Nematode Host Research Articles

Analyses of Xenorhabdus griffiniae genomes reveal two distinct sub-species that display intra-species variation due to prophages

BackgroundNematodes of the genus Steinernema and their Xenorhabdus bacterial symbionts are lethal entomopathogens that are useful in the biocontrol of insect pests, as sources of diverse natural products, and as research models for mutualism and parasitism. Xenorhabdus play a central role in all aspects of the Steinernema lifecycle, and a deeper understanding of their genomes therefore has the potential to spur advances in each of these applications.ResultsHere, we report a comparative genomics analysis of Xenorhabdus griffiniae, including the symbiont of Steinernema hermaphroditum nematodes, for which genetic and genomic tools are being developed. We sequenced and assembled circularized genomes for three Xenorhabdus strains: HGB2511, ID10 and TH1. We then determined their relationships to other Xenorhabdus and delineated their species via phylogenomic analyses, concluding that HGB2511 and ID10 are Xenorhabdus griffiniae while TH1 is a novel species. These additions to the existing X. griffiniae landscape further allowed for the identification of two subspecies within the clade. Consistent with other Xenorhabdus, the analysed X. griffiniae genomes each encode a wide array of antimicrobials and virulence-related proteins. Comparative genomic analyses, including the creation of a pangenome, revealed that a large amount of the intraspecies variation in X. griffiniae is contained within the mobilome and attributable to prophage loci. In addition, CRISPR arrays, secondary metabolite potential and toxin genes all varied among strains within the X. griffiniae species.ConclusionsOur findings suggest that phage-related genes drive the genomic diversity in closely related Xenorhabdus symbionts, and that these may underlie some of the traits most associated with the lifestyle and survival of entomopathogenic nematodes and their bacteria: virulence and competition. This study establishes a broad knowledge base for further exploration of not only the relationships between X. griffiniae species and their nematode hosts but also the molecular mechanisms that underlie their entomopathogenic lifestyle.

'Re-Wilding' an Animal Model With Microbiota Shifts Immunity and Stress Gene Expression During Infection.

The frequency of emerging disease is growing with ongoing human activity facilitating new host-pathogen interactions. Novel infection outcomes can also be shaped by the host microbiota. Caenorhabditis elegans nematodes experimentally colonised by a wild microbiota community and infected by the widespread animal pathogen, Staphylococcus aureus, have been shown to suffer higher mortality than those infected by the pathogen alone. Understanding the host responses to such microbiota-pathogen ecological interactions is key to pinpointing the mechanism underlying severe infection outcomes. We conducted transcriptomic analyses of C. elegans colonised by its native microbiota, S. aureus and both in combination. Correlations between altered collagen gene expression and heightened mortality in co-colonised hosts suggest the microbiota modified host resistance to infection. Furthermore, microbiota colonised hosts showed increased expression of immunity genes and variable expression of stress response genes during infection. Changes in host immunity and stress response could encompass both causes and effects of severe infection outcomes. 'Re-wilding' this model nematode host with its native microbiota indicated that typically commensal microbes can mediate molecular changes in the host that are costly when challenged by a novel emerging pathogen.

Suppression of a transcriptional regulator, HexA, is essential for triggering the bacterial virulence of the entomopathogen, Xenorhabdus hominickii

A nematode-symbiotic bacterium, Xenorhabdus hominickii, exhibits two distinct lifestyles. Upon infection of its host nematode into a target insect, X. hominickii is released into the insect hemocoel and becomes pathogenic. This study examines the critical transformation in bacterial life forms concerning the activity of a transcriptional regulator, HexA. When X. hominickii was cultured in tryptic soy broth, HexA was expressed during the stationary phase of bacterial growth. Conversely, HexA was expressed in the early growth stage within the insect host, Spodoptera exigua, when infected with X. hominickii. The transient expression of HexA was succeeded by the expression of another transcriptional regulator, Lrp, which led to the production of bacterial virulent factors. Expression of HexA was manipulated by replacing its promoter with an inducible promoter controlled by the inducer, l-arabinose. In the absence of the inducer, the mutant bacteria expressed HexA at a low level, resulting in a bacterial culture broth that was more effective at suppressing insect immune responses than the wild type. When the inducer was added, HexA was expressed at high levels, rendering the culture broth ineffective in immunosuppression. Interestingly, expression of HexA inhibited the expression of another transcriptional regulator, Lrp, which in turn induced the expression of a non-ribosomal peptide synthetase, gxpS, leading to the production of an immunosuppressive metabolite, GXP. Suppression of HexA expression in mutant bacteria augmented GXP levels in secondary metabolites. This indicates that infection of X. hominickii into the insect host represses HexA expression and upregulates Lrp expression, leading to GXP production. The GXP metabolites inhibit insect immunity, thus protecting the bacteria-nematode complex. Therefore, the suppression of HexA expression in the insect hemocoel is crucial for the bacteria’s transition from a symbiotic to a pathogenic life form.

The marine environmental microbiome mediates physiological outcomes in host nematodes.

Nematodes are the most abundant metazoans in marine sediments, many of which are bacterivores; however, how habitat bacteria affect physiological outcomes in marine nematodes remains largely unknown. RESULTS: Here, we used a Litoditis marina inbred line to assess how native bacteria modulate host nematode physiology. We characterized seasonal dynamic bacterial compositions in L. marina habitats and examined the impacts of 448 habitat bacteria isolates on L. marina development, then focused on HQbiome with 73 native bacteria, of which we generated 72 whole genomes sequences. Unexpectedly, we found that the effects of marine native bacteria on the development of L. marina and its terrestrial relative Caenorhabditis elegans were significantly positively correlated. Next, we reconstructed bacterial metabolic networks and identified several bacterial metabolic pathways positively correlated with L. marina development (e.g., ubiquinol and heme b biosynthesis), while pyridoxal 5'-phosphate biosynthesis pathway was negatively associated. Through single metabolite supplementation, we verified CoQ10, heme b, acetyl-CoA, and acetaldehyde promoted L. marina development, while vitamin B6 attenuated growth. Notably, we found that only four development correlated metabolic pathways were shared between L. marina and C. elegans. Furthermore, we identified two bacterial metabolic pathways correlated with L. marina lifespan, while a distinct one in C. elegans. Strikingly, we found that glycerol supplementation significantly extended L. marina but not C. elegans longevity. Moreover, we comparatively demonstrated the distinct gut microbiota characteristics and their effects on L. marina and C. elegans physiology. Given that both bacteria and marine nematodes are dominant taxa in sedimentary ecosystems, the resource presented here will provide novel insights to identify mechanisms underpinning how habitat bacteria affect nematode biology in a more natural context. Our integrative approach will provide a microbe-nematodes framework for microbiome mediated effects on host animal fitness.

Discovery of a novel Wolbachia in Heterodera expands nematode host distribution.

Bioinformatics sequence data mining can reveal hidden microbial symbionts that might normally be filtered and removed as contaminants. Data mining can be helpful to detect Wolbachia, a widespread bacterial endosymbiont in insects and filarial nematodes whose distribution in plant-parasitic nematodes (PPNs) remains underexplored. To date, Wolbachia has only been reported a few PPNs, yet nematode-infecting Wolbachia may have been widespread in the evolutionary history of the phylum based on evidence of horizontal gene transfers, suggesting there may be undiscovered Wolbachia infections in PPNs. The goal of this study was to more broadly sample PPN Wolbachia strains in tylenchid nematodes to enable further comparative genomic analyses that may reveal Wolbachia's role and identify targets for biocontrol. Published whole-genome shotgun assemblies and their raw sequence data from 33 Meloidogyne spp. assemblies, seven Globodera spp. assemblies, and seven Heterodera spp. assemblies were analyzed to look for Wolbachia. No Wolbachia was found in Meloidogyne spp. and Globodera spp., but among seven genome assemblies for Heterodera spp., an H. schachtii assembly from the Netherlands was found to have a large Wolbachia-like sequence that, when re-assembled from reads, formed a complete, circular genome. Detailed analyses comparing read coverage, GC content, pseudogenes, and phylogenomic patterns clearly demonstrated that the H. schachtii Wolbachia represented a novel strain (hereafter, denoted wHet). Phylogenomic tree construction with PhyloBayes showed wHet was most closely related to another PPN Wolbachia, wTex, while 16S rRNA gene analysis showed it clustered with other Heterodera Wolbachia assembled from sequence databases. Pseudogenes in wHet suggested relatedness to the PPN clade, as did the lack of significantly enriched GO terms compared to PPN Wolbachia strains. It remains unclear whether the lack of Wolbachia in other published H. schachtii isolates represents the true absence of the endosymbiont from some hosts.

Wolbachia of phylogenetic supergroup K identified in oribatid mite Nothrus anauniensis (Acari: Oribatida: Nothridae).

Heritable endosymbionts widely occur in arthropod and nematode hosts. Among these endosymbionts, Wolbachia has been extensively detected in many arthropods, such as insects and crustaceans. Maternal inheritance is the most basic and dominant mode of transmission of Wolbachia, and it might regulate the reproductive system of the host in four ways: feminization, parthenogenesis, male killing, and cytoplasmic incompatibility. There is a relatively high percentage (10%) of thelytokous species in Oribatida, a suborder under the subclass Acari of arthropods, but the study of the endosymbionts in oribatid mites is almost negligible. In this paper, we detected endosymbiotic bacteria in two parthenogenetic oribatid species, Nothrus anauniensis Canestrini and Fanzago, 1877, which has never been tested for endosymbionts, and Oppiella nova, in which Wolbachia and Cardinium have been reported before. The results showed that Wolbachia was first found in N. anauniensis with an infection rate of 100% across three populations. Phylogenetic analysis showed that Wolbachia in N. anauniensis belonged to the supergroup K, marking the second supergroup of Wolbachia found in oribatid mites. Unlike previous studies, our study did not detect Wolbachia in O. nova, leading to the exclusion of Wolbachia's role in mediating thelytoky in this species.

Soil properties predict below-ground community structure, but not nematode microbiome patterns in semi-arid habitats.

Microbial and microeukaryotic communities are extremely abundant and diverse in soil habitats where they play critical roles in ecosystem functioning and services that are essential to soil health. Soil biodiversity is influenced by above-ground (vegetation) and below-ground factors (soil properties), which together create habitat-specific conditions. However, the compound effects of vegetation and soil properties on soil communities are less studied or often focused on one component of the soil biota. Here, we integrate metabarcoding (16S and 18S rRNA genes) and nematode morphology to assess the effects of habitat and soil properties shaping microbial and microeukaryotic communities as well as nematode-associated microbiomes. We show that both vegetation and soil properties (soil bulk density) were major factors structuring microbial and microeukaryotic communities in semi-arid soil habitats. Despite having lower nutrients and lower pH, denser soils displayed significantly higher alpha diversity than less dense soils across datasets. Nematode-associated microbiomes have lower microbial diversity, strongly differ from soil microbes and are more likely to respond to microscale variations among samples than to vegetation or soil bulk density. Consequently, different nematode lineages and trophic groups are likely to display similar associated microbiomes when sharing the same microhabitat. Different microbiome taxa were enriched within specific nematode lineages (e.g. Mycobacterium, Candidatus Cardinium) highlighting potentially new species-specific associations that may confer benefits to their soil nematode hosts. Our findings highlight the importance of exploring above- and below-ground effects to assess community structure in terrestrial habitats, and how fine-scale analyses are critical for understanding patterns of host-associated microbiomes.

Functional Characterization of Ao4g24: An Uncharacterized Gene Involved in Conidiation, Trap Formation, Stress Response, and Secondary Metabolism in Arthrobotrys oligospora.

Arthrobotrys oligospora is a typical nematode-trapping (NT) fungus, which can secrete food cues to lure, capture, and digest nematodes by triggering the production of adhesive networks (traps). Based on genomic and proteomic analyses, multiple pathogenic genes and proteins involved in trap formation have been characterized; however, there are numerous uncharacterized genes that play important roles in trap formation. The functional studies of these unknown genes are helpful in systematically elucidating the complex interactions between A. oligospora and nematode hosts. In this study, we screened the gene AOL_s00004g24 (Ao4g24). This gene is similar to the SWI/SNF chromatin remodeling complex, which was found to play a potential role in trap formation in our previous transcriptome analysis. Here, we characterized the function of Ao4g24 by gene disruption, phenotypic analysis, and metabolomics. The deletion of Ao4g24 led to a remarkable decrease in conidia yield, trap formation, and secondary metabolites. Meanwhile, the absence of Ao4g24 influenced the mitochondrial membrane potential, ATP content, autophagy, ROS level, and stress response. These results indicate that Ao4g24 has crucial functions in sporulation, trap formation, and pathogenicity in NT fungi. Our study provides a reference for understanding the role of unidentified genes in mycelium growth and trap formation in NT fungi.

Unraveling the interactions of Esteya vermicola, pinewood nematode, and pine hosts: Insights into population dynamics and molecular responses

Esteya vermicola has shown promise as an efficient biological control agent against pine wilt disease, a devastating disease in pine forests caused by the pinewood nematode (PWN, Bursaphelenchus xylophilus). However, the in vivo interactions among E. vermicola, PWN, and pine hosts are less understood, both at the population and molecular levels. In this study, we performed a series of bioassays to investigate E. vermicola colonization patterns in pine xylem and its population responses to PWN invasion in healthy and PWN-induced wilting trees. Our results demonstrated that although E. vermicola exhibits slow growth, its conidia germinate and grew along the pine tracheid, even producing lunate conidia capable of initiating PWN infections within the xylem. Interestingly, while fungal hyphae became undetectable in pine sapling xylem after inoculation, the E. vermicola population increased immediately in response to PWN invasion. Furthermore, we observed a “leap-frog” dispersal pattern of fungal colonization in PWN-induced wilting pines, facilitated by the migration of fungal-infected nematodes. Moreover, we explored the molecular mechanisms underlying fungal tolerance to pine defense systems using transcriptomic analysis. Comparative transcriptomics revealed that carbohydrate metabolism and abiotic stress-induced oxidoreductive activities are involved in the fungal tolerance to the pine defense compound β-pinene. This study enhances our understanding of how E. vermicola colonizes and persists within pine xylem, its molecular responses to plant defense compounds, and its population dynamics upon PWN invasion, validating its efficacy as a biocontrol agent against pine wilt disease.

The Xenorhabdus nematophila LrhA transcriptional regulator modulates production of γ-keto-N-acyl amides with inhibitory activity against mutualistic host nematode egg hatching.

Xenorhabdus bacteria are of interest due to their symbiotic relationship with Steinernema nematodes and their ability to produce a variety of natural bioactive compounds. Despite their importance, the regulatory hierarchy connecting specific natural products and their regulators is poorly understood. In this study, comparative metabolomic profiling was utilized to identify the secondary metabolites modulated by the X. nematophila global regulator LrhA. This analysis led to the discovery of three metabolites, including an N-acyl amide that inhibited the egg hatching rate and length of Steinernema carpocapsae nematodes. These findings support the notion that X. nematophila LrhA influences the symbiosis between X. nematophila and S. carpocapsae through N-acyl amide signaling. A deeper understanding of the regulatory hierarchy of these natural products could contribute to a better comprehension of the symbiotic relationship between X. nematophila and S. carpocapsae.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Unveiling the Diversity: Plant Parasitic Nematode Effectors and Their Plant Interaction Partners.

Root-knot and cyst nematodes are two groups of plant parasitic nematodes that cause the majority of crop losses in agriculture. As a result, these nematodes are the focus of most nematode effector research. Root-knot and cyst nematode effectors are defined as secreted molecules, typically proteins, with crucial roles in nematode parasitism. There are likely hundreds of secreted effector molecules exuded through the nematode stylet into the plant. The current research has shown that nematode effectors can target a variety of host proteins and have impacts that include the suppression of plant immune responses and the manipulation of host hormone signaling. The discovery of effectors that localize to the nucleus indicates that the nematodes can directly modulate host gene expression for cellular reprogramming during feeding site formation. In addition, plant peptide mimicry by some nematode effectors highlights the sophisticated strategies the nematodes employ to manipulate host processes. Here we describe research on the interactions between nematode effectors and host proteins that will provide insights into the molecular mechanisms underpinning plant-nematode interactions. By identifying the host proteins and pathways that are targeted by root-knot and cyst nematode effectors, scientists can gain a better understanding of how nematodes establish feeding sites and subvert plant immune responses. Such information will be invaluable for future engineering of nematode-resistant crops, ultimately fostering advancements in agricultural practices and crop protection. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Host status and susceptibility of Cannabis sativa cultivars to root-knot nematodes.

Root-knot nematode host status of hemp cultivars of different uses (fiber, dual, CBD/CBG) and from different regions (Europe, China, US) were evaluated in five different greenhouse trials. None of the tested cultivars showed resistance to any of the tested root-knot nematode species, and all tested hemp cultivars were good hosts for root-knot nematodes, especially to mixed populations of M. javanica and M. incognita. Root gall symptoms on hemp were less severe than on cucumber (and tomato), but reproduction rates were similar. Lower infection and reproduction rates were noted for M. hapla and M. enterolobii, which were probably due to the colder temperatures at the time of the trial, as the same effect was noted for the cucumber control plants. While no negative impact on hemp shoot growth was seen in trials where nematodes were added to pasteurized soil, a significant and visible negative effect on hemp growth was noted when two CBG hemp cultivars were planted in heavily naturally root-knot infested soil. This result indicates that hemp is not only a good host to root-knot nematodes, but also that root-knot can be a limiting factor for hemp production in Florida and other places with high abundance and pressure of root-knot nematodes.

Genetic variation in parasite avoidance, yet no evidence for constitutive fitness costs.

Behavioral avoidance of parasites is a widespread strategy among animal hosts and in human public health. Avoidance has repercussions for both individual and population-level infection risk. Although most cases of parasite avoidance are viewed as adaptive, there is little evidence that the basic assumptions of evolution by natural selection are met. This study addresses this gap by testing whether there is a heritable variation in parasite avoidance behavior. We quantified behavioral avoidance of the bacterial parasite Serratia marcescens for 12 strains of the nematode host Caenorhabditis elegans. We found that these strains varied in their magnitude of avoidance, and we estimated the broad-sense heritability of this behavior to be in the range of 11%-26%. We then asked whether avoidance carries a constitutive fitness cost. We did not find evidence of one. Rather, strains with higher avoidance had higher fitness, measured as population growth rate. Together, these results direct future theoretical and empirical work to identify the forces maintaining genetic variation in parasite avoidance.

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

Isolation of Pasteuria penetrans, an obligate hyper-parasite, infecting root knot nematode, Meloidogyne spp. from the rhizosphere of pulses in India

BackgroundPasteuria penetrans is a mycelial, endospore forming, obligate, bacterial parasite that has shown enormous potential for biological control of root-knot nematode, Meloidogyne spp. In the present study, the effort has been made to isolate effective and high temperature tolerant indigenous strains from pulse rhizosphere in India.ResultsSurveys were carried out in four different agro-climatic zones and collected 106 root-knot nematode infected soil and plant samples from pulses rhizosphere. Of these, we observed Pasteuria spore attachment on root knot nematode juveniles from seven samples: three from Varanasi, two from Salem and one each from Deoria and Kushinagar. The pure cultures of these seven isolates (named as IIPR-Pp-1 to IIPR-Pp-7 from single infected female, respectively) were developed in 7 cm pot and soil-less CYG medium. The nematode host species was identified as Meloidogyne incognita by perineal pattern and confirmed by sequencing of ITS region. The host range study with M. incognita and Heterodera cajani showed the spore attachment was specific to M. incognita revealed that the species of the parasite was Pasteuria penetrans. SEM study on morphometrics of Pasteuria spores belong to two groups. In vitro assay on endospore attachment assay with pre-treated endospores at 20, 30, 40, 50, 60 and 70 °C revealed that maximum number of endospore attachment of all isolates was observed at 30 °C followed by 40 °C, and the least number of them was observed at 60 °C and above. The isolate Pp3 exhibited maximum number of spore attachments among all isolates at 30 °C treatment (14) and 40 °C treatment (10.67). Endospore attachment assay with pre-treated J2 at 20, 25, 30, 35, 40 and 45 °C revealed that maximum number of attachment of all isolates was observed at 30 °C treatment, and no attachment was observed at 40 °C and above.ConclusionPasteuria penetrans isolate IIPR-Pp3 exhibited high temperature tolerance and can be used further to develop Pasteuria-based biopesticide for management of root-knot nematode, M. incognita in pulses.

Green and red fluorescent strains of Xenorhabdus griffiniae HGB2511, the bacterial symbiont of the nematode Steinernema hermaphroditum (India).

Steinernema entomopathogenic nematodes form specific, obligate symbiotic associations with gram-negative, gammaproteobacteria members of the Xenorhabdus genus. Together, the nematodes and symbiotic bacteria infect and kill insects, utilize the nutrient-rich cadaver for reproduction, and then reassociate, the bacteria colonizing the nematodes' anterior intestines before the nematodes leave the cadaver to search for new prey. In addition to their use in biocontrol of insect pests, these nematode-bacteria pairs are highly tractable experimental laboratory models for animal-microbe symbiosis and parasitism research. One advantageous feature of entomopathogenic nematode model systems is that the nematodes are optically transparent, which facilitates direct observation of nematode-associated bacteria throughout the lifecycle. In this work, green- and red-fluorescently labeled X. griffiniae HGB2511 bacteria were created and associated with their S . hermaphroditum symbiotic nematode partners and observed using fluorescence microscopy. As expected, the fluorescent bacteria were visible as a colonizing cluster in the lumen of the anterior intestinal caecum of the infective stage of the nematode. These tools allow detailed observations of X. griffiniae localization and interactions with its nematode and insect host tissues throughout their lifecycles.

Manipulation of GameXPeptide synthetase gene expression by a promoter exchange alters the virulence of an entomopathogenic bacterium, Photorhabdus temperata temperata, by modulating insect immune responses.

An entomopathogenic bacterium, Photorhabdus temperata subsp. temperata, is mutualistic to its host nematode, Heterorhabditis megidis. The infective juvenile nematodes enter target insects through natural openings and release the symbiotic bacteria into the insect hemocoel. The released bacteria suppress the insect immune responses and cause septicemia through their secondary metabolites. GameXPeptide (GXP) is one of the common secondary metabolites of most Photorhabdus species and is produced by the catalytic activity of a specific non-ribosomal peptide synthetase called GxpS encoded by the gxpS gene. This study confirmed gxpS to be encoded in the P. temperata temperata genome and analyzed its expression during bacterial growth. LC-MS/MS analysis of the bacterial culture broth contained at least four different GXPs (GXP-A to GXP-D), in which GXP-A was the most abundant. To investigate GXP synthesis following gxpS expression, the gxpS promoter of P. temperata temperata was replaced with an inducible arabinose promoter by homologous recombination. The gxpS transcript levels in the mutant were altered by the addition of l-arabinose. Without the inducer, the gxpS transcript level was significantly lower compared to the wild type and produced significantly lower amounts of the four GXPs. The addition of the inducer to the mutant significantly increased gxpS expression and produced significantly higher levels of the four GXPs compared to the wild type. The metabolite extracts obtained from wild-type and mutant bacteria showed differential immunosuppressive activities according to their GXP contents against the cellular and humoral immune responses of a lepidopteran insect, Spodoptera exigua. Interestingly, the gxpS-mutant bacteria showed less insecticidal activity compared to the wild type, whereas the addition of GXP to the mutant significantly restored insecticidal activity. These results suggest that the gxpS gene encoded in P. temperata temperata is responsible for the production of at least four different GXPs, which play crucial roles in bacterial virulence.

Natural genetic variation and negative density effects in plant-nematode interactions.

Arabidopsis thaliana is a suitable host for phytoparasitic nematodes of the genus Meloidogyne. Successful nematode infection leads to the formation of root galls. We tested for natural genetic variation and inoculation density effects on nematode reproductive success in the interaction between A. thaliana and Meloidogyne javanica. We inoculated different Arabidopsis genotypes with two sources of nematodes at two different doses, using a mild protocol for inoculum preparation. We counted root galls and egg masses 2 months after inoculation. We obtained a high number of successful nematode infections. Infection success differed among Arabidopsis genotypes in interaction with the nematode source. Overall, infection success and reproductive success of nematodes were lower at a higher inoculum dose of nematodes. Our results indicate that natural genetic variation in both host plants and nematodes, as well as short- and long-term negative density effects, shape nematode reproductive success.