Entomopathogenic Nematodes In Soil Research Articles

Spreyleme ve Toprak Enjeksiyonu Yöntemleri ile Toprağa Uygulanan Entomopatojen Nematodların Şeftali Bahçesinde Kalıcılığının Belirlenmesi

Entomopathogenic nematodes (EPN) are effective biological control agents against underground and cryptic pests. Persistency and survival of EPN in soil after soil application is important for long term success of management programs. In this study, it was aimed to determine the soil persistency of 4 native EPN species after surface spraying and soil injection applications in a peach orchard. In the study, S. feltiae (96), S. carpocapsae 1133, H. bacteriophora 1144 and S. affine 47 species were applied in 30 l water with 140.000 IJ/tree dose per tree by surface spraying and soil injection methods. EPN were applied to soil with a watering can in surface spraying method and with a pulverizator into 5-15 cm depth in soil injection method. After the monthly application, soil samples were collected and EPN presence was tested with G. mellonella larvae and White traps in the laboratory. The study was conducted for 2 times in 2018 and 2019. At the end of the study, EPN persistency in soil was found to be 90 days in 2018 and 150 days in 2019.

A novel tumor-targeting strain of Xenorhabdus stockiae exhibits potent biological activities.

Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema presenting two distinct forms in their life cycle, and can produce a broad range of bioactive compounds. In this study, a novel Xenorhabdus stockiae strain HN_xs01 was isolated from a soil sample via an entrapment method using Galleria melonella nematodes. The supernatants of strain HN_xs01 exhibited antimicrobial properties against Gram-negative and Gram-positive bacteria, and insecticidal properties against Helicoverpa armigera larvae, and antitumor properties as well. Moreover, three linear rhabdopeptides (1, 2 and 3) were identified from strain HN_xs01 using nuclear magnetic resonance analysis, which exhibited significant cytotoxic activity against the human epithelial carcinoma cell line A431 and the human chronic myelogenous leukemia cell line K562. Some bacteria have been reported to colonize the tumor region, and we determined that HN_xs01 could grow in tumor xenografts in this study. HN_xs01 invaded and replicated in B16 melanoma cells grafted into C57BL/6 mice, resulting in tumor inhibition. Moreover, strain HN_xs01 not only merely aggregated in the tumor environment, but also prevented pulmonary metastasis. It caused fragmentation of vessels and cell apoptosis, which contributed to its antitumor effect. In conclusion, X. stockiae HN_xs01 is a novel tumor-targeting strain with potential applications in medicinal and agricultural fields.

Şeftali bahçesinde damla sulama yöntemi ile toprağa uygulanan yerel entomopatojen nematodların kalıcılığının belirlenmesi

The period of persistence of entomopathogenic nematodes (EPNs) in soil is an important factor in the success of biological control efforts. In this study, the persistency of four native Turkish entomopathogenic nematode species, Steinernema feltiae (Filipjev), S. carpocapsae (Weiser), S. affine (Bovien) and Heterorhabditis bacteriophora (Poinar), was investigated in a peach orchard after their application with a drip irrigation system in Çanakkale Province, Turkiye, in 2018 and 2019. The EPNs were applied at the rate of 50 IJs/cm2. After application, the persistency of the EPNs was determined with soil sampling on a monthly basis. EPN persistence in the soil was 90 days and 150 days in 2018 and 2019, respectively. Additionally, the persistency of the EPNs in soil after their application varied, depending on environmental factors. The longer survival of EPNs in soil after application is important for the increase of the efficiency of EPNs against pests. It was concluded that the local EPN species have potential for use in the biological control of agricultural pests.

Invasion of Popillia japonica in Lombardy, Italy: Interactions with soil entomopathogenic nematodes and native grubs

Abstract The recent invasion of the Japanese beetle Popillia japonica Newman (Coleoptera: Scarabaeidae) in northern Italy offered the opportunity to explore the entomopathogenic nematodes (EPNs) associated with the soil of hay meadows. A total of 61 sites were considered for nematode sampling, and from 17 of them (27.9%) EPNs were isolated and further characterized with molecular and morphological techniques as well as with laboratory bioassays. Two main species, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) were recorded with the same frequency together with a few other species (Rhabditida: Oscheius sp. and Phasmarhabditis sp.). The newly isolated EPN populations were characterized for their activity (penetration rate) and infectivity to P. japonica grubs. EPNs occurrence was related to the time of beetle invasion at each sampling site and there was evidence of a density‐dependent response of the EPNs community to P. japonica density. The invasion of P. japonica apparently did not significantly affect the occurrence of native grubs, although a tendency to a decline was observed and should be further investigated.

Comparative Genomics between Two Xenorhabdus bovienii Strains Highlights Differential Evolutionary Scenarios within an Entomopathogenic Bacterial Species.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Within Xenorhabdus bovienii species, the X. bovienii CS03 strain (Xb CS03) is nonvirulent when directly injected into lepidopteran insects, and displays a low virulence when associated with its Steinernema symbiont. The genome of Xb CS03 was sequenced and compared with the genome of a virulent strain, X. bovienii SS-2004 (Xb SS-2004). The genome size and content widely differed between the two strains. Indeed, Xb CS03 had a large genome containing several specific loci involved in the inhibition of competitors, including a few NRPS-PKS loci (nonribosomal peptide synthetases and polyketide synthases) producing antimicrobial molecules. Consistently, Xb CS03 had a greater antimicrobial activity than Xb SS-2004. The Xb CS03 strain contained more pseudogenes than Xb SS-2004. Decay of genes involved in the host invasion and exploitation (toxins, invasins, or extracellular enzymes) was particularly important in Xb CS03. This may provide an explanation for the nonvirulence of the strain when injected into an insect host. We suggest that Xb CS03 and Xb SS-2004 followed divergent evolutionary scenarios to cope with their peculiar life cycle. The fitness strategy of Xb CS03 would involve competitor inhibition, whereas Xb SS-2004 would quickly and efficiently kill the insect host. Hence, Xenorhabdus strains would have widely divergent host exploitation strategies, which impact their genome structure.

Attenuated virulence and genomic reductive evolution in the entomopathogenic bacterial symbiont species, Xenorhabdus poinarii.

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

Wide interguild relationships among entomopathogenic and free-living nematodes in soil as measured by real time qPCR

Entomopathogenic nematodes (EPNs) are promising biological control agents of soil-dwelling insect pests of many crops. These nematodes are ubiquitous in both natural and agricultural areas. Their efficacy against arthropods is affected directly and indirectly by food webs and edaphic conditions. It has long been suggested that a greater understanding of EPN ecology is needed to achieve consistent biological control by these nematodes and the development of molecular tools is helping to overcome obstacles to the study of cryptic organisms and complex interactions. Here we extend the repertoire of molecular tools to characterize soil food webs by describing primers/probe set to quantify certain free-living, bactivorous nematodes (FLBNs) that interact with EPNs in soil. Three FLBN isolates were recovered from soil baited with insect larvae. Morphological and molecular characterization confirmed their identities as Acrobeloides maximum (RT-1-R15C and RT-2-R25A) and Rhabditis rainai (PT-R14B). Laboratory experiments demonstrated the ability of these FLBNs to interfere with the development of Steinernema diaprepesi, Steinernema riobrave and Heterorhabditis indica parasitizing the weevil Diaprepes abbreviatus (P<0.001), perhaps due to resource competition. A molecular probe was developed for the strongest competitor, A. maximum. We selected the highly conserved SSU rDNA sequence to design the primers/probe, because these sequences are more abundantly available for free-living nematodes than ITS sequences that can likely provide better taxonomic resolution. Our molecular probe can identify organisms that share ⩾98% similarity at this locus. The use of this molecular probe to characterize soil communities from samples of nematode DNA collected within a citrus orchard revealed positive correlations (P<0.01) between Acrobeloides-group nematodes and total numbers of EPNs (S. diaprepesi, H. indica and Heterorhabditis zealandica) as well as a complex of nematophagous fungi comprising Catenaria sp. and Monachrosporium gephyropagum that are natural enemies of EPNs. These relationships can be broadly interpreted as supporting Linford’s hypothesis, i.e., decomposition of organic matter (here, insect cadavers) greatly increases bactivorous nematodes and their natural enemies.

Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae

Abstract Xenorhabdus nematophila, a Gram‐negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells of X. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis (PAGE). Seven polypeptides ranging 50∼250 kDa were well separated from the protein complex (named Xnpt) by sodium dodecyl sulfate (SDS)‐PAGE, five of which are identified as XptA2, xptC1, XptB1, GroEL and hypothetical protein by matrix‐assisted laser desorption‐time‐of‐flight mass spectrometry (MALDI‐TOFMS). Xnpt showed high oral virulence to larvae of diamondback moth (DBM), Plutella xylostella L. (Lepidoptera, Plutellidae) as its median lethal concentration (LC50) against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnpt‐fed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.

From insects to human hosts: Identification of major genomic differences between entomopathogenic strains of Photorhabdus and the emerging human pathogen Photorhabdus asymbiotica

Pathogenic bacteria of the genus Photorhabdus are naturally found in symbiotic association with soil entomopathogenic nematodes, and are of increasing economic interest in view of their potential for the development of novel biopesticides. This bipartite natural system is currently used for the biological control of crop pests in several countries. However, an increasing number of Photorhabdus strains have recently been isolated from human clinical specimens in both the United States and Australia, associated with locally invasive soft tissue infections and disseminated bacteraemia. In view of their growing use in biological control, which increases the potential rate of exposure of humans to these pathogens, we decided to undertake a comparative study of the genomic differences between insect and human pathogenic strains of Photorhabdus, in an attempt to understand the genetic mechanisms involved in the apparent change of host specificity, presumably responsible for their recently acquired capacity to infect humans. The data presented here demonstrates that major genomic differences exist between strains of Photorhabdus exhibiting virulence against insects or humans. Several individual genes, coding for virulence factors, were isolated and shown to be specific to the Photorhabdus asymbiotica human pathogens. One of these genes, sopB, encoding a host cell invasion factor translocated via the type III secretion system, has been cloned and the comparison of its genomic context in different pathogens strongly indicates that horizontal gene transfer is implicated in the acquisition of these virulence factors specific to the human pathogens. The precise role of this and other virulence factors identified here in the pathogenicity of P. asymbiotica towards humans is currently under investigation.

Estimating the incidence of entomopathogenic nematodes in soil by the use of bait insects

AbstractSoil samples were stored and baited in different ways in order to develop a bait method that could estimate the level of steinernematid nematodes in soil in a time-efficient way. The method was based on incidence (proportion of samples with a given species of Steinernema). Tenebrio molitor (Coleoptera) larvae were used as bait and it was concluded that: i) repeated baiting in a later period improved the bait result; ii) extension of the bait periods from 1–2 weeks was not necessary; iii) the precision of the estimated incidence did not improve by taking more than 60–80 samples; iv) the minimum time required to analyse one soil sample was 6 min.

Application of the most probable number method to estimations of entomopathogenic nematode populations in soil

SummaryThe calculation of most probable numbers (mpn) was used for the estimation of numbers of infective entomopathogenic nematodes in soil. The mpn concept was first introduced in bacteriology as a means of estimating numbers of organisms in a substrate without a direct count, in cases where direct enumeration could not be applied. In the work reported here, soil samples infested with infective juveniles (IJs) of Heterorhabditis megidis (isolate HF85) were diluted with uninfested soil and the diluted soils were baited with mealworm larvae (Tenebrio molitor). The mpn of infective units of IJs in the undiluted soil was calculated. The mpn calculation was found to be applicable to entomopathogenic nematodes in soil under particular conditions. It could be successfully applied to data for the response of soil units but not to the data based on the response of individual insects, as the latter did not confirm to a Poisson series. The calculated mpn represented between 2.9 and 7.1% of the initial inoculum of IJs. It was suggested that IJs might act as a group in infecting an insect host. Using the data for Tenebrio mortality on parasitisation, the mpn based on quantal response of the mealworms would therefore not give the true density of IJs in the soil sample but the effective density, or the quantity of infective units. Although the biological significance of the infective unit needs further clarification, mpn was found to be a useful parameter for use in comparative experiments.

Heterorhabditis marelatusn. sp. (Rhabditida: Heterorhabditidae) from Oregon

A new species of the nematode genusHeterorhabditiswas found during a survey of soil entomopathogenic nematodes near Seaside, Oregon.Heterorhabditis marelatusn. sp. differs from other species ofHeterorhabditisby the length of infective juvenile and gubernaculum, and the shape of spicules, bursa, and genital papillae.Heterorhabditis marelatusn. sp. has distinct random amplified polymorphic DNA (RAPD) fragments, with a minimal RAPD polymorphism frequency of 0.85 to otherHeterorhabditisspecies examined.

Survey of 15 sites over 28 months for entomopathogenic nematodes (Rhabditida: Steinernematidae)

SUMMARYThe presence of entomopathogenic nematodes in soil from 15 sites on 10 sampling occasions over a period of 28 months was documented. Sites were chosen to represent habitats classified as heathland, hedgerow, roadside verge, pasture or woodland. Bioassays utilized Galleria larvae and most of the nematodes obtained were identified asSteinernema (= Neoaplectana) bibionis (Bovien). A sibling species which fails to interbreed with S. bibionis was also found at 2 of the sites. Bioassays were at 15 and 20°C and there was no significant difference in the results at these temperatures. There was no evidence for seasonality in the presence of the nematodes and at 2 sites they were found on every sampling occasion. Otherwise, sites converted unpredictably from positive to negative or negative to positive over the 28-month sampling period.