A Draft Transcriptome Announcement of Anguina tritici.

Anguina tritici, the wheat seed gall nematode, causes the 'ear-cockle' or seed gall disease of wheat (Triticum sp.), leading to an extensive decline of yield (30-70%) in underdeveloped wheat cultivating countries of the world. The nematode is known to survive in anhydrobiotic conditions for up to 32 years. Here, we present the first transcriptome assembly of A. tritici, which will be a valuable resource for understanding the genes responsible for nematode survival and above-ground plant parasitism. The final 133.2 Mb assembly consists of 105606 open reading frames (including isoforms) with the following BUSCO scores against Nematoda database: 80.3% complete (16.4% single copy and 63.9% duplicated), 2.1% fragmented, and 17.6% missing.

Stringent in-silico identification of putative G-protein-coupled receptors (GPCRs) of the entomopathogenic nematode Heterorhabditis bacteriophora.

The infective juveniles (IJs) of entomopathogenic nematode (EPN) Heterorhabditis bacteriophora find and infect their host insects in heterogeneous soil ecosystems by sensing a universal host cue (CO2) or insect/plant-derived odorants, which bind to various sensory receptors, including G protein-coupled receptors (GPCRs). Nematode chemosensory GPCRs (NemChRs) bind to a diverse set of ligands, including odor molecules. However, there is a lack of information on the NemChRs in EPNs. Here we identified 21 GPCRs in the H. bacteriophora genome sequence in a triphasic manner, combining various transmembrane detectors and GPCR predictors based on different algorithms, and considering inherent properties of GPCRs. The pipeline was validated by reciprocal BLAST, InterProscan, GPCR-CA, and NCBI CDD search. Functional classification of predicted GPCRs using Pfam revealed the presence of four NemChRs. Additionally, GPCRs were classified into various families based on the reciprocal BLAST approach into a frizzled type, a secretin type, and 19 rhodopsin types of GPCRs. Gi/o is the most abundant kind of G-protein, having a coupling specificity to all the fetched GPCRs. As the 21 GPCRs identified are expected to play a crucial role in the host-seeking behavior, these might be targeted to develop novel insect-pest management strategies by tweaking EPN IJ behavior, or to design novel anthelminthic drugs. Our new and stringent GPCR detection pipeline may also be used to identify GPCRs from the genome sequence of other organisms.

Exploring Genomic Variations in Nematode-Resistant Mutant Rice Lines.

Rice (Oryza sativa) production is seriously affected by the root-knot nematode Meloidogyne graminicola, which has emerged as a menace in upland and irrigated rice cultivation systems. Previously, activation tagging in rice was utilized to identify candidate gene(s) conferring resistance against M. graminicola. T-DNA insertional mutants were developed in a rice landrace (acc. JBT 36/14), and four mutant lines showed nematode resistance. Whole-genome sequencing of JBT 36/14 was done along with the four nematode resistance mutant lines to identify the structural genetic variations that might be contributing to M. graminicola resistance. Sequencing on Illumina NovaSeq 6000 platform identified 482,234 genetic variations in JBT 36/14 including 448,989 SNPs and 33,245 InDels compared to reference indica genome. In addition, 293,238-553,648 unique SNPs and 32,395-65,572 unique InDels were found in the four mutant lines compared to their JBT 36/14 background, of which 93,224 SNPs and 8,170 InDels were common between all the mutant lines. Functional annotation of genes containing these structural variations showed that the majority of them were involved in metabolism and growth. Trait analysis revealed that most of these genes were involved in morphological traits, physiological traits and stress resistance. Additionally, several families of transcription factors, such as FAR1, bHLH, and NAC, and putative susceptibility (S) genes, showed the presence of SNPs and InDels. Our results indicate that subject to further genetic validations, these structural genetic variations may be involved in conferring nematode resistance to the rice mutant lines.

An improved draft genome assembly of Meloidogyne graminicola IARI strain using long-read sequencing.

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.

Rapid and sensitive detection of potato cyst nematode Globodera rostochiensis by loop-mediated isothermal amplification assay.

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.

A rice root-knot nematode Meloidogyne graminicola-resistant mutant rice line shows early expression of plant-defence genes.

MAIN CONCLUSION: 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.

Identification of Galtox, a new protein toxin from Photorhabdus bacterial symbionts of Heterorhabditis nematodes.

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.

Transcriptomic changes in the pre-parasitic juveniles of Meloidogyne incognita induced by silencing of effectors Mi-msp-1 and Mi-msp-20.

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.

Further observations on Meloidogyne enterolobii (Nematoda: Meloidogynidae) infecting guava (Psidium guajava) in India.

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.

Meloidogyne incognita (Nematoda: Meloidogynidae) sterol-binding protein Mi-SBP-1 as a target for its management.

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.

Draft Genome Sequences for Five Photorhabdus Bacterial Symbionts of Entomopathogenic Heterorhabditis Nematodes Isolated from India.

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.

Nematode Genome Announcement: A Draft Genome for Rice Root-Knot Nematode, Meloidogyne graminicola.

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.

A comprehensive annotation for the root-knot nematode Meloidogyne incognita proteome data.

Root-knot nematodes are devastating pathogens of crop plants. The draft genome of southern root-knot nematode Meloidogyne incognita was published in 2008 and additional genome and transcriptome data became available later on. However, lack of a publically available annotation for M. incognita genome and transcriptome(s) limits the use of this data for functional and comparative genomics by the interested researchers. Here we present a comprehensive annotation for the M. incognita proteome data available at INRA Meloidogyne Genomic Resources page (https://meloidogyne.inra.fr/Downloads/Meloidogyne-incognita-V2-2017) and European Nucleotide Archive (ENA) (accession number: ERP009887) using a multi-pronged approach.

Silencing of a Meloidogyne incognita selenium-binding protein alters the cuticular adhesion of Pasteuria penetrans endospores.

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.

Morphological, Morphometric, and Molecular Characterization of Intraspecific Variations within Indian Populations of Meloidogyne graminicola.

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.

Discovery of a Highly Virulent Strain of Photorhabdus luminescens ssp. akhurstii from Meghalaya, India.

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.

Cloning and expression of ß-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill.

A strain of Bacillus subtilis IARI-SP-1 isolated from soil long term irrigated with effluents of paper and pulp mill showed high ß-1, 4-endoglucanase (2.5 IU/ml) but low activity of ß-1, 4-exoglucanase (0.8 IU/ml) and ß-glucosidase (0.084 IU/ml). The ß-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 ß-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 ß-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 ß-1, 4-endoglucanase enzyme was identical to that of wild strain and was 8 and 50-60 °C, respectively.