Skip to main content Accessibility help

The first report of Xenorhabdus indica from Steinernema pakistanense: co-phylogenetic study suggests co-speciation between X. indica and its steinernematid nematodes

  • A.H. Bhat (a1), A.K. Chaubey (a1) and V. Půža (a2)


During a survey in agricultural fields of the sub-humid region of Meerut district, India, two strains of entomopathogenic nematodes, labelled CS31 and CS32, were isolated using the Galleria baiting technique. Based on morphological and morphometric studies, and molecular data, the nematodes were identified as Steinernema pakistanense, making this finding the first report of this species from India. For the first time, we performed a molecular and biochemical characterization of the bacterial symbiont of S. pakistanense. Furthermore, a co-phylogenetic analysis of the bacteria from the monophyletic clade containing a symbiont of S. pakistanense, together with their nematode hosts, was conducted, to test the degree of nematode–bacteria co-speciation. Both isolates were also tested in a laboratory assay for pathogenicity against two major pests, Helicoverpa armigera and Spodoptera litura. The morphology of the Indian isolates corresponds mainly to the original description, with the only difference being the absence of a mucron in first-generation females and missing epiptygmata in the second generation. The sequences of bacterial recA and gyrB genes have shown that the symbiont of S. pakistanense is closely related to Xenorhabdus indica, which is associated with some other nematodes from the ‘bicornutum’ group. Co-phylogenetic analysis has shown a remarkable congruence between the nematode and bacterial phylogenies, suggesting that, in some lineages within the Steinernema / Xenorhabdus complex, the nematodes and bacteria have undergone co-speciation. In the virulence assay, both strains caused a 100% mortality of both tested insects after 48 h, even at the lowest doses of 25 infective juveniles per insect, suggesting that S. pakistanense could be considered for use in the biocontrol of these organisms in India.


Corresponding author

Author for correspondence: A.H. Bhat, E-mail:


Hide All
Akhurst, RJ (1980) Morphological and functional dimorphism in Xenorhabdus spp., bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis. Journal of General Microbiology 121, 303309.
Altschul, SF, Gish, W, Miller, W, Myers, EW and Lipman, DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403410.
Bedding, RA and Akhurst, RJ (1974) A simple technique for the detection of insect parasitic nematodes in soil. Nematologica 21, 109110.
Bhat, AH, Istkhar, , Chaubey, AK, Půža, V and San-Blas, E (2017) First report and comparative study of Steinernema surkhetense (Rhabditida: Steinernematidae) and its symbiont bacteria from subcontinental India. Journal of Nematology 49, 92102.
Boemare, NE, Akhurst, RJ and Mourant, RG (1993) DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. International Journal of Systematic Bacteriology 43, 249255.
Burnell, AM and Stock, SP (2000) Heterorhabditis, Steinernema and their bacterial symbionts – lethal pathogens of insects. Nematology 2, 3142.
Charleston, MA and Page, RDM (2002) TreeMap v2.0.2. Available at (accessed 15 October 2017).
Cimen, H, Půža, V, Nermuť, J, Hatting, J, Ramakuwela, T and Hazir, S (2016). Steinernema biddulphi n. sp., a new entomopathogenic nematode (Nematoda: Steinernematidae) from South Africa. Journal of Nematology 48, 148158.
Courtney, WD, Polley, D and Miller, VL (1955) TAF, an improved fixative in nematode technique. Plant Disease Reporter 39, 570571.
Dreyer, J, Malan, AP and Dicks, LM (2017) Three novel XenorhabdusSteinernema associations and evidence of strains of X. khoisanae switching between different clades. Current Microbiology 74, 938942.
Ferreira, T, van Reenen, CA, Tailliez, P, Pagès, S, Malan, AP and Dicks, LMT (2014) First report of the symbiotic bacterium Xenorhabdus indica associated with the entomopathogenic nematode Steinernema yirgalemense. Journal of Helminthology 90, 108112.
Gaugler, R and Kaya, HK (1990) Entomopathogenic nematodes in biological control. Boca Raton, FL, USA, CRC Press.
Hall, TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 9598.
Hunt, DJ (2007) Overview of taxonomy and systematics. pp. 2757 in Nguyen, KB and Hunt, DJ (Eds) Entomopathogenic nematodes: Systematics, phylogeny and bacterial symbionts. Nematology monographs and perspectives, vol. 5. Leiden, The Netherlands, Brill.
Hunt, DJ and Nguyen, KB (2016) Advances in entomopathogenic nematode taxonomy and phylogeny. Leiden, Brill.
Joyce, SA, Burnell, AM and Powers, TO (1994) Characterization of Heterorhabditis isolates by PCR amplification of segments of mtDNA and rDNA genes. Journal of Nematology 26, 260270.
Kalia, V, Sharma, G, Shapiro-Ilan, DI and Ganguly, S (2014) Biocontrol potential of Steinernema thermophilum and its symbiont Xenorhabdus indica against lepidopteran pests: virulence to egg and larval stages. Journal of Nematology 46, 1826.
Kaya, HK and Gaugler, R (1993) Entomopathogenic nematodes. Annual Review of Entomology 38, 181206.
Kaya, HK, Aguillera, MM, Alumai, A, Choo, HY, De la Torre, M, Fodor, A, Ganguly, S, Hazir, S, Lakatos, T and Pye, A (2006) Status of entomopathogenic nematodes and their symbiotic bacteria from selected countries or regions of the world. Biological Control 38, 134155.
Kergunteuil, A, Bakhtiari, M, Formenti, L, Xiao, Z, Defossez, E and Rasmann, S (2016) Biological control beneath the feet: a review of crop protection against insect root herbivores. Insects 7, 70. doi: 10.3390/insects7040070
Lacey, LA, Grzywacz, D, Shapiro-Ilan, DI, Frutos, R, Brownbridge, M and Goettel, MS (2015) Insect pathogens as biological control agents: back to the future. Journal of Invertebrate Pathology 132, 141.
Lee, MM and Stock, SP (2010) A multigene approach for accessing evolutionary relationships of Xenorhabdus spp. (gamma-Proteobacteria), the bacterial symbionts of entomopathogenic Steinernema nematodes. Journal of Invertebrate Pathology 104, 6774.
Nei, M and Kumar, S (2000) Molecular evolution and phylogenetics. New York, NY, Oxford University Press.
Půža, V, Chundelová, D, Nermuť, J, Žurovcová, M and Mráček, Z (2015) Intra-individual variability of ITS region in entomopathogenic nematodes (Steinernematidae: Nematoda): implications for their taxonomy. Biocontrol 60, 547554.
Rzhetsky, A and Nei, M (1992) A simple method for estimating and testing minimum evolution trees. Molecular Biology and Evolution 9, 945967.
Saitou, N and Nei, M (1987) The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406425.
Sandstrom, JP, Russel, JA, White, JP and Moran, NA (2001) Independent origins and horizontal transfer of bacterial symbionts of aphids. Molecular Ecology 10, 217228.
Seinhorst, JW (1959) A rapid method for the transfer of nematodes from fixative to anhydrous glycerine. Nematologica 4, 6769.
Shahina, F, Anis, M, Reid, AP, Rowe, J and Maqbool, MA (2001) Steinernema pakistanense sp. n. (Rhabditida: Steinernematidae) from Pakistan. International Journal of Nematology 11, 124133.
Shahina, F, Manzar, H and Tabassum, KA (2004) Symbiotic bacteria Xenorhabdus and Photorhabdus associated with entomopathogenic nematodes in Pakistan. Pakistan Journal of Nematology 22, 117128.
Shapiro-Ilan, D, Han, R and Qiu, X (2014) Production of entomopathogenic nematodes. pp. 321355 in Morales-Ramos, JA, Rojas, MG and Shapiro-Ilan, DI (Eds) Mass production of beneficial organisms. Oxford, Elsevier.
Stock, P (2015) Diversity, biology and evolutionary relationships. pp. 327 in Campos-Herrera, R (Ed.) Sustainability in plant and crop protection: ecology and applied technologies for sustainable plant and crop protection. Basel, Springer.
Tailliez, P, Laroui, C, Ginibre, N, Paule, A, Pages, S and Boemare, N (2010) Phylogeny of Photorhabdus and Xenorhabdus based on universally conserved protein-coding sequences and implications for the taxonomy of these two genera. Proposal of new taxa: X. vietnamensissp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp. tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P. temperata subsp. thracensis comb. nov. International Journal of Systematic and Evolutionary Microbiology 60, 19211937.
Tamura, K, Stecher, G, Peterson, D, Filipski, A and Kumar, S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30, 27252729.
Vrain, TC, Wakarchuk, DA, Levesque, AC and Hamilton, RI (1992) Intraspecific rDNA restriction fragment length polymorphisms in the Xiphinema americanum group. Fundamental and Applied Nematology 15, 563574.
White, GF (1927) A method for obtaining infective nematode larvae from cultures. Science 66, 302303.


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed