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Summary Ditylenchus rafiqi n. sp. was recovered from the rhizosphere of pomegranate in New Delhi (India), and it is characterised by morphological, morphometrical and molecular methods. The new species is characterised by having a body length of 553-901 μm, delicate stylet (7-10 μm long), longer conus length (5.0-5.5 μm in females and 4.5-5.5 μm in males) with small round knobs sloping backward, seven lateral lines at the mid-body, median bulb well developed with thickening of lumen walls, a post-uterine sac well developed (27-47 μm), tail long, elongate conoid with an almost round terminus (45-74 μm) and male spicule of 15-17.5 μm, gubernaculum simple, leptoderan type of bursa enveloping the tail almost >50% of its tail length. A detailed study based on morphological and morphometrical methods clearly demarcates that D. rafiqi n. sp. differs from other known species of Ditylenchus. The molecular characterisation was done using D2-D3 of 28S rRNA and ITS rRNA markers. Molecular characterisation shows that the new species is closely related to D. acutus, D. gilanicus (D2-D3 marker), and D. medicaginis (ITS rRNA markers). Based on phylogenetic analyses using D2-D3 of LSU and ITS rRNA markers, D. rafiqi n. sp. forms an intermediate clade between the D. triformis and D. dipsaci groups.

Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema kill insects with the help of their symbiotic bacteria. They are widely used as biocontrol agents to manage insect pests of crops. The infective juveniles (IJ) of EPNs are isolated from soil by insect baiting technique, which is labour-intensive, time-consuming, wasteful, and inefficient. Here, we present loop-mediated isothermal amplification (LAMP) assays for rapid detection of Heterorhabditis spp. (Het-LAMP) and Steinernema spp. (Ste-LAMP) from total soil DNA. The primers for Het-LAMP and Ste-LAMP were designed using ITS and 18S rDNA regions of genomic DNA. The LAMP reactions could be completed in 60 min, at 66 °C and 68 °C, respectively, followed by termination at 85 °C for 5 min. The assays were highly sensitive and could detect up to 0.02 picograms of Heterorhabditis DNA and 96 picograms of Steinernema DNA in a 25 μl reaction. Both the assays were specific for the target nematode species and detected the presence of a single IJ in the total DNA extracted from 250 mg of soil. The assays developed in this study would be of immense utility for the efficient detection and identification of native EPNs in large-scale surveys. These assays are amenable to automation and could be used to develop convenient detection kits for point-of-service diagnosis of EPNs in the field without the need for a trained and experienced personnel.

The Gram-negative bacteria Photorhabdus lives in a symbiotic relationship with the insect-pathogenic Heterorhabditis nematodes and produces numerous hydrolytic enzymes, secondary metabolites and protein toxins. Seven Photorhabdus strains were previously isolated from the Heterorhabditis nematodes collected from different geographical regions of India. The strains IARI-SGMG3, IARI-SGHR2, IARI-SGHR4, IARI-SGMS1 and IARI-SGGJ2 were identified as P. akhurstii, whereas IARI-SGLDK1 and IARI-SGHP1 were identified as P. laumondii subsp. laumondii and P. laumondii subsp. clarkeii, respectively. A new and previously unreported 35 kDa molecular weight protein toxin 'Galtox' was identified from these Photorhabdus strains. The nucleotide sequences of the toxin gene from seven Photorhabdus strains were PCR amplified, sequenced, cloned into pET protein expression vector, and the protein toxin was expressed and purified. The Galtox sequence from various strains showed variations in sequence and toxicity against Galleria mellonella. The injection of purified Galtox protein into the 4th instar larvae showed median lethal dose (LD50) values of 2.39–26.08 ng toxin/g G. mellonella bodyweight after 48 h. The protein injection killed the insects quickly and exhibited a median lethal time (LT50) of 12–60 h when injected at the rate of 3.1–31.2 ng toxin/g G. mellonella bodyweight. Galtox protein sequence analysis indicated similarity to several bacterial toxin-related protein domains, such as 6rgnA domain of Bordetella membrane targeting toxin BteA, 6gy6 domain of Xenorhabdus α-Xenorhabdolysins, 4mu6A and 4xa9a domains similar to effector protein LegC3 from Legionella pneumophila and 1cv8.1 domain of staphylococcal cysteine proteinase staphopain B. The mode of action of Galtox needs to be understood to enable its use for the management of agricultural insect-pests.

Cultivated fenugreek Trigonella corniculata L, also known as kasuri methi or nagauri paan is a popular spice crop and widely grown in the state of Rajasthan, India. Galls and root-knot nematodes were observed on the roots of T. corniculata collected from Naguar district and its surrounding areas in the state of Rajasthan. The nematode DNA isolated from the sample was subjected to PCR by using species-specific primers for the common root-knot nematode species. Molecular identification confirmed the infestation of Meloidogyne enterolobii on T. corniculata. This is the first report of M. enterolobii infestation on T. corniculata from India.

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.

Root-knot nematodes (Meloidogyne spp.) infect a large number of crops including guava. We investigated a population of Meloidogyne sp. infecting guava in the Coimbatore region of Tamil Nadu, India for identification and species confirmation. Detailed morphological and morphometric observations based on second-stage juveniles, males, females, and perineal patterns showed resemblance of the isolated population with the original and subsequent descriptions of M. enterolobii. Isozyme analysis of the young egg-laying females displayed the characteristic esterase phenotype pattern similar to that of M. enterolobii. Additionally, the identity of the nematode population was further validated by M. enterolobii specific SCAR marker and ITS rDNA. Recently published reports on the occurrence and morphological descriptions of M. enterolobii from India are largely incongruent with the original and subsequent redescriptions of the species. Here, we present the most comprehensive morphology and morphometrics of an Indian population of M. enterolobii for its authentic identification.

Amyna axis (Lepidoptera: Erebidae), Chrysodeixis spp. ( C. acuta and C. eriosoma ) and Spodoptera spp. ( S. exigua and S. litura ) (Lepidoptera: Noctuidae) are important polyphagous insect-pests of leguminous crops. A mermithid (Nematoda: Mermithidae) nematode was found parasitizing the larval stages of these insects in the western Indian state of Rajasthan. Morphological and molecular analysis suggests that the nematode might be a species of the genus Hexamermis . The average length of post-parasitic nematode juveniles was 15 cm, and the average greatest body width was 1.5 mm. The vulva was median (V% = 48) without vulval flap. Approximately 100-125 μm long caudal appendages were present on tail. The cross-section revealed the presence of six hypodermal chords at the mid body region, and stichosome was present. The 18S rDNA sequence showed 99% similarity to mermithid nematode. A phylogenetic analysis of 18S rDNA sequences of mermithids resulted in identification of a new clade “Lineage 3” which included representatives of Mermis sp., Isomermis sp., Limnomermis sp., and Pheromermis sp. This is the first report of natural mermithid parasitism of A. axis and Chrysodeixis spp. along with its molecular characterization.

Summary A new species of pin nematode, Paratylenchus jasmineae sp. n., was isolated and described from the rhizosphere of jasmine in India. The new species can be diagnosed by having a relatively larger body length of 248-355 μm, stylet length of 24.6-28.5 μm, lateral field with four lateral lines, the presence of small post-vulval uterine sac and absence of advulval flaps in the females. Additional morphological characters included continuous lip region with small projected submedian lobes, large and oblong-shaped spermatheca, the presence of prevulval swelling, and arcuate conoid tail with finely rounded terminus. Molecular characterisation of the species was carried out based on internal transcribed spacer sequence (ITS rRNA) and D2-D3 expansion segment of 28S rRNA subunit. Phylogenetic analyses based on ITS and D2-D3 molecular markers resulted in clear separation of P. jasmineae sp. n. from other Paratylenchus species.

Summary A population of a Hemicriconemoides species was found from the rhizosphere of sugarcane in the Meerut district, Uttar Pradesh, India. Preliminary morphological studies led to the identification of the species as H. rosae. Further morphological and morphometric characterisation of the species was carried out by light microscopy. The gross morphology was consistent with the description of H. rosae except for some deviations in measurements. Here we present an amended description of the species together with illustrations and additional morphometric and morphological characters. The diagnostic relationship of H. rosae is given based on morphological and molecular information and a comparative study of paratype and holotype females. In addition, ITS rRNA and D2-D3 sequences were used for the construction of evolutionary relationship by Bayesian inference. The present study provides molecular data and a redescription of H. rosae collected from the rhizosphere of sugarcane in India.

Cyst nematodes of the species Globodera rostochiensis and G. pallida are devastating parasites of the potato crop. Early detection of cyst nematodes in the field is critical for adopting an appropriate management strategy. A specific and sensitive loop-mediated isothermal amplification (LAMP) assay using four oligonucleotide primers has been developed to amplify the internal transcribed spacer region (ITS) of ribosomal DNA of potato cyst nematode G. rostochiensis. The PCN-LAMP reaction could be completed within 75 min at 68 °C followed by termination at 85 °C for 7 min. The primers exhibited specificity for G. rostochiensis and did not detect any other tested genera of plant parasitic or entomopathogenic nematodes. LAMP reaction was highly sensitive, suitable for crude genomic DNA and could successfully detect G. rostochiensis DNA up to femtogram quantity. This assay is rapid, cost effective and requires minimal instrumentation. It will facilitate the detection of G. rostochiensis at field and point-of-care labs and help in the interception of infested plant material/soil samples at quarantine stations independent of a professional nematologist.

Cyst nematodes of the species Globodera rostochiensis and G. pallida are devastating parasites of the potato crop. Early detection of cyst nematodes in the field is critical for adopting an appropriate management strategy. A specific and sensitive loop-mediated isothermal amplification (LAMP) assay using four oligonucleotide primers has been developed to amplify the internal transcribed spacer region (ITS) of ribosomal DNA of potato cyst nematode G. rostochiensis. The PCN-LAMP reaction could be completed within 75 min at 68 °C followed by termination at 85 °C for 7 min. The primers exhibited specificity for G. rostochiensis and did not detect any other tested genera of plant parasitic or entomopathogenic nematodes. LAMP reaction was highly sensitive, suitable for crude genomic DNA and could successfully detect G. rostochiensis DNA up to femtogram quantity. This assay is rapid, cost effective and requires minimal instrumentation. It will facilitate the detection of G. rostochiensis at field and point-of-care labs and help in the interception of infested plant material/soil samples at quarantine stations independent of a professional nematologist. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02830-8.

The rice root-knot nematode Meloidogyne graminicola is a major biotic stress for the rice crop under upland, rain-fed lowland and irrigated cultivation conditions. Here, we present an improved draft genome assembly of M. graminicola IARI strain using the long-read sequencing approach (PacBio Sequel platform). The assembled genome size was 36.86 Mb with 514 contigs and N50 value of 105 kb. BUSCO estimated the genome to be 88.6% complete. Meloidogyne graminicola genome contained 17.83% repeat elements and showed 14,062 protein-coding gene models, 4,974 conserved orthologous genes, 561 putative secreted proteins, 49 RNAi pathway genes, 1,853 proteins involved in pathogen-host interactions, 1,575 carbohydrate-active enzymes, and 32,138 microsatellites. Five of the carbohydrate-active enzymes were found only in M. graminicola genome and were not present in any other analysed root-knot nematode genome. Together with the previous two genome assemblies, this improved genome assembly would facilitate comparative and functional genomics for M. graminicola.

Resistance to rice root-knot nematode Meloidogyne graminicola in a mutant rice line is suggested to be conferred by higher expression of several genes putatively involved in damage-associated molecular pattern recognition, secondary metabolite biosynthesis including phytoalexins, and defence-related genes. Meloidogyne graminicola has emerged as the most destructive plant–parasitic nematode disease of rice (Oryza sativa L.). Genetic resistance to M. graminicola is one of the most effective methods for its management. A M. graminicola-resistant O. sativa ssp. indica mutant line-9 was previously identified through a forward genetic screen (Hatzade et al. Biologia 74:1197–1217, 2019). In the present study, we used RNA-Sequencing to investigate the molecular mechanisms conferring nematode resistance to the mutant line-9 compared to the susceptible parent JBT 36/14 at 24 h post-infection. A total of 674 transcripts were differentially expressed in line-9. Early regulation of genes putatively related to nematode damage-associated molecular pattern recognition (e.g., wall-associated receptor kinases), signalling [Nucleotide-binding, Leucine-Rich Repeat (NLRs)], pathogenesis-related (PR) genes (PR1, PR10a), defence-related genes (NB-ARC domain-containing genes), as well as a large number of genes involved in secondary metabolites including diterpenoid biosynthesis (CPS2, OsKSL4, OsKSL10, Oscyp71Z2, oryzalexin synthase, and momilactone A synthase) was observed in M. graminicola-resistant mutant line-9. It may be suggested that after the nematode juveniles penetrate the roots of line-9, early recognition of invading nematodes triggers plant immune responses mediated by phytoalexins, and other defence proteins such as PR proteins inhibit nematode growth and reproduction. Our study provides the first transcriptomic comparison of nematode-resistant and susceptible rice plants in the same genetic background and adds to the understanding of mechanisms underlying plant-nematode resistance in rice.

Root-knot nematodes (Meloidogyne spp.) infect a large number of crops including guava. We investigated a population of Meloidogyne sp. infecting guava in the Coimbatore region of Tamil Nadu, India for identification and species confirmation. Detailed morphological and morphometric observations based on second-stage juveniles, males, females, and perineal patterns showed resemblance of the isolated population with the original and subsequent descriptions of M. enterolobii. Isozyme analysis of the young egg-laying females displayed the characteristic esterase phenotype pattern similar to that of M. enterolobii. Additionally, the identity of the nematode population was further validated by M. enterolobii specific SCAR marker and ITS rDNA. Recently published reports on the occurrence and morphological descriptions of M. enterolobii from India are largely incongruent with the original and subsequent redescriptions of the species. Here, we present the most comprehensive morphology and morphometrics of an Indian population of M. enterolobii for its authentic identification.

Plant–parasitic root-knot nematode Meloidogyne incognita uses an array of effector proteins to establish successful plant infections. Mi-msp-1 and Mi-msp-20 are two known effectors secreted from nematode subventral oesophageal glands; Mi-msp-1 being a putative secretory venom allergen AG5-like protein, whereas Mi-msp-20 is a pioneer gene with a coiled-coil motif. Expression of specific effector is known to cause disturbances in the expression of other effectors. Here, we used RNA-Seq to investigate the pleiotropic effects of silencing Mi-msp-1 and Mi-msp-20. A total of 25.1–51.9 million HQ reads generated from Mi-msp-1 and Mi-msp-20 silenced second-stage juveniles (J2s) along with freshly hatched J2s were mapped to an already annotated M. incognita proteome to understand the impact on various nematode pathways. As compared to control, silencing of Mi-msp-1 caused differential expression of 29 transcripts, while Mi-msp-20 silencing resulted in differential expression of a broader set of 409 transcripts. In the Mi-msp-1 silenced J2s, cytoplasm (GO:0005737) was the most enriched gene ontology (GO) term, whereas in the Mi-msp-20 silenced worms, embryo development (GO:0009792), reproduction (GO:0000003) and nematode larval development (GO:0002119) were the most enriched terms. Limited crosstalk was observed between these two effectors as a sheer 5.9% of the up-regulated transcripts were common between Mi-msp-1 and Mi-msp-20 silenced nematodes. Our results suggest that in addition to the direct knock-down caused by silencing of Mi-msp-1 and Mi-msp-20, the cascading effect on other genes might also be contributing to a reduction in nematode's parasitic abilities.

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02288-0) contains supplementary material, which is available to authorized users.

Plant–parasitic root-knot nematode Meloidogyne incognita uses an array of effector proteins to establish successful plant infections. Mi-msp-1 and Mi-msp-20 are two known effectors secreted from nematode subventral oesophageal glands; Mi-msp-1 being a putative secretory venom allergen AG5-like protein, whereas Mi-msp-20 is a pioneer gene with a coiled-coil motif. Expression of specific effector is known to cause disturbances in the expression of other effectors. Here, we used RNA-Seq to investigate the pleiotropic effects of silencing Mi-msp-1 and Mi-msp-20. A total of 25.1–51.9 million HQ reads generated from Mi-msp-1 and Mi-msp-20 silenced second-stage juveniles (J2s) along with freshly hatched J2s were mapped to an already annotated M. incognita proteome to understand the impact on various nematode pathways. As compared to control, silencing of Mi-msp-1 caused differential expression of 29 transcripts, while Mi-msp-20 silencing resulted in differential expression of a broader set of 409 transcripts. In the Mi-msp-1 silenced J2s, cytoplasm (GO:0005737) was the most enriched gene ontology (GO) term, whereas in the Mi-msp-20 silenced worms, embryo development (GO:0009792), reproduction (GO:0000003) and nematode larval development (GO:0002119) were the most enriched terms. Limited crosstalk was observed between these two effectors as a sheer 5.9% of the up-regulated transcripts were common between Mi-msp-1 and Mi-msp-20 silenced nematodes. Our results suggest that in addition to the direct knock-down caused by silencing of Mi-msp-1 and Mi-msp-20, the cascading effect on other genes might also be contributing to a reduction in nematode's parasitic abilities. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02353-8) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (10.1186/s12864-018-5230-8) contains supplementary material, which is available to authorized users.

Insect-parasitic nematodes of the genus Heterorhabditis live in a symbiotic relationship with a gram negative Gamma-proteobacteria of the genus Photorhabdus. This nematode-bacteria pair is a simple and genetically tractable model to study animal-microbe symbiosis. Here we investigated the role of Heterorhabditis nematode C-type lectin (clec) genes in context of nematode-bacteria symbiosis. The in silico analysis identified seven clec genes in H. bacteriophora and three clec genes in H. indica. Two of the clec genes, H. bacteriophora clec-1 (Hb-clec-1) and H. bacteriophora clec-78 (Hb-clec-78) were further characterized. Both of these genes were present in a single copy in the H. bacteriophora genome. The phylogenetic analysis revealed that H. bacteriophora CLEC proteins were close to CLEC-1 and CLEC-78 proteins of free living Caenorhabditis but not to the CLEC proteins of insect-parasitic Steinernema nematodes which share a similar symbiotic relationship with Xenorhabdus bacteria. In situ hybridization showed that expression of Hb-clec-1 and Hb-clec-78 was localized to the alimentary canal of infective juveniles (IJs) in the region of terminal bulb, oesophago-intestinal valve and anterior part of intestine. Hb-clec-78 gene expression displayed significant positive correlation to the presence of bacteria during various stages of symbiosis: it was up-regulated during all the nematode developmental stages when Photorhabdus was symbiotically associated, but down-regulated at the post-IJ recovery stage when the developing nematodes were free of bacteria. Hb-clec-1 gene expression did not show any correlation with presence or absence of symbiont bacteria. Subject to genetic validation, our study suggests that Hb-clec-78 might be actively involved in modulation of symbiosis with Photorhabdus symbionts.

The rice root-knot nematode Meloidogyne graminicola has emerged as a devastating pest of rice in South-East Asian countries. Here we present a draft genome sequence for M. graminicola, assembled using data from short and long insert libraries sequenced on Illumina GAIIx sequencing platform.

Plant-parasitic nematodes (PPNs) cause substantial damage to agricultural and horticultural crops around the world. The early, accurate and rapid detection of PPNs at field level is the most important step to manage these pests, but remains a challenge for researchers. Loop-mediated isothermal amplification (LAMP) has emerged as a robust, rapid and sensitive method for point-of-service detection and diagnosis of plant pathogens such as bacteria, fungus, virus and nematodes. The present review summarises and analyses the LAMP assays developed for the detection and diagnosis of various important PPNs. Different gene targets such as SSU, 5.8S, LSU, ITS-1, ITS-2, D2-D3, IGS-1 and IGS-2 regions of the rRNA, syg-2 and pel-3 genes have been used for the development of the highly sensitive, species-specific or genus-specific LAMP assays for various PPNs. The LAMP assays are superior to PCR based assays as they are faster, more sensitive and can detect nematode DNA in up to femtogram quantities from the soil, plant tissue, preserved nematode samples or nematode genomic DNA stabilised on cellulose cards. The results of the LAMP assays can be visualised in the reaction tube by DNA intercalating dyes, observation of sample turbidity, by gel electrophoresis, and through lateral flow assays. Presently the LAMP-based assays are gaining popularity as the method of choice for point-of-care detection and diagnosis of various PPNs.

Meloidogyne incognita is a polyphagous plant-parasitic nematode that causes considerable yield loss in agricultural and horticultural crops. The management options available for M. incognita are extremely limited. Here we identified and characterised a M. incognita homolog of Caenorhabditis elegans sterol-binding protein (Mi-SBP-1), a transcriptional regulator of several lipogenesis pathway genes, and used RNA interference-mediated gene silencing to establish its utility as a target for the management of M. incognita. Mi-sbp-1 is predicted to be a helix-loop-helix domain containing DNA binding transcription factor, and is present in the M. incognita genome in three copies. The RNA-Seq analysis of Mi-sbp-1 silenced second stage juveniles confirmed the key role of this gene in lipogenesis regulation in M. incognita. In vitro and host-induced gene silencing of Mi-sbp-1 in M. incognita second stage juveniles resulted in loss of nematodes' ability to utilise the stored fat reserves, slower nematode development, and reduced parasitism on adzuki bean and tobacco plants. The multiplication factor for the Mi-sbp-1 silenced nematodes on adzuki bean plants was reduced by 51% compared with the control nematodes in which Mi-sbp-1 was not silenced. Transgenic expression of the double-stranded RNA construct of the Mi-sbp-1 gene in tobacco plants caused 40-45% reduction in M. incognita multiplication, 30-43.8% reduction in the number of egg masses, and 33-54% reduction in the number of eggs per egg mass compared with the wild type control plants. Our results confirm that Mi-sbp-1 is a key regulator of lipogenesis in M. incognita and suggest that it can be used as an effective target for its management. The findings of this study can be extended to develop methods to manage other economically important parasitic nematodes.

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.

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.

Photorhabdus bacteria exhibit contrasting lifestyles; they are virulent insect pathogens but symbionts of the entomopathogenic Heterorhabditis nematodes. Photorhabdus genomes encode several secondary metabolites and insecticidal protein toxins., Photorhabdus bacteria exhibit contrasting lifestyles; they are virulent insect pathogens but symbionts of the entomopathogenic Heterorhabditis nematodes. Photorhabdus genomes encode several secondary metabolites and insecticidal protein toxins. Here, we present the draft genome sequences for five Photorhabdus strains isolated from Heterorhabditis nematodes collected from various geographical regions of India.

Entomopathogenic nematodes (EPNs) exhibit complex host-seeking behaviour in soil and utilise the insect- and plant-derived odorants to locate and infect their insect hosts. The odorant response defective (odr) genes were identified in Caenorhabditis elegans through a forward genetic screen for worms defective in odour sensing. It is hypothesised that odr genes could be involved in insect host recognition by the EPNs, but no information is available on these genes or their functions in EPNs. Here, we investigated and characterised odr genes in the EPN Heterorhabditis bacteriophora. Seven orthologs of C. elegans odr genes were identified by in-silico analysis in H. bacteriophora, of which four genes (Hb-odr-2, Hb-odr-3, Hb-odr-4 and Hb-odr-10) could be amplified and cloned from the cDNA. The developmental stage-specific expression in infective juvenile (IJ), fourth stage juvenile (J4) and adult stage of H. bacteriophora showed that Hb-odr-2, Hb-odr-3 and Hb-odr-10 were up-regulated only at the IJ stage, whereas Hb-odr-10 was significantly up-regulated in all the developmental stages. Exposure to insect hemolymph resulted in high transcript accumulation of Hb-odr-3 but did not affect the expression of other Hb-odr genes. The in-situ hybridisation assays showed that Hb-odr-2 mRNA expression was localised at the region of the nerve ring while that of Hb-odr-3 in the region of pharyngointestinal valve and anterior esophagous of the IJs. Our results extend the knowledge of C. elegans odr genes to the entomopathogenic nematode H. bacteriophora. To the best of our information, this is the second investigation on the characterisation of odr genes in a parasitic nematode after the plant-parasitic species Meloidogyne incognita.

Photorhabdus is an insect-pathogenic Gram negative enterobacterium found in the gut of Heterorhabditis nematodes. Photorhabdus is highly virulent to insects, and can kill insects rapidly upon injection at very low concentrations of one to few cells. We characterized the virulence of Photorhabdus symbionts isolated from the Heterorhabditis nematodes collected from various parts of India by injecting different concentrations of bacterial cells into fourth instar larval stage of insect Galleria mellonella. Photorhabdus luminescens ssp. akhurstii strain IARI-SGMG3 from Meghalaya was identified as the most virulent of all the tested strains on the basis of LT50 and LC50 values. This study forms a basis for further investigations on the genetic basis of virulence in Photorhabdus bacteria.

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.

Potato cyst nematodes (PCNs), Globodera rostochiensis (Woll.) and G. pallida (Stone), are globally important pests of potato prevalent in the Nilgiri area of South India. PCNs were first intercepted from North India at Shimla, Himachal Pradesh (HP) in 2010. Subsequent surveys in the hilly areas of North India have revealed the occurrence of PCNs in several districts of HP, Uttarakhand and Jammu & Kashmir, despite domestic quarantine against this nematode. High populations of PCN were mainly encountered in the Potato Seed Multiplication Farms that supply seed potato to farmers. Here, a hypothesis on the dissemination of PCNs within India is proposed.

Pasteuria penetrans is an endospore forming hyperparasitic bacterium of the plant-pathogenic root-knot nematode, Meloidogyne incognita. For successful parasitization, the first step is adherence of bacterial endospores onto the cuticle surface of nematode juveniles. The knowledge of molecular intricacies involved during this adherence is sparse. Here, we identified a M. incognita selenium-binding protein (Mi-SeBP-1) differentially expressed during the initial interaction of M. incognita and P. penetrans, and show that it is involved in modulating parasitic adhesion of bacterial endospores onto nematode cuticle. Selenium-binding proteins (SeBPs) are selenium associated proteins important for growth regulation, tumor prevention and modulation of oxidation/reduction in cells. Although reported to be present in several nematodes, the function of SeBPs is not known in Phylum Nematoda. In situ hybridization assay localized the Mi-SeBP-1 mRNA to the hypodermal cells. RNAi-mediated silencing of Mi-SeBP-1 significantly increased the adherence of P. penetrans endospores to the nematode juvenile cuticle. Silencing of Mi-SeBP-1 did not change the nematode's ability to parasitize plants and reproduction potential within the host. These results suggest that M. incognita Mi-SeBP-1 might be involved in altering the attachment of microbial pathogens on the nematode cuticle, but is not involved in nematode-host plant interaction. This is the first report for a function of SeBP in Phylum Nematoda.

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.

Entomopathogenic nematodes (EPNs) have been successfully used in the USA and Europe for the biological management of insect pests, for example, invasive mole crickets, white grubs and other scarab larvae in lawns and turf, fungus gnats in mushroom, black vine weevil and Diaprepes root weevil in nursery plants and citrus, respectively. The use of EPNs in India is still in the preliminary stages. To find the best EPN isolates for use in local insect pest management programs, we previously collected 35 strains of EPNs from different parts of India, and screened them for insect infectivity at broad temperature range. Here we quantitated the virulence of seven EPN strains showing infectivity at broad temperature range against Galleria mellonella and Spodoptera litura. Three isolates of Steinernema and four isolates of Heterorhabditis were tested. The Heterorhabditis sp. Hmg3 was the most virulent EPN isolate among all the tested nematodes (LC50 = 16.1 infective juveniles (IJs)/insect larvae at 24 h), whereas t...

Fourteen populations of Meloidogyne graminicola were collected from different agroecological regions of India. Morphological and morphometrical comparisons were made for various nematode life stages. Three populations (Hisar, New Delhi, and Samastipur) were different from typical M. graminicola on the basis of the length of eggs; J2 length, a-value, hyaline tail portion; male length, distance up to excretory pore, spicule and gubernaculum lengths; female length and width, stylet length, distance up to excretory pore, EPST (distance of excretory pore from anterior end / stylet length [females]) ratio, and vulval length. Morphological and morphometrical comparison with closely related species M. graminis, M. oryzae, M. salasi, M. triticoryzae, and M. lini clustered these populations into two groups: Anand, Bhubaneswar, Hyderabad, Jammu, Jorhat, Kalyani, Kanpur, Ludhiana, Mandya, Palampur, Vellayani grouped with M. graminicola, M. triticoryzae and M. salasi; whereas, Hisar, New Delhi, Samastipur grouped with M. oryzae and M. graminis. Molecular phylogenetic analysis using internal transcribed spacer (ITS) suggested that in spite of morphological differences, these populations belonged to M. graminicola.

A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high β-1, 4-endoglucanase (2.5 IU/ml) but low activity of β-1, 4-exoglucanase (0.8 IU/ml) and β-glucosidase (0.084 IU/ml). The β-1, 4-endoglucanase gene of IARI-SP-1 was amplified using degenerate primers designed based on sequences already available in NCBI GenBank. A full length gene of β-1, 4-endonuclease consisting of 1499 nucleotides was identified through sequence analysis of the amplified product. The ORF encoded for a protein of 500 amino acids with a predicted molecular weight of 55 kDa. The gene was cloned in pET-28a and over expressed in Escherichia coli BL21 (DE3). In comparison to wild strain ( B. subtilis ), the transformed E. coli exhibited four times increase in cellulase production. Higher enzyme activity was observed in supernatant (8.2 IU/ml) than cell pellet (2.8 IU/ml) suggesting more extracellular production of β-1, 4-endoglucanase. SDS-PAGE and CMC plate assay also confirmed the overproduction by the transformed E. coli . The pH and temperature optima of expressed β-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50–60 °C, respectively.

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.

Root-knot nematode, Meloidogyne incognita is a key global pest of agricultural and horticultural crops. The soil dwelling second-stage juveniles of the nematode species enter the plant roots and develop specialized feeding cells by modifying host plant physiology. Being obligate in nature the nematode cannot be multiplied in artificial media, and the traditional culturing is usually on soil-based tritrophic microcosm. The alternative use of CYG germination pouches provides a clean and contamination free culturing of the nematode species, where adzuki bean is used as a susceptible host. The present study introduces rice bean as an alternative to adzuki bean for maintenance of M. incognita in CYG germination pouches. The rice bean plants were found to harbour comparatively higher number of females leading to higher eggmass production than the adzuki plants. The reproductive fitness of M. incognita was not compromised in rice bean, and the maintenance of plants was easier due to determinate plant type with sturdy stems. This provides additional advantage of using rice bean in the germination pouches. Lastly, the rice bean plants offer clean and contamination free nematode culture which is highly suitable for downstream basic research to understand compatible/incompatible plant nematode interactions.

Photorhabdus is an insect-pathogenic Gram negative enterobacterium found in the gut of Heterorhabditis nematodes. Photorhabdus is highly virulent to insects, and can kill insects rapidly upon injection at very low concentrations of one to few cells. We characterized the virulence of Photorhabdus symbionts isolated from the Heterorhabditis nematodes collected from various parts of India by injecting different concentrations of bacterial cells into fourth instar larval stage of insect Galleria mellonella. Photorhabdus luminescens ssp. akhurstii strain IARI-SGMG3 from Meghalaya was identified as the most virulent of all the tested strains on the basis of LT50 and LC50 values. This study forms a basis for further investigations on the genetic basis of virulence in Photorhabdus bacteria.