Nematode Isolates Research Articles

Blood Parasites of Vangas and Other Corvoidea on Madagascar

Madagascar hosts a great diversity of bird species. This study focuses on the description of the diversity and prevalence of blood parasites (Haemosporida, trypanosomes and filarioid nematodes) in 131 blood samples of 14 species of Corvoidea, namely vangas (Vangidae), Coracina cinerea (Campephagidae), Dicrurus forficatus (Dicruridae) and Terpsiphone mutata (Monarchidae) found in primary rainforests on Madagascar. Blood parasites were detected using both molecular and microscopic methods. Multiplex PCR was used to detect mixed haemosporidian infections and nested PCR was used to describe a 479 bp fragment of the haemosporidian cytochrome b (cytb) gene. Furthermore, a 770 bp SSU rRNA fragment of trypanosomes, and, for microfilariae, a 690 bp fragment of 28S rRNA, as well as a 770 bp fragment of 28S rRNA, were amplified for identification using nested PCRs. Phylogenetic analyses were carried out for all sequences obtained from all blood parasite taxa. Over half of the samples (54.2%; n = 71) were infected with Haemosporida, whereas only 21.4% (n = 28) were infected with Trypanosoma and 5.3% (n = 7) contained filarioid nematode DNA. Fourteen of 56 blood smears contained some of the above-mentioned parasite taxa. The results corroborate the great diversity of blood parasites in the different bird species studied, especially in vangas. Vangas had the greatest diversity of parasites found, as well as the highest number of multiple infections, which may be due to their morphological diversity and resulting habitat use. Fifteen haemosporidian lineages, seven Trypanosoma and five filarioid nematode isolates were newly discovered in the avian species studied, particularly in the vangas. Members of the other Corvoidea families on Madagascar showed a lower susceptibility for avian haemosporidian parasites than vangas, which could be attributed to possible resistance against those parasites. The study confirmed the host specificity of some Haemosporida and microfilariae; however, it demonstrated that this was not the case for Trypanosoma.

Molecular Diagnosis And Genetic Variability of New Isolates of Root – Knot Nematodes Meloidogyne Spp.on Cucumber Plants

Results of the amplified DNA products of the ITS revealed the presence of two bands among the six amplified products,in position at about 500 bp as illustrated by agarose gel electrophoresis,then sequencing of the amplification products showed the presence of two species out of a total of six nematode isolates including Meloidogyne incognita and M. javanica which displayed some nucleotide differences through comparing the DNA sequence alignment of  their ITS and emphasized by a DNA Dot  plot for each species against the other,where a genetic similarity was 98.84%. Phylogenetic tree analysis for M.incognita showed that the closest species to it is M.incognita isolate CN 36 from China with 99.55% genetic similarity whilst more nucleotide differences were observed by two isolates (A-1and B-2) of M.incognita from Iraq with a genetic similarity of 97.99%%. As for M.javanica,the closest species to it is a group of three nematode species including: M.javaniac voucher M J- 21, M.incognita  isolate Nem Mi TG2 and Meloidogyne sp.Mj – c3b from  Brazil, India and New Zealand  respectively, after which nucleotide differences increased more,as it reached its maximum level with M. incognita isolate C N 36 with a  genetic similarity of 98.84%

Insecticidal potential of entomopathogenic nematodes of Northern Kazakhstan

The goal of this research experiment is to create a biological preparation based on predatory nematodes to control potato pests. 15 isolates of entomopathogenic nematodes were isolated: 13 were isolated from coniferous and mixed forests; 2 from soils of agricultural fields: 1 from a potato field, 1 from a field sown with perennial grasses. Nematodes were identified as Steinernematids based on the color of the corpses of larvae of Galleria mellonella and the morphology of males. Isolates of entomopathogenic nematodes from various habitats (forests, agricultural fields, grasslands) of Akmola and Pavlodar regions were isolated. Screening of isolates on the larvae of the seed nutcracker (Agriotes obscurus) was carried out. It is established that the wireworm is sensitive to all isolates. Of the 15 isolates tested, 12 of these isolates showed high mortality ability in relation to wireworm: about 50–70 % mortality in laboratory biotests; 3 isolates (AF 15, AF 22, and AS 36) could not cause more than 40 % of the average mortality of larvae. The screening results showed that the isolates AF 29, AF57, and KP76 are good isolates for further studies as a possible bioinsecticide agent against A. obscurus.

Molecular Phylogenetic Characterization of Anguina Tritici (Steinbuch, 1799) Filipjev, 1936 (Rhabditida: Anguinidae) on Barley from Iraq.

During a nematological survey in Iraq, in the Bashika area, Ninevah province, an anguinid nematode population was isolated from galls of infected barley plants. The morphological characteristics indicated that the recovered species is identical to Anguina tritici. The barley population of A. tritici was molecularly characterized by sequencing two ribosomal regions (ITS and 18S rRNA genes), and their phylogenetic analyses revealed the newly generated sequences are in sister relation to corresponding sequences of A. tritici from wheat in the Bayesian tree, providing further evidence that the host plant can contribute to the separation of new isolates of plant parasitic nematodes.

Evaluation of entomopathogenic nematode (EPN) isolate, Heterorhabditis indica of Vidarbha region, against the tobacco cutworm, Spodoptera litura

The present study on pathogenicity of entomopathogenic nematodes against Spodoptera litura in laboratory conditions was undertaken during 2020-21, with the aim to ascertain the effectiveness of entomopathogenic nematodes, against an obnoxious cosmopolitan pest S. litura. Experiments were conducted by using entomopathogenic nematode (EPN) isolate Heterorhabditis indica (CICR-Guava), on filter paper, against Galleria mellonella and S. litura at the treatment dose of 10, 20, 30, 40, 60, 80 and 100 IJs/100µl along with control (Sterile distilled water). The results of our study revealed that, EPN isolate H. indica (CICR-Guava) caused 100% mortality at the treatment dose of 40 IJs/100µl within 72 h of infection in 5th instar larvae of G. mellonella and in case of S. litura, 100% mortality was recorded within 72 h of infection at the treatment dose of 100 IJs/100µl in 3rd instar larvae, which was found more susceptible. The median lethal concentration of H. indica (CICR-Guava) for 5th instar larvae was 2.29 IJs/100µl. The result of reproductive potential of isolates of entomopathogenic nematodes revealed that the highest yield was obtained from 5th instar larvae of G. mellonella at treatment dose of 100 IJs/100µl 278667 IJs per larva. In case of S. litura, the highest yield obtained was 152533 IJs. It could be concluded that, there was a positive correlation between nematode treatment concentration, time of exposure and the insect mortality of the tobacco cut worm and multiplication rate of IJs increased with increase of exposure time and size of larvae. This EPN isolate, H. indica (CICR-Guava) can be suggested as biocontrol agents for the control of S. litura in the Vidarbha region.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-labgrowth in microbially diverse and natural-like environments. Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Efficacy of entomopathogenic nematode isolates from Turkey against wheat stink bug, Aelia rostrata Boheman (Hemiptera: Pentatomidae) adults under laboratory conditions

BackgroundThe wheat stink bug, Aelia rostrata Boheman (Hemiptera: Pentatomidae), is one of the harmful insects that caused significant product losses in cereals due to outbreaks. Entomopathogenic nematodes (EPNs) are natural enemies of soil insect pests whose effects as a biocontrol agent against many harmful pests have been demonstrated by many laboratories and field/garden studies in the world. The present laboratory studies, using native EPNs [Steinernema carpocapsae (Black sea isolate), S. feltiae (isolate 09-31) (Aydin isolate) and Heterorhabditis bacteriophora (isolate 09-43) (Aydin isolate)] against the wheat stink bug adults, were carried out.ResultsIn the application of EPNs to the soil in plastic cups, the highest mortality rates (75 and 70%) on wheat stink bug occurred at 200 IJs cm−2 concentration of S. carpocapsae isolate at 15 and 12 °C, respectively. In other tested nematode species, the highest insect mortality was observed at 200 IJs cm−2 concentration of S. feltiae and H. bacteriophora isolates, with mortality rates of 65–35% and 55–25%, at 15 and 12 °C, respectively. S. carpocapsae (Black sea isolate) was the most pathogenic among the three nematodes tested.ConclusionsIt was concluded that S. carpocapsae had the potential as a biocontrol agent against the wheat stink bug, A. rostrata. The applications of S. carpocapsae were the great potential for the management of the pest. Effective use of EPNs should be evaluated in integrated pest management strategies between other biological control agents. According to EPNs laboratory experiments results, field trials should be conducted in future studies.

Cold Tolerance in Pinewood Nematode Bursaphelenchus xylophilus Promoted Multiple Invasion Events in Mid-Temperate Zone of China

Pinewood nematode (Bursaphelenchus xylophilus) is a highly destructive invasive species, causing extensive economic and ecological losses across Eurasia. It has recently invaded mid-temperate zone of northern China, threating large areas of coniferous forests. Herein, we evaluated the physiological and molecular basis of cold tolerance in pinewood nematode isolates from different temperature zones of China. After exposure to −5 °C and −10 °C, the survival rates of five pinewood nematode isolates from different temperature zones were 93.94%–94.77% and 43.26%–45.58% after 8 h, and 93.04%–94.85% and 9.93%–10.56% after 24 h, without significant differences among isolates. In a comparison of an isolate from a mid-temperate zone and an isolate from a subtropical zone under gradient cooling, the survival rates remained steady at nearly 95% when minimum temperature ranged from −5 °C to −15 °C, with no significant difference between isolates. In addition, phylogenetic and population structure analyses based on whole genome resequencing data suggested that isolates from mid-temperate and warm temperate zones are clustered with different isolates from subtropical zone, with no obvious geographic pattern. We did not detect significant variation in cold tolerance ability and selected gene among pinewood nematode isolates from different temperature zones. The recently invaded pinewood nematodes in the mid-temperate zone of northern China may spread by multiple invasion events from southern China, without adaptive revolution. Our research implies that it is important to reinforce quarantine inspection to control the rapid spread of pinewood nematode.

Natural association of the entomopathogenic nematode Heterorhabditis indica (Rhabditida: Heterorhabditidae) from the Philippines with the non-symbiotic bacterium Ochrobactrum anthropi (Proteobacteria: Brucellaceae)

BackgroundEntomopathogenic nematodes of the genera Heterorhabditis and Steinernema are widely used as biocontrol agents against diverse insect pests. Their association with symbiont bacteria is found to be the primary cause of insect mortality. The present study reported a comprehensive characterization of a local Philippine EPN isolate using congruent morpho-taxometrical and molecular data and the associated bacterium in the infective dauer juveniles.ResultsUsing an insect-baiting technique, a nematode isolate labeled F2H was recovered from the sandy soils in the coastal area of southern Philippines. Based on morphology and morpho-taxometrics, the F2H isolate collected from Iligan City was identified as Heterorhabditis indica. Analysis of the ITS region of rDNA revealed the highest sequence match to H. indica. The sequences of the bacterial 16S rDNA gene showed that the symbiont of H. indica presented 100% similitude with the sequences of Ochrobactrum anthropi. Further, pairwise alignment and phylogenetic analysis demonstrated that O. anthropi, O. lupini and O. cytisi shared 100% similarity and form a monophyletic clade. These strains have not presented any differences in the phylogenetic and 16S rDNA data, and O. lupini and O. cytisi should be considered a later heterotypic synonym of O. anthropi.ConclusionsThe description of H. indica-O. anthropi association was the first report in the Philippines. This provides additional account and collection of naturally isolated EPN-bacteria, contributing to limited knowledge on its diversity in the Philippines, which can be utilized in the biocontrol of pests after further comprehensive assessments.

Potential of Oscheius tipulae nematodes as biological control agents against Ceratitis capitata.

A survey to collect soil nematodes with potential to control Ceratitis capitata flies was carried out in different locations in Tunisia. Several nematode isolates were recovered, laboratory colonies were established, and their taxonomic identities were determined based on molecular methods. Among all the recovered nematode isolates, two of them, Oscheius tipulae TC2 and OC2, were evaluated for their capacity to control C. capitata flies and for their ability to kill and reproduce on Galleria mellonella larvae. Our results show a great potential of these two isolates as biocontrol agents as they kill C. capitata eggs and pupae and interfere with the metamorphosis of C. capitata larvae. More specifically, TC2 and OC2 nematodes killed 39 and 31% of C. capitata eggs, respectively, impaired the metamorphosis of up to 77% and up to 67% of C. capitata larvae, respectively, and killed up to 66% and up to 58% of C. capitata pupae, respectively. The efficacy of TC2 and OC2 nematodes was particularly high on C. capitata pupae, and significant insect mortalities were observed even at concentrations of 1 and 5 nematodes/pupae, respectively. We also found that TC2 and OC2 nematodes efficiently kill and reproduce in G. mellonella larvae, suggesting that these insects could be used for mass-multiplication of these nematodes. These results reveal the potential of O. tipulae to complement integrated pest management programs against C. capitata flies.

Trade-offs in defence to pathogen species revealed in expanding nematode populations.

Many host organisms live in polymicrobial environments and must respond to a diversity of pathogens. The degree to which host defences towards one pathogen species affect susceptibility to others is unclear. We used a panel of Caenorhabditis elegans nematode isolates to test for natural genetic variation in fitness costs of immune upregulation and pathogen damage, as well as for trade-offs in defence against two pathogen species, Staphylococcus aureus and Pseudomonas aeruginosa. We examined the fitness impacts of transient pathogen exposure (pathogen damage and immune upregulation) or exposure to heat-killed culture (immune upregulation only) by measuring host population sizes, which allowed us to simultaneously capture changes in reproductive output, developmental time and survival. We found significant decreases in population sizes for hosts exposed to live versus heat-killed S. aureus and found increased reproductive output after live P. aeruginosa exposure, compared with the corresponding heat-killed challenge. Nematode isolates with relatively higher population sizes after live P. aeruginosa infection produced fewer offspring after live S. aureus challenge. These findings reveal that wild C. elegans genotypes display a trade-off in defences against two distinct pathogen species that are evident in subsequent generations.

Distribution of Phasmarhabditis (Nematode: Rhabditidae) and Their Gastropod Hosts in California Plant Nurseries and Garden Centers.

Three species of Phasmarhabditis were recovered from 75 nurseries and garden centers in 28 counties in California during fall and winter 2012–2021. A total of 18 mollusk species were recovered, most of them invasive. Nematodes were identified by sequencing the D2-D3 expansion segments of the large subunit (LSU or 28S) rRNA. Based on these surveys, P. californica was the most widespread species (37 isolates, 53.6% recovery); followed by P. hermaphrodita (26 isolates; 37.7% recovery); P. papillosa and a closely related P. papillosa isolate (6 isolates; 8.7% recovery). Nematode isolates were mainly collected from four invasive slugs (Deroceras reticulatum, D. laeve, Arion hortensis agg, Ambigolimax valentianus) and snails (Oxychilus spp. and Discus spp.). Results suggest that P. californica and P. hermaphrodita share an ecological niche in Northern, Central, Coastal, and Southern California, north of Los Angeles County.

Selection of Genome-Wide SNPs for Pooled Allelotyping Assays Useful for Population Monitoring.

Parasitic worms are serious pests of humans, livestock, and crops worldwide. Multiple management strategies are employed in order to reduce their impact, and some of these may affect their genome and population allelic frequency distribution. The evolution of chemical resistance, ecological changes, and pest dispersal has allowed an increasing number of pests to become difficult to control with current management methods. Their lifestyle limits the use of ecological and individual-based management of populations. There is a need to develop rapid, affordable, and simple diagnostics to assess the efficacy of management strategies and delay the evolution of resistance to these strategies. This study presents a multilocus, equal-representation, whole-genome pooled single nucleotide polymorphisms (SNPs) selection approach as a monitoring tool for the ovine nematode parasite Haemonchus contortus. The SNP selection method used two reference genomes of different quality, then validated these SNPs against a high-quality recent genome assembly. From over 11 million high-quality SNPs identified, 334 SNPs were selected, of which 262 were species-specific, yielded similar allele frequencies when assessed as multiple individuals or as pools of individuals, and suitable to distinguish mixed nematode isolate pools from single isolate pools. As a proof-of-concept, 21 Australian H. contortus populations with various phenotypes and genotypes were screened. This analysis confirmed the overall low level of genetic differentiation between populations collected from the field, but clearly identifying highly inbred populations, and populations showing genetic signatures associated with chemical resistance. The analysis showed that 66% of the SNPs were necessary for stability in assessing population genetic patterns, and SNP pairs did not show linkage according to allelic frequencies across the 21 populations. This method demonstrates that ongoing monitoring of parasite allelic frequencies and genetic changes can be achieved as a management assessment tool to identify drug-treatment failure, population incursions, and inbreeding signatures due to selection. The SNP selection method could also be applied to other parasite species.

Isolation, Identification, and Biocontrol Potential of Entomopathogenic Nematodes and Associated Bacteria against Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae).

Simple SummaryFor sustainable agriculture, there is a need to identify and evaluate more biocontrol agents, including entomopathogenic nematodes (EPNs). In this study, EPNs and their associated entomopathogenic bacteria symbionts (EPBs) were isolated and identified from 320 soil samples collected in Taif, Saudi Arabia. The biocontrol efficacy of EPNs and EPBs was also evaluated against third instar larvae of the pomegranate butterfly, Virachola livia, and the carob moth, Ectomyelois ceratoniae, two important insect pests of pomegranate, under laboratory conditions. Our results show that the EPNs Steinernema spp. were more virulent than Heterorhabditis spp. against the two pomegranate insects. In addition, the EPB Stenotrophomonas maltophilia CQ1, isolated from Steinernema spp., surpassed Pseudomonas mosselii SJ10, associated with Heterorhabditis spp., in their ability to kill V. livia or E. ceratoniae larvae. We conclude that either application of EPNs’ infective juveniles (IJs) or their associated EPBs could serve as potential biocontrol agents for V. livia and E. ceratoniae.Virachola livia (Lepidoptera: Lycaenidae) and Ectomyelois ceratoniae (Lepidoptera: Pyralidae) are the key pests of pomegranates in Saudi Arabia that are managed mainly using broad-spectrum pesticides. Interactions between the entomopathogenic nematodes (EPNs) Steinernematids, and Heterorhabditids, and their entomopathogenic bacterial symbionts (EPBs) have long been considered monoxenic 2-partner associations responsible for killing insects and, therefore, are widely used in insect pest biocontrol. However, there are limited reports identifying such organisms in Taif, Saudi Arabia. The current study aimed to identify the EPNs and their associated bacteria isolated from Taif, Saudi Arabia, and evaluate their biocontrol potential on third instar larvae of V. livia and E. ceratoniae under laboratory conditions. A total of 35 EPN isolates belonging to Steinernema (20) and Heterorhabditis (15) were recovered from 320 soil samples. Twenty-six isolates of symbiotic or associated bacteria were isolated from EPNs and molecularly identified as Xenorhabdus (6 isolates), Photorhabdus (4 isolates), Pseudomonas (7), or Stenotrophomonas (9). A pathogenicity assay revealed that Steinernema spp. were more virulent than Heterorhabditis spp. against the two pomegranate insects, with LC50 values of 18.5 and 13.6 infective juveniles (IJs)/larva of V. livia for Steinernema spp. and 52 and 32.4 IJs/larva of V. livia for Heterorhabditis spp. at 48 and 72 h post-treatment, respectively. Moreover, LC50 values of 9 and 6.6 IJs/larva (Steinernema spp.) and 34.4 and 26.6 IJs/larva (Heterorhabditis spp.) were recorded for E. ceratoniae larvae at 48 and 72 h post-treatment. In addition, the EPB Stenotrophomonas maltophilia CQ1, isolated from Steinernema spp., surpassed Pseudomonas mosselii SJ10, associated with Heterorhabditis spp., in their ability to kill V. livia or E. ceratoniae larvae within 6 h post-application, resulting in 100% mortality in both insects after 24 and 48 h of exposure. We conclude that either application of EPNs’ IJs or their associated EPBs could serve as potential biocontrol agents for V. livia and E. ceratoniae.

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

Rapid Isolation of Wild Nematodes by Baermann Funnel.

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

Isolation and morphometric characterization of entomopathogenic nematodes Steinernema kushidai from Jorhat district of Assam, India

A random survey was conducted for the natural occurrence of EPNs from vegetable growing areas ol the district Jorhat, Assam. Only four of the 200 soil samples examined tested positive for entomopathogenic nematodes and contained one Steinernema isolate. To characterise the isolate, morphological and morphometrical examinations were carried out. Steinernema kushidai was identified as the Steinernema isolate.

Virulence of entomopathogenic nematode isolate Steinernema bicornutum against diamondback moth (Plutella xylostella L.)

Plutella xylostella L., commonly known as the diamondback moth, is a significant pest of cole crops. It has established resistance to a range of pesticides, including chemical insecticides and Bt toxins. The use of appropriate bio-agents to manage the diamondback moth is a viable alternative to chemical pesticides. The current study was carried out with the goal of using entomopathogenic nematode (EPN) isolate Steinernema bicornutum to manage diamondback moths. Laboratory studies were carried out to know the virulence and reproductive potential of an indigenous population of Steinernema bicornutum at five doses i.e., 2IJs, 5 IJs, 8 IJs, 10 IJs and 12 IJs per third instar larvae of diamondback moth. Percent mortalities were recorded after 24h, 48h and 72h of post inoculation. Maximum percent mortality (81.25) was recorded after 72h with the highest dose i.e., 12 IJs per larva and the LC50 value was 7.99 IJs/100µl. The result of reproduction and recovery of isolates of entomopathogenic nematodes revealed that the highest IJs was yielded (2255.7) from dose of 12 IJs/larva. The overall results strongly suggested that P. xylostella is more susceptible to the entomopathogenic nematode and can be the host for multiplication of entomopathogenic nematodes under laboratory conditions.

Developing a Real-Time PCR Assay for Direct Identification and Quantification of Soybean Cyst Nematode, Heterodera glycines, in Soil and Its Discrimination from Sugar Beet Cyst Nematode, Heterodera schachtii.

The soybean cyst nematode (SCN) Heterodera glycines continues to be a major threat to soybean production worldwide. Morphological discrimination between SCN and other nematodes of the Heterodera schachtii sensu stricto group is not only difficult and time-consuming but also requires high expertise in nematode taxonomy. Molecular assays were developed to differentiate SCN from sugar beet cyst nematode (SBCN) and other nematodes and to quantify SCN directly from DNA extracts of field soils. SCN- and SBCN-specific quantitative real-time PCR (qPCR) primers were designed from a nematode-secreted CLAVATA gene and used for these assays. The primers were evaluated on the basis of target specificity to SCN or SBCN using DNA from 20 isolates of SCN and 32 isolates of other plant-parasitic nematodes. A standard curve relating threshold cycle and log values of nematode numbers was generated from artificially infested soils and was used to quantify SCN in naturally infested field soils. There was a high correlation between the SCN numbers estimated from naturally infested field soils by conventional methods, and the numbers quantified using the SYBR Green I-based qPCR assay. The qPCR assay is highly specific and sensitive and provides improved SCN detection sensitivity down to 1 SCN egg in 20 g of soil (10 eggs/200 g soil). This assay is useful for efficient detection and quantification of SCN directly from field soil. Species-specific conventional PCR assays were also developed each for SCN and SBCN, alongside a qPCR assay that simultaneously discriminates SCN from SBCN. These assays require no expertise in nematode taxonomy and morphology, and they may serve as useful diagnostic tools in research, diagnostic laboratories, and extension services for SCN management. Sensitive and accurate detection and quantification of SCN are essential for recommending effective management measures against SCN. We also investigated the impact of soil texture and nematode life stage on molecular quantification of SCN.

Efficacy of Thai indigenous entomopathogenic nematodes for controlling fall armyworm (Spodoptera frugiperda)\xa0(J. E. Smith)(Lepidoptera; Noctuidae)

BackgroundUnder laboratory and greenhouse conditions, the virulence of 2 isolates of Thai indigenous entomopathogenic nematodes (EPNs) in controlling the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Lepidoptera; Noctuidae), was demonstrated. Six EPNs dosages were tested against 2 larval instars of FAW under the laboratory conditions, while 2 different concentrations were tested under the greenhouse conditions.ResultsThe results of a laboratory experiment revealed that 2 Thai indigenous EPNs isolates (Heterorhabditis indica isolate AUT 13.2 and Steinernema siamkayai isolate APL 12.3) were efficient against the FAW, 2nd and 5th larval instars. Six different nematode concentrations (50,100, 150, 200, 250 and 300 infectious juveniles (IJs) ml−1) were evaluated, and all were proven to be effective, with the mortality rate associated with concentration. Inoculated larvae in the 2nd instar was more vulnerable than that in the 5th instar. H. indica isolate AUT 13.2 was more destructive than S. siamkayai isolate APL 12.3. The greatest mortality rate of 2nd instar larvae was 83% when H. indica AUT 13.2 was applied at the concentration of 250 IJs ml−1, and 68% when the nematode S. siamkayai APL 12.3 was used at the concentration of 300 IJs ml−1. At 250 IJsml−1, the highest mortality rate of the 5th instar larvae was 45% for H. indica AUT 13.2 and 33% for S. siamkayai APL 12.3, respectively. To customize the concentration and volume of nematodes suspension evaluated in the greenhouse settings, the most sensitive stage of FAW and the optimum concentration that caused the highest mortality were used. The concentrations of both indigenous nematodes’ isolates were 20,000 and 50,000 IJsml−1 per pot, respectively, and the results showed that the mortality rates were lower than that in the laboratory. FAW mortality rate was the highest (58%) in case of the nematode H. indica isolate AUT 13.2, against (45%) in case of S. siamkayai isolate APL 12.3, at the 50,000 IJs ml−1 concentrations.ConclusionsThe study revealed the 2 Thai indigenous EPNs isolates (H. indica isolate AUT 13.2 and S. siamkayai isolate APL 12.3) were capable of controlling the FAW in both laboratory and greenhouse environments. The 2 Thai EPNs showed the potential to be considered as a biological control agent.