Xenorhabdus bovienii subsp. africana subsp. nov., isolated from Steinernema africanum entomopathogenic nematodes.

Four Gram-negative bacterial strains isolated from Steinernema africanum entomopathogenic nematodes were biochemically and molecularly characterized to determine their taxonomic position. Results of 16S rRNA gene sequencing indicated that they belong to the class Gammaproteobacteria, family Morganellaceae, genus Xenorhabdus, and that they are conspecific. The average 16S rRNA gene sequence similarity between the newly isolated strains and the type strain of its more closely related species, Xenorhabdus bovienii T228T, is 99.4 %. We therefore selected only one of them, XENO-1T, for further molecular characterization using whole genome-based phylogenetic reconstructions and sequence comparisons. Phylogenetic reconstructions show that XENO-1T is closely related to the type strain of X. bovienii, T228T, and to several other strains that are thought to belong to this species. To clarify their taxonomic identities, we calculated average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values. We observed that the ANI and dDDH values between XENO-1T and X. bovienii T228T are 96.3 and 71.2 %, respectively, suggesting that XENO-1T represents a novel subspecies within the X. bovienii species. Noteworthy, the dDDH values between XENO-1T and several other X. bovienii strains are between 68.7 and 70.9 % and ANI values are between 95.8 and 96.4 %, which could be interpreted, in some instances, as that XENO-1T represents a new species. Considering that for taxonomic description the genomic sequences of the type strains are compared, and to avoid future taxonomic conflicts, we therefore propose to assign XENO-1T to a new subspecies within X. bovienii. ANI and dDDH values between XENO-1T and any other of the species with validly published names of the genus are lower than 96 and 70 %, respectively, supporting its novel status. Biochemical tests and in silico genomic comparisons show that XENO-1T exhibit a unique physiological profile that differs from all the Xenorhabdus species with validly published names and from their more closely related taxa. Based on this, we propose that strain XENO-1T represents a new subspecies within the X. bovienii species, for which we propose the name X. bovienii subsp. africana subsp. nov, with XENO-1T (=CCM 9244T=CCOS 2015T) as the type strain.

Steinernema Africanum n. Sp. (Rhabditida, Steinernematidae), a New Entomopathogenic Nematode Species Isolated in the Republic of Rwanda.

Alternatives to hazardous insecticides are urgently needed for an environmentally friendly and effective management of insect pests. One such option is the use of entomopathogenic nematodes (EPN). To increase the availability of EPN with potential for biocontrol, we surveyed agricultural soils in the Republic of Rwanda and collected two Steinernema isolates. Initial molecular characterization showed that they represent a new species, for which we propose the name S. africanum n. sp. To describe this new species, we reconstructed phylogenetic relationships, calculated sequence similarity scores, characterized the nematodes at the morphological level, conducted crossing experiments, and isolated and characterized their symbiotic bacteria. At the molecular level, S. africanum n. sp. is closely related to S. litorale and S. weiseri. At the morphological level, S. africanum n. sp. differs from closely related species by the position of the nerve ring and also because the stoma and pharynx region is longer. The first-generation males have ventrally curved spicules with lanceolate manubrium and fusiform gubernaculum and the second-generation males have rounded manubrium and anteriorly hook-like gubernaculum. Steinernema africanum n. sp. does not mate or produce fertile progeny with any of the closely related species.

Multi-locus phylogenetic analyses uncover species boundaries and reveal the occurrence of two new entomopathogenic nematode species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp.

Species of the nematode genus Heterorhabditis are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on phylogenetic reconstructions based on a few genetic markers with little resolutive power, specially of closely related species. To fill this knowledge gap, we sequenced several phylogenetically relevant genetic loci and used them to reconstruct phylogenetic trees, to calculate sequence similarity scores, and to determine signatures of species- and population-specific genetic polymorphism. In addition, we revisited the current literature related to the description, synonymisation, and declaration as species inquirendae of Heterorhabditis species to compile taxonomically relevant morphological and morphometric characters, characterized new nematode isolates at the morphological and morphometrical level, and conducted self-crossing and cross-hybridization experiments. The results of this study show that the sequences of the mitochondrial cytochrome C oxidase subunit I (COI) gene provide better phylogenetic resolutive power than the sequences of nuclear rRNA genes and that this gene marker can phylogenetically resolve closely related species and even populations of the same species with high precision. Using this gene marker, we found two new species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp. A detailed characterization of these species at the morphological and morphometric levels and nematode reproduction assays revealed that the threshold for species delimitation in this genus, using COI sequences, is 97% to 98%. Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture.

Entomopathogenic nematodes as an effective and sustainable alternative to control the fall armyworm in Africa.

The recent invasion of the fall armyworm (FAW), a voracious pest, into Africa and Asia has resulted in unprecedented increases in insecticide applications, especially in maize cultivation. The health and environmental hazards posed by these chemicals have prompted a call for alternative control practices. Entomopathogenic nematodes are highly lethal to the FAWs, but their application aboveground has been challenging. In this study, we report on season-long field trials with an innocuous biodegradable gel made from carboxymethyl cellulose containing local nematodes that we specifically developed to target the FAW. In several Rwandan maize fields with distinct climatic conditions and natural infestation rates, we compared armyworm presence and damage in control plots and plots that were treated with either our nematode gel formulation, a commercial liquid nematode formulation, or the commonly used contact insecticide cypermethrin. The treatments were applied to the whorl of each plant, which was repeated three to four times, at 2-week intervals, starting when the plants were still seedlings. Although all three treatments reduced leaf damage, only the gel formulation decreased caterpillar infestation by about 50% and yielded an additional ton of maize per hectare compared with untreated plots. Importantly, we believe that the use of nematodes can be cost-effective, since we used nematode doses across the whole season that were at least 3-fold lower than their normal application against belowground pests. The overall results imply that precisely formulated and easy-to-apply nematodes can be a highly effective, affordable, and sustainable alternative to insecticides for FAW control.

Comparative Screening of Mexican, Rwandan and Commercial Entomopathogenic Nematodes to Be Used against Invasive Fall Armyworm, Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae) is an important pest of maize originating from the Americas. It recently invaded Africa and Asia, where it causes severe yield losses to maize. To fight this pest, tremendous quantities of synthetic insecticides are being used. As a safe and sustainable alternative, we explore the possibility to control FAW with entomopathogenic nematodes (EPN). We tested in the laboratory whether local EPNs, isolated in the invasive range of FAW, are as effective as EPNs from FAW native range or as commercially available EPNs. This work compared the virulence, killing speed and propagation capability of low doses of forty EPN strains, representing twelve species, after placing them with second-, third- and sixth-instar caterpillars as well as pupae. EPN isolated in the invasive range of FAW (Rwanda) were found to be as effective as commercial and EPNs from the native range of FAW (Mexico) at killing FAW caterpillars. In particular, the Rwandan Steinernema carpocapsae strain RW14-G-R3a-2 caused rapid 100% mortality of second- and third-instar and close to 75% of sixth-instar FAW caterpillars. EPN strains and concentrations used in this study were not effective in killing FAW pupae. Virulence varied greatly among EPN strains, underlining the importance of thorough EPN screenings. These findings will facilitate the development of local EPN-based biological control products for sustainable and environmentally friendly control of FAW in East Africa and beyond.