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.

CABI Switzerland Delémont Switzerland

Departamento de Biología Animal Biología Vegetal y Ecología Universidad de Jaén Campus 'Las Lagunillas' Jaén Spain

Department of Crop Innovations and Technology Transfer Rwanda Agriculture and Animal Resources Development Board Kigali Rwanda

Department of Plant Production Soil Science and Agricultural Engineering University of Limpopo Sovenga South Africa

Department of Soil Plant and Food Sciences University of Bari Aldo Moro Bari Italy

Experimental Biology Research Group Institute of Biology Faculty of Sciences University of Neuchâtel Neuchâtel Switzerland

Innovative Institute for Plant Health College of Agriculture and Biology Zhongkai University of Agriculture and Engineering Guangzhou China

Institute of Entomology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Fundamental and Applied Research in Chemical Ecology Institute of Biology Faculty of Sciences University of Neuchâtel Neuchâtel Switzerland

MARA CABI Joint Laboratory for Biosafety Institute of Plant Protection Chinese Academy of Agricultural Sciences Beijing China

Zobrazit více v PubMed

Abolafia J.. A low-cost technique to manufacture a container to process meiofauna for scanning electron microscopy. Microscopy Research and Technique. 2015;78:771–776. PubMed

Abolafia J.. Extracción y procesado de nematodos de muestras de suelos de cuevas y otros hábitats. Monografías Bioespeleológicas. 2022;16(1):6–17.

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J.. Basic local alignment search tool. Journal of Molecular Biology. 1990;215(3):403–410. PubMed

Askary T. H., Bhat A. H., Ahmad M. J., Chaubey A. K., Spiridonov S. E.. Steinernema feltiae (Rhabditida: Steinernematidae) from hilly areas of Kashmir valley, India with a note on its geographical distribution. Russian Journal of Nematology. 2020;28(2):99–106.

Auch A. F., Jan M.. Klenk H. P., Göker M.. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Standards in genomic sciences. 2010a;2:117–134. von. doi/10.4056/sigs.531120. PubMed PMC

Auch A. F., Klenk H. P., Göker M.. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Standards in genomic sciences. 2010b;2:142–148. doi/10.4056/sigs.541628. PubMed PMC

Bedding R. A., Akhurst R. J.. A simple technique for the detection of insect parasitic rhabditid nematodes in soil. Nematologica. 1975;21:109–110.

Bhat A. H., Chaubey A. K., Askary T. H.. Global distribution of entomopathogenic nematodes, Steinernema and Heterorhabditis. Egyptian Journal of Biological Pest Control. 2020;30:1–15.

Chevenet F., Brun C., Bañuls A. L., Jacq B., Christen R.. TreeDyn: Towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics. 2006;7:439. doi/10.1186/1471-2105-7-439. PubMed PMC

Edgar R. C.. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research. 2004;32:1792–1797. doi/10.1093/ nar/gkh340. PubMed PMC

Fallet P., Gianni L., de Machado R. A. R., Bruno P., Bernal J. S., Karangwa P., Kajuga J., Waweru B., Bazagwira D., Degen T.. Comparative screening of mexican, rwandan and commercial entomopathogenic nematodes to be used against invasive fall armyworm, Spodoptera frugiperda. Insects. 2022;13:205. PubMed PMC

Grisse A. T. de.. Rediscription ou modification de quelques techniques utilisees dans letude des nematodes phytoparasitaries. Meddelingen Rijksfauculteit landbouwweteschappen Bull. 1969;34:351–356.

Hasegawa M., Kishino H., Yano T.. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution. 1985;22:160–174. PubMed

Hunt D. J., Nguyen K. B. Hunt D. J., Nguyen K. B. Advances in Entomopathogenic Nematode Taxonomy and Phylogeny. Nematology Monographs and Perspectives 12. (Series Editors: Hunt, D.J. & Perry, R.N.) Brill; Leiden: 2016. Taxonomy and systematics. DOI

Joyce S. A., Reid A., Driver F., Curran J. Burnell A. M., Ehlers R.- U., Masson J. P. COST 812 Biotechnology: Genetics of Entomopathogenic Nematode-bacterium Complexes. Proceedings of Symposium & workshop, St. Patrick’s College, Maynooth, Co. Kildare. Ireland, Luxembourg: European Commission; 1994. Application of polymerase chain reaction (PCR) methods to identification of entomopathogenic nematodes; pp. 178–87. DG XII, pp.

Kajuga J., Hategekimana A., Yan X., Waweru B. W., Li H., Li K., Yin J., Cao L., Karanja D., Umulisa C.. Management of white grubs (Coleoptera: Scarabeidae) with entomopathogenic nematodes in Rwanda. Egyptian Journal of Biological Pest Control. 2018;28:1–13.

Kaya H. K., Stock S. P. Lacey L.A. Manual of techniques in insect pathology. Academic Press; London (England): 1997. Techniques in insect nematology; pp. 281–324. DOI

Kimura M.. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution. 1980;16:111–120. PubMed

Kumar S., Stecher G., Tamura K.. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution. 2016;33:1870–1874. PubMed PMC

Letunic I., Bork P.. Interactive tree of life (iTOL) v3: An online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Research. 2016;44:W242–W2455. doi/10.1093/nar/gkw290. PubMed PMC

Lis M., Sajnaga E., Skowronek M., Wiater A., Rachwał K.. Kazimierczak W.. Steinernema sandneri n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Poland. Journal of Nematology. 2021;53(1):1–24. &. PubMed PMC

Liu J.. Berry R. E.. Steinernema oregonensis n. sp. (Rhabditida: Steinernematidae) from Oregon, USA. Fundamental and Applied Nematology. 1996;19:375–380. &.

Ma J., Chen S., Li X., Han R., Khatri-Chhetri H. B., De Clercq P.. Moens M.. A new entomopathogenic nematode, Steinernema tielingense n. sp.(Rhabditida: Steinernematidae), from north China. Nematology. 2012;14(3):321–338. &.

Machado R. A. R., Bhat A. H., Abolafia J., Muller A., Bruno P., Fallet P., Arce C. C. M., Turlings T. C. J., Bernal J. S., Kajuga J.. 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. Journal of Nematology. 2021a;53:1–42. PubMed PMC

Machado R. A. R., Bruno P., Arce C. C. M., Liechti N., Köhler A., Bernal J., Bruggmann R., Turlings T. C. J.. Photorhabdus khanii subsp. guanajuatensis subsp. nov., isolated from Heterorhabditis atacamensis, and Photorhabdus luminescens subsp. mexicana subsp. nov., isolated from Heterorhabditis mexicana entomopathogenic nematodes. International Journal of Systematic and Evolutionary Microbiology. 2019;69:652–661. PubMed

Machado R. A. R., Muller A., Ghazal S. M., Thanwisai A., Pagès S., Bode H. B., Hussein M. A., Khalil K. M., Tisa L. S.. Photorhabdus heterorhabditis subsp. aluminescens subsp. nov., Photorhabdus heterorhabditis subsp. heterorhabditis subsp. nov., Photorhabdus australis subsp. thailandensis subsp. nov., Photorhabdus australis subsp. australis subsp. nov., and Photorhabdus aegyptia sp. nov. isolated from Heterorhabditis entomopathogenic nematodes. International Journal of Systematic and Evolutionary Microbiology. 2021b;71:4610. PubMed

Machado R. A. R., Somvanshi V. S., Muller A., Kushwah J., Bhat C. G.. Photorhabdus hindustanensis sp. nov., Photorhabdus akhurstii subsp. akhurstii subsp. nov., and Photorhabdus akhurstii subsp. bharatensis subsp. nov., isolated from Heterorhabditis entomopathogenic nematodes. International Journal of Systematic and Evolutionary Microbiology. 2021c;71:4998. PubMed

Machado R. A. R., Wüthrich D., Kuhnert P., Arce C. C. M., Thönen L., Ruiz C., Zhang X., Robert C. A. M., Karimi J., Kamali S., Ma J., Bruggmann R., Erb M.. Whole-genome-based revisit of Photorhabdus phylogeny: Proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov. International Journal of Systematic and Evolutionary Microbiology. 2018;68:2664–2681. PubMed

Maeseneer J. de., d’Herde J.. Méthodes utilisées pour l’étude des anguillules libres du sol. Revue d’Agriculture. 1963;16:441–447.

Mamiya Y.. Steinernema kushidai n. sp. (Nematoda: Steinernematidae) associated with scarabaeid beetle larvae from Shizuoka, Japan. Applied Entomology and Zoology. 1988;23:313–320.

Meier-Kolthoff J. P., Auch A. F., Klenk H. P., Göker M.. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14:60. doi/10.1186/1471-2105-14-60. PubMed PMC

Meier-Kolthoff J. P., Hahnke R. L., Petersen J., Scheuner C., Michael V., Fiebig A., Rohde C., Rohde M., Fartmann B., Goodwin L. A., Chertkov O., Reddy T., Pati A., Ivanova N. N., Markowitz V., Kyrpides N. C., Woyke T., Göker M., Klenk H. P.. Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy. Standards in Genomic Sciences. 2014;9:2. doi/10.1186/1944-3277-9-2. PubMed PMC

Nei M., Kumar S. Molecular evolution and phylogenetics. Oxford University Press.; USA: 2000.

Nguyen K. B., Stuart R. J., Andalo V., Gozel U., Rogers M. E.. Steinernema texanum n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Texas, USA. Nematology. 2007;9:379–396.

Phan K. L., Nguyen N. C., Moens M.. Steinernema sangi sp. n. (Rhabditida: Steinernematidae) trom Vietnam. Russian Journal of Nematology. 2001;9:1–7.

Půža V., Nermuť J., Konopická J., Skoková Habuštová O.. Efficacy of the applied natural enemies on the survival of colorado potato beetle adults. Insects. 2021;12:1030. PubMed PMC

Qiu L., Hu X., Zhou Y., Mei S., Nguyen K. B., Pang Y.. Steinernema akhursti sp. n. (Nematoda: Steinernematidae) from Yunnan, China. Journal of Invertebrate Pathology. 2005;90:151–160. PubMed

Seinhorst J. W.. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica. 1959;4:67–69.

Siddiqi M. R.. Studies on Discolaimus spp. (Nematoda: Dorylaimidae) from India. Journal of Zoological Systematics and Evolutionary Research. 1964;2:174–184.

Smart G. C.. Entomopathogenic nematodes for the biological control of insects. Journal of Nematology. 1995;27:529. PubMed PMC

Spiridonov S. E., Krasomil-Osterfeld K., Moens M.. Steinernema jollieti sp n. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from the American midwest. Russian Journal of Nematology. 2004;12:85–95.

Spiridonov S. E., Subbotin S. A. Hunt D.J. Nguyen K.B. Advances in entomopathogenic nematode taxonomy and phylogeny. Nematology Monographs and Perspectives 12 (Series Editors: Hunt, D.J. & Perry, R.N.) Leiden, The Netherlands: Brill; 2016. Phylogeny and phylogeography of Heterorhabditis and Steinernema; pp. 413–427. &. DOI

Sturhan D., Spiridonov S., Mráček Z.. Steinernema silvaticum sp. n. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Europe. Nematology. 2005;7:227–241. PubMed

Subbotin S. A., Sturhan D., Chizhov V. N., Vovlas N., Baldwin J. G.. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology. 2006;8:455–474.

Tamura K.. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+ C-content biases. Molecular Biology and Evolution. 1992;9:678–687. PubMed

Tarasco E., Mráček Z., Nguyen K. B., Triggiani O.. Steinernema ichnusae sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from Sardinia Island (Italy). Journal of Invertebrate Pathology. 2008;99:173–185. PubMed

Tian C. L., Zhu F., Li X. Y., Zhang J. H., Půža V., Shapiro-Ilan D., Zhao D., Liu J. W., Zhou J. J., Ding Y., Wang J. C., Ma J., Zhu X. F., Li M. H., Li J. P.. Steinernema populi n. sp. (Panagrolaimomorpha, Steinernematidae), a new entomopathogenic nematode species from China. Journal of Helminthology. 2022;96:E57. doi: 10.1017/S0022149X22000426.. PubMed DOI

URIBE-LORÍO L., MORA M., STOCK S. P.. Steinernema costaricense n. sp. and S. puntauvense n. sp. (Rhabditida: Steinernematidae), two new entomopathogenic nematodes from Costa Rica. Systematic Parasitology. 2007;68:167–182. PubMed

White G. F.. A method for obtaining infective nematode larvae from cultures. Science. 1927;66:302–303. PubMed

Yan X., Waweru B., Qiu X., Hategekimana A., Kajuga J., Li H., Edgington S., Umulisa C., Han R., Toepfer S.. New entomopathogenic nematodes from semi-natural and small-holder farming habitats of Rwanda. Biocontrol Science and Technology. 2016;26:820–834.

Yoshida M.. Steinernema litorale n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Japan. Nematology. 2004;6:819–838.

Systematics and phylogeny of the entomopathogenic nematobacterial complexes Steinernema-Xenorhabdus and Heterorhabditis-Photorhabdus

Xenorhabdus aichiensis sp. nov., Xenorhabdus anantnagensis sp. nov., and Xenorhabdus yunnanensis sp. nov., Isolated from Steinernema Entomopathogenic Nematodes

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