Induced knockdown of Mg-odr-1 and Mg-odr-3 perturbed the host seeking behavior of Meloidogyne graminicola in rice.

Root-knot nematode Meloidogyne graminicola is one of the most destructive plant parasites in upland as well as direct seeded rice. As an integral part of nematode biology, host finding behavior involves perceiving and responding to different chemical cues originating from the rhizosphere. A sustainable management tactic may include retardation of nematode chemoreception that would impair them to detect and discriminate the host stimuli. Deciphering the molecular basis of nematode chemoreception is vital to identify chokepoints for chemical or genetic interventions. However, compared to the well-characterized chemoreception mechanism in model nematode Caenorhabditis elegans, plant nematode chemoreception is yet underexplored. Herein, the full-length cDNA sequences of two chemotaxis-related genes (Mg-odr-1 and Mg-odr-3) were cloned from M. graminicola. Both the genes were markedly upregulated in the early developmental stages of M. graminicola suggesting their involvement in host finding processes. RNAi-induced independent knockdown of Mg-odr-1 and Mg-odr-3 caused behavioral aberration in second-stage juveniles of M. graminicola which in turn perturbed the nematodes' host finding ability and parasitic success inside rice roots. Additionally, nematodes' chemotactic response to different host root exudates, volatile and nonvolatile compounds was affected. Our results demonstrating the role of specific chemosensory genes in modulating M. graminicola host seeking behavior can enrich the existing knowledge of plant nematode chemoreception mechanism, and these genes can be targeted for novel nematicide development or in planta RNAi screens.

Novel moisture retaining dustable powder containing Steinernema abbasi effectively controls damage of subterranean termite in wheat and chickpea.

The application of biocontrol agents in farm operations for pest control programs is gaining priority and preference globally. Effective delivery, infectivity of the biocontrol agents, and quality shelf-life products containing these bioagents are vital parameters responsible for the success of biopesticides under field conditions. In the present study, moisture-retaining bio-insecticidal dustable powder formulation (SaP) of Steinernema abbasi (Sa) infective juveniles (IJs) was developed and assessed for its shelf life, physicochemical profile, and bio-efficacy against subterranean termite under field conditions. Formulation exhibited free-flowing character, with pH of 6.50-7.50, and apparent density in the range 0.50-0.70 g cm-3. The bioefficacy study for two rabi seasons (2020-2021, and 2021-2022) in wheat and chickpea grown in an experimental farm heavily infested with subterranean termites (Odontotermes obesus) revealed a significant reduction in plant damage due to pest attack in formulation-treated plots, monitored in terms of relative number of infested tillers in wheat and infested plants in chickpea fields. The reduced damage to the crop caused by termite was reflected in the relative differences in the growth and yield attributes as well. The study establishes the potential of the developed product as a biopesticide suitable for organic farming and integrated pest management operations.

RNA-Sequencing of Heterorhabditis nematodes to identify factors involved in symbiosis with Photorhabdus bacteria.

BACKGROUND: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria. RESULTS: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified. CONCLUSIONS: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

Photorhabdus hindustanensis sp. nov., Photorhabdus akhurstii subsp. akhurstii subsp. nov., and Photorhabdus akhurstii subsp. bharatensis subsp. nov., isolated from Heterorhabditis entomopathogenic nematodes.

Two Gram-negative, rod-shaped bacteria, H1T and H3T, isolated from the digestive tract of Heterorhabditis entomopathogenic nematodes were biochemically and molecularly characterized to determine their taxonomic positions. The 16S rRNA gene sequences of these strains indicate that they belong to the Gammaproteobacteria, to the family Morganellaceae, and to the Photorhabdus genus. Deeper analyses using whole genome-based phylogenetic reconstructions show that strains H1T and H3T are closely related to P. akhurstii DSM 15138T, to P. hainanensis DSM 22397T, and to P. namnaonensis PB45.5T. In silico genomic comparisons confirm these observations and show that strain H1T shares 70.6, 66.8, and 63.5 % digital DNA-DNA hybridization (dDDH) with P. akhurstii DSM 15138T, P. hainanensis DSM 22397T, and P. namnaonensis PB45.5T, respectively, and that strain H3T shares 76.6, 69.4, and 59.2 % dDDH with P. akhurstii DSM 15138T, P. hainanensis DSM 22397T, and P. namnaonensis PB45.5T, respectively. Physiological and biochemical characterization reveals that these two strains differ from most of the validly described Photorhabdus species and from their more closely related taxa. Given the clear phylogenetic separations, that the threshold to discriminate species and subspecies is 70 and 79% dDDH, respectively, and that strains H1T and H3T differ physiologically and biochemically from their more closely related taxa, we propose to classify H1T and H3T into new taxa as follows: H3T as a new subspecies within the species P. akhurstii, and H1T as a new species within the Photorhabdus genus, in spite that H1T shares 70.6 % dDDH with P. akhurstii DSM 15138T, score that is slightly higher than the 70 % threshold that delimits species boundaries. The reason for this is that H1T and P. akhurstii DSM 15138T cluster apart in the phylogenetic trees and that dDDH scores between strain H1T and other P. akhurstii strains are lower than 70 %. Hence, the following names are proposed: Photorhabdus hindustanensis sp. nov. with the type strain H1T (=IARI-SGMG3T,=KCTC 82683T=CCM 9150T=CCOS 1975T) and P. akhurstii subsp. bharatensis subsp. nov. with the type strain H3T (=IARI-SGHR2T=KCTC 82684T=CCM 9149T=CCOS 1976T). These propositions automatically create P. akhurstii subsp. akhurstii subsp. nov. with DSM 15138T as the type strain (currently classified as P. akhurstii).

Nematode genome announcement: The draft genome sequence of entomopathogenic nematode Heterorhabditis indica.

Heterorhabditis indica is one of the most widely used entomopathogenic nematodes for the biological control of agricultural insect pests worldwide. The draft genome of H. indica was sequenced using three genomic libraries of 300 bp, 600 bp and 5 kb sizes by Illumina HiSeq platform. The size of the draft genome assembly was 91.26 Mb, comprising 3,538 scaffolds. Genome completeness analysis by BUSCO (Benchmarking Universal Single-Copy Orthologs) showed 84% complete, and 6.5% fragmented BUSCOs. Further, 10,494 protein-coding genes were predicted. The H. indica draft genome will enable comparative and functional genomic studies in Heterorhabditis nematodes.

The differential strain virulence of the candidate toxins of Photorhabdus akhurstii can be correlated with their inter-strain gene sequence diversity.

Photorhabdus akhurstii is an insect-parasitic bacterium that symbiotically associates with the nematode, Heterorhabditis indica. The bacterium possesses several pathogenicity islands that aids in conferring toxicity to different insects. Herein, we constructed the plasmid clones of coding sequences of four toxin genes (pirA, tcaA, tccA and tccC; each was isolated from four P. akhurstii strains IARI-SGMG3, IARI-SGGJ2, IARI-SGHR2 and IARI-SGMS1) in Escherichia coli and subsequently, their biological activity were investigated against the fourth-instar larvae of the model insect, Galleria mellonella via intra-hemocoel injection. Bioinformatics analyses indicated inter-strain amino acid sequence difference at several positions of the candidate toxins. In corroboration, differential insecticidal activity of the identical toxin protein (PirA, TcaA, TccA and TccC conferred 15-59, 27-100, 25-100 and 33-98% insect mortality, respectively, across the strains) derived from the different bacterial strains was observed, suggesting that the diverse gene pool in Indian strains of P. akhurstii leads to strain-specific virulence in this bacterium. These toxin candidates appear to be an attractive option to deploy them in biopesticide development for managing the insect pests globally.

Txp40, a protein from Photorhabdus akhurstii, conferred potent insecticidal activity against the larvae of Helicoverpa armigera, Spodoptera litura and S. exigua.

BACKGROUND: Txp40, a 37 kDa protein, previously characterized from the Gram-negative bacterium Photorhabdus akhurstii (symbiotically associates with insect-parasitic nematode, Heterorhabditis indica), conferred insecticidal activity against Galleria mellonella. Here, the biological activity of Txp40 was evaluated against economically important insects, including Helicoverpa armigera, Spodoptera litura and S. exigua. RESULTS: When both intra-hemocoel injected and orally fed to test insects, comparatively greater oral LD50 (187.7-522 ng g-1 ) than injection LD50 (32.33-150.6 ng g-1 ) was obtained with Txp40 derived from P. akhurstii strain IARI-SGMG3. Injection of purified Txp40 caused a dose-dependent reduction in the total circulatory hemocytes and hemocyte viability of fourth-instar larvae of the test insects at 12 h post incubation; unlike healthy cells toxin-treated ones displayed aggregated distribution. Injection of Txp40 significantly elevated the phenoloxidase activity of insect hemolymph, which potentially led to unrestrained melanization reaction and ultimately larval death. Histological analyses showed the primary site of action of Txp40 in the insect midgut. Extensive damage to midgut epithelium 24 h after injection of the Txp40 explains the access of the toxin from hemocoel to midgut via leaky septate junctions. In silico analyses suggested that Txp40 can potentially interact with H. armigera midgut receptor proteins cadherin, ATP-binding cassettes, aminopeptidase N1 and alkaline phosphatase to exert toxicity. CONCLUSION: We propose Txp40 as an attractive alternative to Cry toxins of Bacillus thuringiensis, the transgenic expression of which is reported to cause resistance development in insects. © 2019 Society of Chemical Industry.

TcaB, an insecticidal protein from Photorhabdus akhurstii causes cytotoxicity in the greater wax moth, Galleria mellonella.

Photorhabdus akhurstii can produce a variety of proteins that aid this bacterium and its mutualistic nematode vector, Heterorhabditis indica to kill the insect host. Herein, we characterized (by heterologously expressing in E. coli) an open reading frame (1713 bp) of the toxin complex protein, TcaB from P. akhurstii strains IARI-SGHR2 and IARI-SGMS1 and assessed its toxic effect on G. mellonella larvae. The intra-hemocoel injection of purified TcaB (molecular weight-63 kDa) caused fourth instar larval bodies to blacken and die with LD50 values of 67.25 (IARI-SGHR2) and 52.08 (IARI-SGMS1) ng per larva at 12 h. Additionally, oral administration of the toxin caused larval mortality with LD50 values of 709.55 (IARI-SGHR2) and 598.44 (IARI-SGMS1) ng per g diet per larva at 7 days post feeding. Injection of purified TcaB caused loss of viability of fourth instar G. mellonella hemocytes at 6 h post incubation; cells displayed morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and disintegration. Injection of TcaB also elevated the phenoloxidase activity in insect hemolymph which triggers an extensive immune response that potentially leads to larval death. Similar to other bacterial toxins TcaB possesses potent biological activity which may enable it to be used as an efficient agent for pest management.

Description of Oscheius indicus n. sp. (Rhabditidae: Nematoda) from India.

A new amphimictic species Oscheius indicus n. sp. is described and illustrated with morphological and molecular data. The species is characterized by a medium-sized and slender body (female: L = 1.1 to 1.5 mm; a = 16.8 to 20.6; b = 5.7 to 7.1; c = 7.5 to 10.4; c' = 5.0 to 7.6; V = 45 to 51%), presence of four incisures each in the lateral fields with three minute warts, long rectum (2 to 3 anal body diameters), nine pairs of papillae arranged as 1+1+1/3+3 pattern, a prominent double-flapped epipytigma on vulval opening, presence of open leptoderan bursa and crochet needle-shaped spicules place it in the insectivora group. Morphologically, O. indicus n. sp. closely resembles O. carolinensis, O. chongmingensis, O. colombiana, and O. nadarajani. Molecular phylogenetic analysis carried out using ITS and D2/D3 expansion region of 28S rDNA sequences suggests that O. indicus n. sp. is closer to O. chongmingensis and O. rugaonensis. In summary, the morphometrical data, morphological observations and molecular phylogenetic analysis suggested that O. indicus n. sp. is sufficiently different from any known species and is therefore proposed as a new species within the insectivora group.A new amphimictic species Oscheius indicus n. sp. is described and illustrated with morphological and molecular data. The species is characterized by a medium-sized and slender body (female: L = 1.1 to 1.5 mm; a = 16.8 to 20.6; b = 5.7 to 7.1; c = 7.5 to 10.4; c' = 5.0 to 7.6; V = 45 to 51%), presence of four incisures each in the lateral fields with three minute warts, long rectum (2 to 3 anal body diameters), nine pairs of papillae arranged as 1+1+1/3+3 pattern, a prominent double-flapped epipytigma on vulval opening, presence of open leptoderan bursa and crochet needle-shaped spicules place it in the insectivora group. Morphologically, O. indicus n. sp. closely resembles O. carolinensis, O. chongmingensis, O. colombiana, and O. nadarajani. Molecular phylogenetic analysis carried out using ITS and D2/D3 expansion region of 28S rDNA sequences suggests that O. indicus n. sp. is closer to O. chongmingensis and O. rugaonensis. In summary, the morphometrical data, morphological observations and molecular phylogenetic analysis suggested that O. indicus n. sp. is sufficiently different from any known species and is therefore proposed as a new species within the insectivora group.

A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita.

BACKGROUND: Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. RESULTS: A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. CONCLUSIONS: Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.

A 37 kDa Txp40 protein characterized from Photorhabdus luminescens sub sp. akhurstii conferred injectable and oral toxicity to greater wax moth, Galleria mellonella.

Photorhabdus luminescens is a gram-negative bacterium that symbiotically associates with insect-parasitic nematode, Heterorhabditis indica. Herein, we have characterized an insecticidal gene, Txp40 (1008 bp) from the indigenous isolates of P. luminescens, and tested its bioefficacy against Galleria mellonella via injectable and oral bioassay. The recombinant protein characterized from P. luminescens strain H3 exhibited comparatively greater insect toxicity than strain H1 in terms of LD50 and LT50 values. Txp40 holds great potential to replace Bt toxins in global agriculture.

Proteomic Investigation of Photorhabdus Bacteria for Nematode-Host Specificity.

Majority of animals form symbiotic relationships with bacteria. Based on the number of bacterial species associating with an animal, these symbiotic associations can be mono-specific, relatively simple (2-25 bacterial species/animal) or highly complex (>10(2)-10(3) bacterial species/animal). Photorhabdus (family-Enterobacteriaceae) forms a mono-specific symbiotic relationship with the entomopathogenic nematode Heterorhabditis. This system provides a tractable genetic model for animal-microbe symbiosis studies. Here, we investigated the bacterial factors that may be responsible for governing host specificity between nematode and their symbiont bacteria using proteomics approach. Total protein profiles of P. luminescens ssp. laumondii (host nematode- H. bacteriophora) and P. luminescens ssp. akhurstii (host nematode- H. indica) were compared using 2-D gel electrophoresis, followed by identification of differentially expressed proteins by MALDI-TOF MS. Thirty-nine unique protein spots were identified - 24 from P. luminescens ssp. laumondii and 15 from P. luminescens ssp. akhurstii. These included proteins that might be involved in determining host specificity directly (for e.g. pilin FimA, outer membrane protein A), indirectly through effect on bacterial secondary metabolism (for e.g. malate dehydrogenase Mdh, Pyruvate formate-lyase PflA, flavo protein WrbA), or in a yet unknown manner (for e.g. hypothetical proteins, transcription regulators). Further functional validation is needed to establish the role of these bacterial proteins in nematode-host specificity.

A transcriptomic insight into the infective juvenile stage of the insect parasitic nematode, Heterorhabditis indica.

BACKGROUND: Nematodes are the most numerous animals in the soil. Insect parasitic nematodes of the genus Heterorhabditis are capable of selectively seeking, infecting and killing their insect-hosts in the soil. The infective juvenile (IJ) stage of the Heterorhabditis nematodes is analogous to Caenorhabditis elegans dauer juvenile stage, which remains in 'arrested development' till it finds and infects a new insect-host in the soil. H. indica is the most prevalent species of Heterorhabditis in India. To understand the genes and molecular processes that govern the biology of the IJ stage, and to create a resource to facilitate functional genomics and genetic exploration, we sequenced the transcriptome of H. indica IJs. RESULTS: The de-novo sequence assembly using Velvet-Oases pipeline resulted in 13,593 unique transcripts at N50 of 1,371 bp, of which 53 % were annotated by blastx. H. indica transcripts showed higher orthology with parasitic nematodes as compared to free living nematodes. In-silico expression analysis showed 30 % of transcripts expressing with ≥100 FPKM value. All the four canonical dauer formation pathways like cGMP-PKG, insulin, dafachronic acid and TGF-ß were active in the IJ stage. Several other signaling pathways were highly represented in the transcriptome. Twenty-four orthologs of C. elegans RNAi pathway effector genes were discovered in H. indica, including nrde-3 that is reported for the first time in any of the parasitic nematodes. An ortholog of C. elegans tol-1 was also identified. Further, 272 kinases belonging to 137 groups, and several previously unidentified members of important gene classes were identified. CONCLUSIONS: We generated high-quality transcriptome sequence data from H. indica IJs for the first time. The transcripts showed high similarity with the parasitic nematodes, M. hapla, and A. suum as opposed to C. elegans, a species to which H. indica is more closely related. The high representation of transcripts from several signaling pathways in the IJs indicates that despite being a developmentally arrested stage; IJs are a hotbed of signaling and are actively interacting with their environment.

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana, India.

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b' = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c' = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b' = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c' = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.

Nitazoxanide: nematicidal mode of action and drug combination studies.

Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections, we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections, was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy.

A single promoter inversion switches Photorhabdus between pathogenic and mutualistic states.

Microbial populations stochastically generate variants with strikingly different properties, such as virulence or avirulence and antibiotic tolerance or sensitivity. Photorhabdus luminescens bacteria have a variable life history in which they alternate between pathogens to a wide variety of insects and mutualists to their specific host nematodes. Here, we show that the P. luminescens pathogenic variant (P form) switches to a smaller-cell variant (M form) to initiate mutualism in host nematode intestines. A stochastic promoter inversion causes the switch between the two distinct forms. M-form cells are much smaller (one-seventh the volume), slower growing, and less bioluminescent than P-form cells; they are also avirulent and produce fewer secondary metabolites. Observations of form switching by individual cells in nematodes revealed that the M form persisted in maternal nematode intestines, were the first cells to colonize infective juvenile (IJ) offspring, and then switched to P form in the IJ intestine, which armed these nematodes for the next cycle of insect infection.

The type 2 secretion Pseudopilin, gspJ, is required for multihost pathogenicity of Burkholderia cenocepacia AU1054.

Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.

Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis.

Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little is known of their function in mutualistic symbioses. Photorhabdus colonization of Heterorhabditis bacteriophora nematodes is essential for the pair's insect pathogenic lifestyle. Maternal nematodes acquire Photorhabdus symbionts as a persistent intestinal biofilm prior to transmission to infective juvenile (IJ) stage offspring developing inside the maternal body. Screening 8000 Photorhabdus mutants for defects in IJ colonization revealed that a single fimbrial locus, named mad for maternal adhesion defective, is essential. The mad genes encode a novel usher/chaperone assembled fimbria regulated by an ON/OFF invertible promoter switch. Adherent Photorhabdus cells in maternal nematode intestines had the switch ON opposite to the OFF orientation of most other cells. A ΔmadA mutant failed to adhere to maternal intestines and be transmitted to the IJs. Mad fimbriae were detected on TT01 phase ON cells but not on ΔmadA phase ON cells. Also required for transmission is madJ, predicted to encode a transcriptional activator related to GrlA. Expression of madA-K or madIJK restored the ability of madJ mutant to adhere. The Mad fimbriae were not required for insect pathogenesis, indicating the specialized function of Mad fimbriae for symbiosis.

Genetic diversity and multihost pathogenicity of clinical and environmental strains of Burkholderia cenocepacia.

A collection of 54 clinical and agricultural isolates of Burkholderia cenocepacia was analyzed for genetic relatedness by using multilocus sequence typing (MLST), pathogenicity by using onion and nematode infection models, antifungal activity, and the distribution of three marker genes associated with virulence. The majority of clinical isolates were obtained from cystic fibrosis (CF) patients in Michigan, and the agricultural isolates were predominantly from Michigan onion fields. MLST analysis resolved 23 distinct sequence types (STs), 11 of which were novel. Twenty-six of 27 clinical isolates from Michigan were genotyped as ST-40, previously identified as the Midwest B. cenocepacia lineage. In contrast, the 12 agricultural isolates represented eight STs, including ST-122, that were identical to clinical isolates of the PHDC lineage. In general, pathogenicity to onions and the presence of the pehA endopolygalacturonase gene were detected only in one cluster of related strains consisting of agricultural isolates and the PHDC lineage. Surprisingly, these strains were highly pathogenic in the nematode Caenorhabditis elegans infection model, killing nematodes faster than the CF pathogen Pseudomonas aeruginosa PA14 on slow-kill medium. The other strains displayed a wide range of pathogenicity to C. elegans, notably the Midwest clonal lineage which displayed high, moderate, and low virulence. Most strains displayed moderate antifungal activity, although strains with high and low activities were also detected. We conclude that pathogenicity to multiple hosts may be a key factor contributing to the potential of B. cenocepacia to opportunistically infect humans both by increasing the prevalence of the organism in the environment, thereby increasing exposure to vulnerable hosts, and by the selection of virulence factors that function in multiple hosts.

Patenting drought tolerance in organisms.

Dehydration is a major form of osmotic stress in cells. Physiological and molecular basis of dehydration stress responses in cells and organisms has been intensively researched over past years. Almost all of the patented dehydration stress tolerance genes from different organisms were used in engineering drought tolerance in crop plants. In spite of the moral, religious and ethical controversies surrounding use of foreign DNA sequences in crop plants, the numbers of such patents has grown tremendously in recent years. In future, we might witness another rise in patents on use of dehydration stress related gene sequences in creating environmental stress tolerant biological control agents for plant disease and insect pest management in agriculture. This review summarizes some of the recent published patents related to drought tolerance genes and their use.