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.

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.

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.