Tarique Hassan Askary

Background: Nematodes of the genus Heterorhabditis are important biocontrol agents as they form a lethal combination with their symbiotic Photorhabdus bacteria against agricultural insect pests. This study describes a new species of Heterorhabditis. Methods: Six Heterorhabditis nematode populations were recovered from agricultural soils in Jammu and Kashmir, India. An initial examination using mitochondrial and nuclear genes showed that they belong to a new species. To describe this new species, a variety of analyses were conducted, including reconstructing phylogenetic relationships based on multiple genes, characterizing the nematodes at the morphological and morphometric levels, performing self-crossing and cross-hybridization experiments, and isolating and characterizing their symbiotic bacteria. Results: The newly discovered species, Heterorhabditis casmirica n. sp., shares 94% mitochondrial cytochrome C oxidase subunit I gene (COI) sequence identity with Heterorhabditis bacteriophora and Heterorhabditis ruandica, and 93% with Heterorhabditis zacatecana. Morphologically, it differs from H. bacteriophora in its infective juvenile phasmids (present vs. inconspicuous) and bacterial pouch visibility in the ventricular portion of the intestine (invisible vs. visible); genital papilla 1 (GP1) position (at manubrium level vs. more anterior), and in its b ratio (body length/neck length), c ratio (tail length/bulb width), and D% [(excretory pore/neck length) × 100]. Other morphological differences include anterior end to the nerve ring distance (77–100 vs. 121–130 μm), V% [(anterior end of vulva/body length) × 100] (46–57 vs. 41–47) in hermaphroditic females; rectum size (slightly longer than the anal body diameter vs. about three times longer), phasmids (smaller vs. inconspicuous), body length (0.13–2.0 vs. 0.32–0.39 mm), body diameter (73–150 vs. 160–220 μm), anterior end to the excretory pore distance (135–157 vs. 174–214 μm), and demanian ratios in amphimictic females. Morphological differences with H. ruandica and H. zacatecana were also observed. Furthermore, H. casmirica n. sp. did not mate or produce fertile progeny with other Heterorhabditis nematodes reported from India. It was also discovered that H. casmirica n. sp. is associated with 'Photorhabdus laumondii subsp. clarkei symbiotic bacteria. Conclusions: The discovery of H. casmirica n. sp. provides novel insights into the diversity and evolution of Heterorhabditis nematodes and their symbiotic bacteria. This new species adds to the catalog of entomopathogenic nematodes in India. Graphical

A population of entomopathogenic nematodes, belonging to the Feltiae-clade and labelled J13, was discovered in the agricultural soils of the hilly regions of the Union territory of Jammu and Kashmir, India. Based on morphological, morphometric, and molecular analyses, the nematodes were identified as Steinernema feltiae. The J13 nematode isolate was tested in a laboratory assay for its pathogenicity against six major pests of vegetable crops: Pieris brassicae, Plutella xylostella, Helicoverpa armigera, Agrotis iplison, Trichoplusia ni, and Exelastis atomosa. The morphology of the isolated nematode closely matched the original description, except for the adult females, which had prominent epiptygmata instead of the weakly developed, double-flapped epiptygmata described in the original report. Analysis of the internal transcribed spacer and large subunit rRNA data from the J13 nematodes showed 100% similarity to sequences of the type population, indicating that they are conspecific. The virulence assays revealed that the nematode caused 100% mortality in the tested insect pests within 48–72 hours, even at the lowest concentration of 50 infective juveniles per insect. The calculated median lethal concentration varied among the pests, with the lowest number of infective juveniles needed to achieve 50% larval killing being 117 for P. xylostella, 181.74 for P. brassicae, 226.35 for H. armigera, and 202.07 for T. ni at 24 hours post-inoculation. These findings suggest that S. feltiae isolated during the present investigation, may be a viable option for the biocontrol of these insect pests in Kashmir valley, India.

The native isolates of entomopathogenic nematodes (EPNs) Heterorhabditis bacteriophora MK256358, H. bacteriophora MK263023, H. bacteriophora MK256378 and Steinernema cholashanense MK128155 were tested in laboratory for their virulence and reproductive potential against 4^th instar larvae of Oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae), the serious pest of rice in Kashmir valley. All the EPN isolates were highly effective against 4^th instar larvae of M. separata. Insect mortalities reached 100% within 24-48 hours at nematode concentrations of 200 infective juveniles (IJs)/larva. The trend was similar at lower concentrations of IJs, but more time was required. The best performance was shown by H. bacteriophora MK256358 and S. cholashanense MH128155 followed by H. bacteriophora MK256378 and H. bacteriophora MK263023 in terms of killing and reproductive potential. The values obtained for lethal concentration (LC₅₀) and lethal time (LT₅₀) of the tested EPN isolates differed significantly. Our results demonstrate the potential of the native EPN isolates against M. separata but before recommendation multi-location field trials need to be conducted to confirm their validity at farm level.

Indigenous entomopathogenic nematode, Heterorhabditis pakistanensis, NBAIR H-05 strain was evaluated against diapausing larvae of the Codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae) at 3 different dosages, i.e., at 15, 20, and 25 gm/ l of water in apple orchards at the district Kargil of Ladakh Region, India, during 2017 and 2018.Two year’s pooled average density of diapausing ranged 34.6 to 56.8 larvae/trunk band before treatment, which declined ranging 43.85 to 86.27 % with respect to different treatments of entomopathogenic nematode at concentrations between 7.5 × 10⁵ IJs to 1.25 × 10⁶ IJs/tree. Percent reduction in larvae over control varied from 41.78 to 85.77% for 7.5 × 10⁵ IJs and 1.25 × 10⁶ IJs respectively. Two-year pooled data indicated larval mortality between 39.85 and 73.38% and 4.0 to 12.89% with respect to different treatments at 48 and 72 h respectively, with statistically significant difference (P = ˂ 0.001). Increase in dosage of nematode formulation from 15 gm to 25 gm resulted in increased larval mortality (r = 0.92**). Post wetting of trunk band after 24 h in each treatment resulted in significantly higher larval mortality than non-post wetting. There was non-significant difference (t = 0.83) between larval mortality with respect to treatments during 2017 and 2018.

Experiments were conducted to test the pathogenicity of entomopathogenic nematode,Heterorhabditis pakistanensisalone and in combination with the chemical insecticides, viz., Dichlorvos and Quinalphos and Neem product, viz., Azadirachtin against the cabbage butterfly,Pieris brassicae(L.) (Lepidoptera: Pieridae) infesting cabbage plants under laboratory and field conditions. In laboratory test, inoculum level of 200 infective juveniles (IJs) was most effective causing mortality to both 3rd and 4th instars larvae at 48 h. Nematode multiplication rate in insect cadaver was inversely proportional to the size of initial inoculum level of IJs, whereas IJs yield was directly proportional to the size of larva. The 4th instar larva resulted in a highest yield of IJs, followed by 3rd, 2nd, and 1st instars larvae at all the inoculum levels used in the study. IJs were found compatible with the chemical insecticides and Neem product at lower than half of the optimum single dose. The cumulative mean survival percentage of IJs recorded the highest (77.5%) in the combination of 100 IJs + Azadirachtin (0.25%) and lowest (70.6%) in 100 IJs + Dichlorvos 76 EC (0.05%). Under field conditions,H. pakistanensis(3.0 lakh IJs/m²) + Dichlorvos 76 EC (0.025%) resulted to the highest larval mortality (79.65%), followed byH. pakistanensis(3.0 lakh IJs/m²) + Azadirachtin (0.25%), where it was 76.22%. The larval mortality was the least (61.16%) in case ofH. pakistanensis(3.0 lakh IJs/m²) when applied alone.H. pakistanensis+ Dichlorvos resulted to the highest increase (31.42%) in cabbage yield over untreated control.

Entomopathogenic nematodes (EPNs) in the families’ Steinernematidae and Heterorhabditidae are obligate insect parasites. Their easy multiplication, broad host range, compatibility with chemical pesticides, and ease in application has grabbed interest among research practitioners to work on these beneficial microorganisms. Till date, around 100 valid species ofSteinernemaand 21 species ofHeterorhabditishave been identified from different countries of the world. Extensive surveys have been conducted across the globe to isolate locally adapted EPN species and exploit them to suppress soil-dwelling and foliar insect pests in agricultural fields. Most of the new species have been described from Asia, whereas research in some Asian countries are still at infancy. Some new species have been recorded from Australia but very few surveys have been conducted in New Zealand. Likewise, less information about these tiny creatures is from Central America; however, in North America many new species have been described, some of which have been commercialized for insect pest control, whereas in South America, several native nematode species have been described and exploited as biological control agents. European countries have also been explored for EPN diversity and new species have been reported, exploited under field condition, and commercialized. Many new species and other previously described species have been reported from Africa. Despite frequent surveys in different continents of the world, number of sites touched are low and, therefore, further surveys are still needed to explore untouched geographic areas and climatic conditions, both in plantations and indigenous forests with an aim to identify and exploit additional EPN species.