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.
Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.
Four bacterial strains, A-IN1T, A-TC2T, E-TC7T, and K-TC2T, isolated from soil-borne nematodes of the species Oscheius tipulae and Acrobeloides bodenheimeri, were found to represent new species of the genera Acinetobacter, Alcaligenes, Enterobacter, and Kaistia, respectively. In this study, we described these new species using a polyphasic taxonomic approach that included whole-genome and whole-proteome phylogenomic reconstructions, core genome sequence comparisons, and phenotypic characterization. Phylogenomic reconstructions using whole-genome and whole-proteome sequences show that A-IN1T is closely related to Acinetobacter guillouiae DSM 590T and to Acinetobacter bereziniae LMG 1003T. The dDDH values between A-IN1T and these latest strains are 25.1 and 39.6%, respectively, which are below the 70% divergence threshold for prokaryotic species delineation. A-TC2T is closely related to Alcaligenes faecalis subsp. faecalis DSM 30030T and to Alcaligenes faecalis subsp. phenolicus DSM 16503T. The dDDH values between A-TC2T and these latest strains are 47.0 and 66.3%, respectively. In addition, the dDDH values between Alcaligenes faecalis subsp. faecalis DSM 30030T, Alcaligenes faecalis subsp. phenolicus DSM 16503T, and Alcaligenes faecalis subsp. parafaecalis are always lower than 70%, demonstrating that the three strains represent species within the genus Alcaligenes rather than subspecies within Alcaligenes faecalis. E-TC7T is closely related to Enterobacter kobei DSM 13645T, Enterobacter chuandaensis 090028T, and to Enterobacter bugandensis STN0717-56T. The dDDH values between E-TC7T and these strains are 43.5, 42.9, and 63.7%, respectively. K-TC2T is closely related to Kaistia terrae DSM 21341T and to Kaistia defluvii JCM 18034T. The dDDH values between these strains are 29.2 and 30.7%, respectively. Several biochemical tests allow to differentiate the type strains of the newly described species from the type strains of their more closely related species. Based on the results of this polyphasic taxonomic approach, the following new species are proposed: Acinetobacter nematophilus sp. nov. with A-IN1T (=CCM 9231T =CCOS 2018T) as the type strain, Alcaligenes nematophilus sp. nov. with A-TC2T (=CCM 9230T =CCOS 2017T) as the type strain, Enterobacter nematophilus sp. nov. with E-TC7T (=CCM 9232T =CCOS 2020T) as the type strain, and Kaistia nematophila sp. nov. with K-TC2T (=CCM 9239T =CCOS 2022T) as the type strain. In addition, we propose the elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the species level. Therefore, we propose the creation of Alcaligenes parafaecalis sp. nov. with DSM 13975T as the type strain, and Alcaligenes phenolicus sp. nov. with DSM 16503T as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of bacteria associated with soil-borne nematodes.
Summary Four populations of Acrobeloides nematodes were isolated from Tunisian soils. Based on morphological and morphometric data, two populations, TC9 and HWO, were identified as A. tricornis and the other two, TC7 and K18g, as A. bodenheimeri. Acrobeloides tricornis is distinguished from its closely related species by its high labial probolae with arcuate termini, inconspicuous post-uterine sac, and five lateral incisures, while A. bodenheimeri is distinguished from other acrobeloids by having a low and rounded labial probolae, distinct post-uterine sac, five lateral incisures, and vulva frequently with vaginal plug. The A. tricornis and A. bodenheimeri reported in this study are distinguished by the shape of labial probolae (high with acute distal part vs low and rounded), post-uterine sac (inconspicuous vs distinct), position of the reproductive system with respect to the intestine (sinistral vs dextral), vulva with vaginal plug (present vs absent) and some morphometric characters (such as body length). Further, these species were also molecularly characterised using the sequences of the D2-D3 region of the 28S rRNA gene, the 18S rRNA gene, the ITS rRNA gene, and of the mitochondrial cytochrome c oxidase subunit I (mtCO1) gene. Phylogenetic trees using ribosomal RNA gene sequences clearly separate species of the ‘Maximus’ group, but these sequences appear to be identical in several other Acrobeloides species, and thus are unlikely to be useful as molecular markers for species-level molecular identification in the genus Acrobeloides. The mtCO1 gene sequences of two A. tricornis populations differ in 58 and 64 nucleotide bases from A. varius and A. nanus, respectively, indicating that the mtCO1 gene sequences have greater phylogenetic resolving power than nuclear rRNA gene sequences, and that this gene marker can differentiate closely related species in the genus Acrobeloides.
The red palm weevil, Rhynchophorus ferrugineus (RPW) (Olivier, 1790) Herbst, 1795 (Coleoptera, Curculionidae), is one of the most voracious pest of palm trees, including Phoenix canariensis Hort. ex Chabaud (Liliopsida: Phoeniceae) date palms, worldwide. It is originally from southern India, but it has currently invaded Mediterranean countries including Tunisia,
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