Multigene Sequence-Based and Phenotypic Characterization Reveals the Occurrence of a Novel Entomopathogenic Nematode Species, Steinernema anantnagense n. sp.

Three entomopathogenic nematode populations were isolated from agricultural fields in the Anantnag district of Jammu and Kashmir (India). Sequences of multiple gene regions and phenotypic features show that they are conspecific and represent a novel species. Molecular and morphological features provided evidence for placing the new species into the "Kushidai" clade. Within this clade, analysis of sequence data of the internal transcribed spacer (ITS) gene, the D2D3 region of the 28S rRNA gene, the mitochondrial cytochrome oxidase I (mtCOI) gene, and the mitochondrial 12S (mt12S) gene depicted the novel species as a distinctive entity closely related to Steinernema akhursti, S. kushidai, and S. populi. Phylogenetic analyses also show that the new species is a sister species to S. akhursti, and these two species are closely related to S. kushidai and S. populi. Additionally, the new species does not mate or produce fertile progeny with any of the closely related species, reinforcing its uniqueness from a biological species concept standpoint. The new species is further characterized by the third-stage infective juveniles with almost straight bodies (0.7-0.8 mm length), poorly developed stoma and pharynx, and conoid-elongate tail (49-66 µm) with hyaline posterior part. Adult females are characterized by short and conoid tails bearing a short mucron in the first generation and long conoid tails with thin mucron in the second generation. Adult males have ventrally curved spicules in both generations. Moreover, the first-generation male has rounded manubrium, fusiform gubernaculum, conoid and slightly ventrally curved tails with minute mucron, and the second generation has rhomboid manubrium anteriorly ventrad bent, and tails with long and robust mucron. The morphological, morphometrical, molecular, and phylogenetic analyses support the new species status of this nematode, which is hereby described as Steinernema anantnagense n. sp. The bacterial symbiont associated with S. anantnagense n. sp. represents a novel species, closely related to Xenorhabdus japonica. These findings shed light on the diversity of entomopathogenic nematodes and their symbiotic bacteria, providing valuable information for future studies in this field.

Morphological and molecular characterization of Acrobeloides saeedi Siddiqi, De Ley and Khan, 1992 (Rhabditida, Cephalobidae) from India and comments on its status.

Two cultured populations of Acrobeloides saeedi are described from India. Morphologically and morphometrically this material agrees with other species of the Maximus-group (A. bodenheimeri, A. longiuterus, and A. maximus), especially with A. longiuterus. However, molecular studies based on 18 S, 28 S and ITS rDNA confirmed the Indian material is well differentiated from all of these species. According to this, A. saeedi is considered a valid taxon distinguished mainly from A. bodenheimeri by having dextral female reproductive system (vs sinistral), from A. longiuterus by having larger females (1.03-1.57 vs 0.57-0.88 mm) and from A. maximus by having seta-like labial processes (vs absent) and males as frequent as females (vs males very infrequent). Molecular and phylogenetic studies revealed the present specimens to be conspecific to undescribed Acrobeloides sp. population from Iran, and hence, both regarded to be conspecific to each other. In addition, other similar species are revised: Acrobeloides ishraqi is considered new junior synonym of A. saeedi, Acrobeloides mushtaqi is considered new junior synonym of A. bodenheimeri, while Acrobeloides gossypia is also considered junior synonym of A. saeedi.Two cultured populations of Acrobeloides saeedi are described from India. Morphologically and morphometrically this material agrees with other species of the Maximus-group (A. bodenheimeri, A. longiuterus, and A. maximus), especially with A. longiuterus. However, molecular studies based on 18 S, 28 S and ITS rDNA confirmed the Indian material is well differentiated from all of these species. According to this, A. saeedi is considered a valid taxon distinguished mainly from A. bodenheimeri by having dextral female reproductive system (vs sinistral), from A. longiuterus by having larger females (1.03-1.57 vs 0.57-0.88 mm) and from A. maximus by having seta-like labial processes (vs absent) and males as frequent as females (vs males very infrequent). Molecular and phylogenetic studies revealed the present specimens to be conspecific to undescribed Acrobeloides sp. population from Iran, and hence, both regarded to be conspecific to each other. In addition, other similar species are revised: Acrobeloides ishraqi is considered new junior synonym of A. saeedi, Acrobeloides mushtaqi is considered new junior synonym of A. bodenheimeri, while Acrobeloides gossypia is also considered junior synonym of A. saeedi.

Morphological, morphometrical, and molecular characterization of Metarhabditis amsactae (Ali, Pervez, Andrabi, Sharma and Verma, 2011) Sudhaus, 2011 (Rhabditida, Rhabditidae) from India and proposal of Metarhabditis longicaudata as a junior synonym of M. amsactae.

A new population of Metarhabditis amsactae from India is morphologically, morphometrically, and molecularly characterized. This material is characterized by having 0.65 to 1.14 mm length, lips rounded, and grouped in pairs, stoma with metastegostoma bearing setose denticles, pharynx with metacorpus slightly swollen and fusiform, nerve ring, and excretory pore located at isthmus level, female reproductive system didelphic-amphidelphic with vulva equatorial, female tail conical-elongate with acute tip, male tail conical with large and robust posterior filiform part, spicules free with hooked manubrium slightly bent ventrad, gubernaculum with narrow corpus, bursa open leptoderan with eight genital papillae and phasmids posterior to the GP8. Molecular studies based on 18S and 28S rDNA genes are provided for the first time for the species. In addition, integrated morphological, morphometrical, and molecular characters are compared with other previous records of the species. According to our analysis, Metarhabditis longicaudata and other material described as different species are proposed as new junior synonyms of M. amsactae.

In situ chlorpyrifos (CPF) degradation by Acrobeloides maximus: Insights from chromatographic analysis.

The objective of this study was to evaluate the efficiency of nematodes in zooremediation of chlorpyrifos (CPF), an organophosphate pesticide. The nematode population Acrobeloides maximus (A. maximus) was employed for bioremediation, converting CPF into non-toxic residues. Optimal growth conditions for mass production of A. maximus were achieved by maintaining a temperature of 25 °C, pH 8, and supplementing the culture medium with plant nutrients. The nematodes were then immobilized within sodium alginate beads. The efficacy of the degradation process was assessed using various analytical techniques, including UV-Visible spectroscopy, HPTLC, FTIR, and LC-MS, confirming the successful breakdown of CPF. The bioreactor demonstrated a complete degradation efficiency of CPF exceeding 99%. Additionally, LC-MS analysis was conducted to elucidate the degradation pathway based on the formation of intermediates. These results underscore the potential of A. maximus as a sustainable organism for addressing environmental contamination arising from CPF pesticide.

From soil to plant: strengthening carrot defenses against Meloidogyne incognita with vermicompost and arbuscular mycorrhizal fungi biofertilizers.

Introduction: Sustainable agricultural practices for controlling crop pests are urgently needed to reduce the reliance on chemical pesticides, which have long-term detrimental effects on ecosystems. In this study, we assessed the effectiveness of arbuscular mycorrhizal fungi (AMF) and vermicompost (Vc) supplementation, alone and in combination, in mitigating the negative impacts of Meloidogyne incognita infestation on carrot (Daucus carota L.) growth, development, and physiology. Methods: We measured different plant growth parameters such as plant height and biomass accumulation, several plant physiological parameters such as the levels of photosynthetic pigments, phenolics, and the activity of defense enzymes such as peroxidases and polyphenol oxidases, and evaluated the severity of Meloidogyne incognita nematode infestation on plants treated or not treated with vermicompost (Vc) and/or arbuscular mycorrhizal fungi (AMF). Results: Our findings show that M. incognita significantly affects plant growth, biomass accumulation, and photosynthetic pigment and carotenoid content. The incorporation of Vc and AMF into the soil, either individually or in combination, significantly alleviates the negative effects of nematode infestation on carrot plants. This was accompanied by the induction of phenolic compounds and defense enzymes such as peroxidases (+15.65%) and polyphenol oxidases (29.78%), and by a reduction in the severity of nematode infestation on Vc and AMF-treated plants compared to nematode-infested plants. Principal component analysis (PCA) shows significant correlations between various of the studied parameters. In particular, we observed negative correlations between the application of AMF and Vc alone and in combination and disease severity, and positive correlations between plant growth, photosynthetic pigments phenol content, and activity of defense enzymes. Discussion: Our study highlights the relevance of cultural practices and beneficial microorganisms for the sustainable and environmentally friendly management of agricultural pests.

Corrigendum: From soil to plant: strengthening carrot defenses against Meloidogyne incognita with vermicompost and arbuscular mycorrhizal fungi biofertilizers.

[This corrects the article DOI: 10.3389/fmicb.2023.1206217.].

Morphological and molecular profiling of an entomopathogenic nematode Steinernema feltiae: Unlocking its biocontrol potential against vegetable insect pests.

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 4872 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.

Morphological, Morphometrical and Molecular Characterization of Oscheius siddiqii Tabassum and Shahina, 2010 (Rhabditida, Rhabditidae) from India with Its Taxonomic Consequences for the Subgenus Oscheius Andrássy, 1976.

An insect parasitic nematode belonging to the genus Oscheius was recovered from the agricultural soils from the Hapur district in western Uttar Pradesh, India. Morphological studies on this species exhibited its high resemblance with two Pakistani species: Oscheius siddiqii and O. niazii. No molecular data are available for these taxa but, morphologically, both species do not differ significantly from our strains and each other. Hence, these nematodes can be considered conspecific, and the correct name for this taxon is O. siddiqii, the first described species. The phylogenetic analyses of the ITS-, 18S-, and the 28S rDNA sequences showed that O. siddiqii is a sister taxon to the group formed by Oscheius microvilli, O. myriophilus, O. safricanus, and several unidentified Oscheius species. Additionally, our analyses show that based on molecular and morphological data, the species Oscheius rugaoensis and O. microvilli cannot be distinguished from O. chongmingensis and O. myriophilus, respectively, and are thus considered junior synonyms of these taxa. Furthermore, the available data are not sufficient to evaluate the status of Oscheius basothovii and O. safricanus, which are, in consequence, considered species inquirendae. These findings highlight the necessity of the proper morphological and molecular characterisation of the described Oscheius species.

Morphological and molecular characterization of Heterorhabditis bacteriophora isolated from Indian soils and their biocontrol potential.

Agricultural soils and open fields from Western Uttar Pradesh (India) were surveyed to determine the presence of entomopathogenic nematodes. From the entomopathogenic nematodes isolated, Heterorhabditis isolates were selected and further characterized using morphological, morphometrical and molecular approaches. The results showed that three isolated nematodes were Heterorhabditis bacteriophora and were associated with Photorhabdus laumondii subsp. clarkei bacteria, while the rests were identified as Heterorhabditis indica. The biocontrol potential of H. bacteriophora against three agricultural pests was evaluated. Nematode infectivity experiments showed that the nematode isolates DH7 and DH8 were highly pathogenic against cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura), and less pathogenic against white grub (Holotrichia serrata) larvae. This study sets the basis for establishing new biocontrol agents to be used in pest management programs in India.