins (xenorhabdicins), the functions of these molecules during competition in a host are not well understood. Xenorhabdus bovienii (Xb-Sj), the symbiont of Steinernema jollieti, possesses a remnant P2-like phage tail cluster, xbp1, that encodes genes for xenorhabdicin production. We show that inactivation of either tail sheath (xbpS1) or tail fibre (xbpH1) genes eliminated xenorhabdicin production. Preparations of Xb-Sj xenorhabdicin displayed a narrow spectrum of activity towards other Xenorhabdus and Photorhabdus species. One species, Xenorhabdus szentirmaii (Xsz-Sr), was highly sensitive to Xb-Sj xenorhabdicin but did not produce xenorhabdicin that was active against Xb-Sj. Instead, Xsz-Sr produced high-level antibiotic activity against Xb-Sj when grown in complex medium and lower levels when grown in defined medium (Grace's medium). Conversely, Xb-Sj did not produce detectable levels of antibiotic activity against Xsz-Sr. To study the relative contributions of Xb-Sj xenorhabdicin and Xsz-Sr antibiotics in interspecies competition in which the respective Xenorhabdus species produce antagonistic activities against each other, we co-inoculated cultures with both Xenorhabdus species. In both types of media Xsz-Sr outcompeted Xb-Sj, suggesting that antibiotics produced by Xsz-Sr determined the outcome of the competition. In contrast, Xb-Sj outcompeted Xsz-Sr in competitions performed by co-injection in the insect Manduca sexta, while in competition with the xenorhabdicin-deficient strain (Xb-Sj:S1), Xsz-Sr was dominant. Thus, xenorhabdicin was required for Xb-Sj to outcompete Xsz-Sr in a natural host environment. These results highlight the importance of studying the role of antagonistic compounds under natural biological conditions.

Xenorhabdus bovienii strain jolietti uses a type 6 secretion system to kill closely related Xenorhabdus strains.

Xenorhabdus bovienii strain jolietti (XBJ) is a Gram-negative bacterium that interacts with several organisms as a part of its life cycle. It is a beneficial symbiont of nematodes, a potent pathogen of a wide range of soil-dwelling insects and also has the ability to kill soil- and insect-associated microbes. Entomopathogenic Steinernema nematodes vector XBJ into insects, releasing the bacteria into the insect body cavity. There, XBJ produce a variety of insecticidal toxins and antimicrobials. XBJ's genome also encodes two separate Type Six Secretion Systems (T6SSs), structures that allow bacteria to inject specific proteins directly into other cells, but their roles in the XBJ life cycle are mostly unknown. To probe the function of these T6SSs, we generated mutant strains lacking the key structural protein Hcp from each T6SS and assessed phenotypes related to different parts of XBJ's life cycle. Here we demonstrate that one of the T6SSs is more highly expressed in in vitro growth conditions and has antibacterial activity against other Xenorhabdus strains, and that the two T6SSs have a redundant role in biofilm formation.

Draft Genome Assembly of the Entomopathogenic Bacterium Photorhabdus luminescens subsp. sonorensis Caborca.

Photorhabdus luminescens subsp. sonorensis strain Caborca is an entomopathogenic bacterium with a dual lifestyle, namely, as a mutualist of the Heterorhabditis sonorensis nematode and a pathogen to a wide range of insect species. The genome assembly, in 231 contigs, is 5.2 Mbp long and includes 25 putative gene clusters for secondary metabolism.

Mild thermal stress affects Steinernema carpocapsae infective juvenile survival but not protein content.

Steinernema nematodes and their Xenorhabdus symbionts are a malleable model system to study mutualistic relations. One of the advantages they possess is their ability to be disassociated under in vitro rearing conditions. Various in vitro methods have been developed to produce symbiont colonized and aposymbiotic (symbiont-free) nematodes. Until now, there has been no investigation on how in vitro rearing conditions may have an impact on the storage ability and the protein content of the infective juvenile at different storage temperatures. Thus, in this study, we investigated how infective juvenile longevity and protein content are impacted when the nematodes were reared with two in vitro methods (lipid and liver kidney agar) considering colonized and uncolonized nematodes, and under two different temperatures: 15 °C and 20 °C (mild stress). Infective juveniles reared in vitro (with or without their symbionts) had lower 8-week survival rates. No in vitro reared, colonized IJs survived to the desired 16-week time point. Survival of infective juveniles stored under mild stress temperature (20 °C) was lower than that observed at 15 °C. However, when comparing the interaction between rearing condition and storage temperature, there were not significant differences. With respect to protein content, in vivo, colonized infective juveniles maintained a static protein content over time, suggesting symbiont colonization may influence protein metabolism and/or turnover in infective juveniles.

Influence of symbiotic and non-symbiotic bacteria on pheromone production in Steinernema nematodes (Nematoda, Steinernematidae).

In this study, we assessed the effect of symbiotic (cognate and non-cognate) and non-symbiotic bacteria on ascaroside production of first-generation adults in two Steinernema spp.: S. carpocapsae All strain and S. feltiae SN strain. Each nematode species was reared under three bacterial scenarios: (1) cognate symbiotic, (2) non-cognate symbiotic strain and (3) non-cognate symbiotic species. Our results showed S. carpocapsae produced four quantifiable ascaroside molecules: asc-C5, asc-C6, asc-C7 and asc-C11, whereas in S. feltiae only three molecules were detected: asc-C5, asc-C7 and asc-C11. Bacterial conditions did not significantly affect the quantity of the secreted ascarosides in first-generation adults of S. carpocapsae However, in S. feltiae, Xenorhabdus nematophila All strain influenced the production of two ascaroside molecules: asc-C5 and asc-C11.

Ecological characterization of Heterorhabditis sonorensis (Caborca strain) (Nematoda: Heterorhabditidae), an entomopathogenic nematode from the Sonoran Desert.

Heterorhabditis nematodes are parasites of a wide range of soil-dwelling insect species. Although these nematodes have been exploited as biological control agents since the last half of the 20th century, much research remains to be done to understand how these organisms function in agricultural and other ecosystems. In this study, we present some ecological traits of Heterorhabditis sonorensis, a natural parasite of the cicada Diceroprocta ornea, from the Sonoran Desert. Specifically, we evaluated its infectivity across a diverse panel of insect groups and assessed its fitness (infectivity and reproduction) considering different temperatures, and soil moisture levels. Three other Heterorhabditis species served as points of comparison for temperature and soil moisture assays. Host range experiments indicate that H. sonorensis, although isolated from seasonal cicada nymphs, is more virulent and reproductively fit in the lepidopteran hosts tested. This nematode has an optimum temperature range at 25-30 °C but can also successfully reproduce at temperatures ranging from 15 to 35 °C. Additionally, this nematode is adapted to a variety of soil moisture conditions with successful infections across the tested moisture range (3%-20%). Finally, we demonstrate that H. sonorensis infective juveniles have a high survival rate (over 80%) at various storage temperatures (10-25 °C) after 24 weeks of storage and remain infective as revealed by the post-storage infection assays.

Partners in crime: symbiont-assisted resource acquisition in Steinernema entomopathogenic nematodes.

Entomopathogenic nematodes in the genus Steinernema (Nematoda: Steinernematidae) have a mutualistic relationship with Xenorhabdus bacteria (Gram-negative Enterobacteriaceae). This partnership however, is pathogenic to a wide range of insect species. Because of their potent insecticidal ability, they have successfully been implemented in biological control and integrated pest management programs worldwide. Steinernema-Xenorhabdus-insect partnerships are extremely diverse and represent a model system in ecology and evolution to investigate symbioses between invertebrates and microbes. The reproductive fitness of the nematode-bacterium partnership is tightly associated, and maintenance of their virulence is critical to the conversion of the insect host as a suitable environment where this partnership can be perpetuated.

Entomopathogenic nematology in Latin America: A brief history, current research and future prospects.

Since the 1980s, research into entomopathogenic nematodes (EPNs) in Latin America has produced many remarkable discoveries. In fact, 16 out of the 117 recognized species of EPNs have been recovered and described in the subcontinent, with many more endemic species and/or strains remaining to be discovered and identified. In addition, from an applied perspective, numerous technological innovations have been accomplished in relation to their implementation in biocontrol. EPNs have been evaluated against over 170 species of agricultural and urban insects, mites, and plant-parasitic nematodes under laboratory and field conditions. While much success has been recorded, many accomplishments remain obscure, due to their publication in non-English journals, thesis dissertations, conference proceedings, and other non-readily available sources. The present review provides a brief history of EPNs in Latin America, including current findings and future perspectives.

Secretion Systems and Secreted Proteins in Gram-Negative Entomopathogenic Bacteria: Their Roles in Insect Virulence and Beyond.

Many Gram-negative bacteria have evolved insect pathogenic lifestyles. In all cases, the ability to cause disease in insects involves specific bacterial proteins exported either to the surface, the extracellular environment, or the cytoplasm of the host cell. They also have several distinct mechanisms for secreting such proteins. In this review, we summarize the major protein secretion systems and discuss examples of secreted proteins that contribute to the virulence of a variety of Gram-negative entomopathogenic bacteria, including Photorhabdus, Xenorhabdus, Serratia, Yersinia, and Pseudomonas species. We also briefly summarize two classes of exported protein complexes, the PVC-like elements, and the Tc toxin complexes that were first described in entomopathogenic bacteria.

Influence of Xenorhabdus (Gamma-Proteobacteria: Enterobacteriaceae) symbionts on gonad postembryonic development in Steinernema (Nematoda: Steinernematidae) nematodes.

Steinernema nematodes and their Xenorhabdus partners form an obligate mutualistic association. This partnership is insecticidal to a wide range of insects. Steinernema rely on their Xenorhabdus partner to produce toxins inside the insect cadaver to liberate nutrients from the insect, as well as antimicrobials to sterilize the cadaver, thus creating a suitable environment for reproduction. In return, Steinernema vector their Xenorhabdus between insect hosts. Disruption of this partnership may affect the success of both partners. For instance, when Steinernema associates with non-cognate symbionts, their virulence and reproductive fitness are affected. In this study, we examined the effect of symbiotic (cognate and non-cognate) and non-symbiotic bacteria on maturation time, gonad postembryonic development, and sex ratio of first-generation Steinernema adults. Two Steinernema spp. were considered: S. feltiae SN and S. carpocapsae All. In vitro assays were carried out by pairing each nematode sp. with symbiotic (cognate and non-cognate) Xenorhabdus, and with non-symbiotic bacteria (Serratia proteamaculans). Additionally, for comparative purposes, we also considered adult nematodes reared in vivo in Galleria mellonella larvae to assess nematode development under natural conditions. Results from this study showed non-symbiotic Serratia proteamaculans did not support adult development of S. feltiae but it allowed development of S. carpocapsae adults. Sex ratio decreased from 2:1 to 1:1 (female: male) when S. carpocapsae adults were reared with the non-symbiotic S. proteamaculans. Cognate or non-cognate Xenorhabdus spp. and/or strains did not change the sex ratio of any of either Steinernema spp. tested. Morphometric analysis also revealed that bacterial conditions influenced adult size and gonad postembryonic development in both Steinernema species. Body size (length and width), and gonad length in both S. feltiae males and females, were significantly reduced when reared with a non-cognate Xenorhabdus species. In S. carpocapsae, males exhibited an enhanced body size (length and width) and gonad length when reared with a non-cognate X. nematophila strain. S. carpocapsae females also exhibited an enhanced gonad length when reared with a non-cognate X. nematophila strain. S. carpocapsae males and females were underdeveloped when reared with the non-symbiotic S. proteamaculans, and exhibited reduced body sizes and gonad lengths. We conclude that development of first-generation adults of both Steinernema spp. tested, in particular time to adult maturation as well as body and gonad size were directly influenced by the bacterial symbionts they were cultured with. However, response to the culture conditions was species specific.

Fitness costs of symbiont switching using entomopathogenic nematodes as a model.

BACKGROUND: Steinernematid nematodes form obligate symbioses with bacteria from the genus Xenorhabdus. Together Steinernema nematodes and their bacterial symbionts successfully infect, kill, utilize, and exit their insect hosts. During this process the nematodes and bacteria disassociate requiring them to re-associate before emerging from the host. This interaction can be complicated when two different nematodes co-infect an insect host. RESULTS: Non-cognate nematode-bacteria pairings result in reductions for multiple measures of success, including total progeny production and virulence. Additionally, nematode infective juveniles carry fewer bacterial cells when colonized by a non-cognate symbiont. Finally, we show that Steinernema nematodes can distinguish heterospecific and some conspecific non-cognate symbionts in behavioral choice assays. CONCLUSIONS: Steinernema-Xenorhabdus symbioses are tightly governed by partner recognition and fidelity. Association with non-cognates resulted in decreased fitness, virulence, and bacterial carriage of the nematode-bacterial pairings. Entomopathogenic nematodes and their bacterial symbionts are a useful, tractable, and reliable model for testing hypotheses regarding the evolution, maintenance, persistence, and fate of mutualisms.

Secondary Metabolites Produced by Heterorhabditis Symbionts and Their Application in Agriculture: What We Know and What to Do Next.

Gram-negative Photorhabdus bacteria have a dual lifestyle: they are mutualists of Heterorhabditis nematodes and are pathogens of insects. Together, this nematode-bacterium partnership has been used to successfully control a wide range of agricultural insect pests. Photorhabdus produce a diverse array of small molecules that play key biological roles in regulating their dual roles. In particular, several secondary metabolites (SM) produced by this bacterium are known to play a critical role in the maintenance of a monoxenic infection in the insect host and are also known to prevent contamination of the cadaver from soil microbes and/or predation by arthropods. A few of the SM this bacteria produce have been isolated and identified, and their biological activities have also been tested in laboratory assays. Over the past two decades, analyses of the genomes of several Photorhabdus spp. have revealed the presence of SM numerous gene clusters that comprise more than 6% of these bacteria genomes. Furthermore, genome mining and characterization of biosynthetic pathways, have uncovered the richness of these compounds, which are predicted to vary across different Photorhabdus spp. and strains. Although progress has been made in the identification and function of SM genes and gene clusters, the targeted testing for the bioactivity of molecules has been scarce or mostly focused on medical applications. In this review, we summarize the current knowledge of Photorhabdus SM, emphasizing on their activity against plant pathogens and parasites. We further discuss their potential in the management of agricultural pests and the steps that need to be taken for the implementation of Photorhabdus SM in pest management.

R-type bacteriocins in related strains of Xenorhabdus bovienii: Xenorhabdicin tail fiber modularity and contribution to competitiveness.

R-type bacteriocins are contractile phage tail-like structures that are bactericidal towards related bacterial species. The C-terminal region of the phage tail fiber protein determines target-binding specificity. The mutualistic bacteria Xenorhabdus nematophila and X. bovienii produce R-type bacteriocins (xenorhabdicins) that are selectively active against different Xenorhabdus species. We analyzed the P2-type remnant prophage clusters in draft sequences of nine strains of X. bovienii The C-terminal tail fiber region in each of the respective strains was unique and consisted of mosaics of modular units. The region between the main tail fiber gene (xbpH1) and the sheath gene (xbpS1) contained a variable number of modules encoding tail fiber fragments. DNA inversion and module exchange between strains was involved in generating tail fiber diversity. Xenorhabdicin-enriched fractions from three different X. bovienii strains isolated from the same nematode species displayed distinct activities against each other. In one set of strains, the strain that produced highly active xenorhabdicin was able to eliminate a sensitive strain. In contrast, xenorhabdicin activity was not a determining factor in the competitive fitness of a second set of strains. These findings suggest that related strains of X. bovienii use xenorhabdicin and additional antagonistic molecules to compete against each other.

Transcript Abundance of Photorhabdus Insect-Related (Pir) Toxin in Manduca sexta and Galleria mellonella Infections.

In this study, we assessed pirAB toxin transcription in Photorhabdus luminescens laumondii (strain TT01) (Enterobacteriaceae) by comparing mRNA abundance under in vivo and in vitro conditions. In vivo assays considered both natural and forced infections with two lepidopteran hosts: Galleria mellonella and Manduca sexta. Three portals of entry were utilized for the forced infection assays: (a) integument; (b) the digestive route (via mouth and anus); and (c) the tracheal route (via spiracles). We also assessed plu4093-2 transcription during the course of a natural infection; this is when the bacteria are delivered by Heterorhabditis bacteriophora nematodes. Transcript abundance in G. mellonella was higher than in M. sexta at two of the observed time points: 15 and 18 h. Expression of pirAB plu4093-2 reached above endogenous control levels at 22 h in G. mellonella but not in M. sexta. Overall, pirAB plu4093-2 transcripts were not as highly expressed in M. sexta as in G. mellonella, from 15 to 22 h. This is the first study to directly compare pirAB plu4093-2 toxin transcript production considering different portals of entry.

Bioprospecting for secondary metabolites in the entomopathogenic bacterium Photorhabdus luminescens subsp. sonorensis.

Crude extracts of in vitro and in vivo cultures of two strains of Photorhabdus l. sonorensis (Enterobacteriaceae) were analyzed by TLC, HPLC-UV and LC-MS. Nine unique compounds with mass/charge ratios (m/z) ranging from 331.3 to 713.5 were found in MS analyses. Bioactivity of extracts was assessed on a selection of plant pathogens/pests and non-target species. Caborca strain extracts showed the highest activity against Helicoverpa zea (Lepidoptera: Noctuidae) neonates at all concentrations tested. Mortality ranged from 11% (at 10µg/ml) to 37% (at 40µg/ml). Strain CH35 extracts showed the highest nematicidal activity on Meloidogyne incognita (Tylenchida: Meloidogynidae) at 40µg/ml. Low to no nematicidal activity was observed against the non-target species Steinernema carpocapsae (Rhabditida: Steinernematidae) and Caenorhabditis elegans (Rhabditida: Rhabditidae). Caborca extracts exhibited a strong antibiotic effect on Pseudomonas syringae (Pseudomonadales: Pseudomonadacedae) at 40µg/ml, while both Caborca and CH35 extracts inhibited the growth of Bacillus subitillis (Bacillales: Bacillaceae) at 40µg/ml. All extracts strongly inhibited the growth of the fungus Fusarium oxysporum (Hypocreales: Nectriceae) but not that of Alternaria alternata (Pleosporales: Pleosporaceae). Contrastingly, a moderate to high inhibitory effect was denoted on the non-target biocontrol fungus Beauveria bassiana (Hypocreales: Clavivipitaceae).