Detection of specific endosymbionts of mealybugs infesting cassava (Manihot esculenta Crantz) using diagnostic PCR and Sanger sequencing
Keywords:
cassava mealybug, insecticide resistant endosymbiotic bacteria, Paracoccus marginatus, Ferrisia virgata, diagnostic PCRAbstract
Cassava is one of the most produced food materials and constitutes the major energy source for about 800 million people worldwide. Mealybugs have been reported to be the main concern in cassava production for the past few years in all the major tuber cultivated ecosystems of India. Endosymbiotic bacteria found in mealybugs have been observed to interact and provide specific advantages to the host and they can be one of the reasons for the pests’ insecticidal resistance. In the present study, endosymbiotic bacteria were isolated from cassava mealybugs, allowed to grow in media after insecticidal treatments, and after observing colony morphology, distinct bacterial colonies were sub-cultured and selected for further molecular identification procedures. PCR analysis was carried out for bacteria using 16S rRNA primer, with an annealing temperature of 49o C, and yielded fragments at 1500 bp. Based on the sequencing report, the observations were that the culturable endosymbionts Pseudomonas oryzihabitans, Staphylococcus sp. which are resistant to the insecticide thiamethoxam are present in Paracoccus marginatus, and malathion resistant Paenibacillus alvei, Ralstonia sp., and thiamethoxam resistant Clostridium lundense are present in Ferrisia virgata. The sequences were submitted to NCBI, and the accession numbers obtained were OP572218, OP572193, OP572215, OP572194, and OP572097, respectively. Also, through diagnostic PCR using specific bacterial primers, the presence of Wolbachia is confirmed in mealybug F. virgata at a fragment length of 650 bp.References
Abaca, A., Kiryowa, M., Awori, E., Andema, A., Dradiku, F., Moja, A.S. and Mukalazi J. 2014. Cassava pests and diseases’ prevalence and performance as revealed by adaptive trial sites in North Western AgroEcological Zone of Uganda. J. Agric. Sci., 6(1), 116–122.
Abinaya, K., Kennedy, J.S. and Anandham, R. 2019. Biochemical characterization of culturable bacterial endosymbionts of papaya mealybug, Paracoccus marginatus Williams and Granara de Willink (Hemiptera: Pseudococcidae) from papaya plants. J. Entomol. Zool. Stud., 7(5), 1324–1327.
Anderson, K.E., Sheehan, T.H., Eckholm, B.J., Mott, B.M. and Degrandi–Hoffman, G. 2011. An emerging paradigm of colony health: Microbial balance of the honey bee and hive (Apis mellifera). Insectes Soc., 58(4), 431–444.
FAO, 2013. Save and Grow: Cassava. A guide to sustainable production intensification, Rome, 1–129.
Anusree Padmanabhan, P.S., Chellappan, M. and Kadarkutty, H.M. 2019. Characterization of endosymbionts of cotton mealybug (Phenacoccus solenopsis Tinsley) on okra using metagenomics approach. Int. J. Chem. Stud., 7(1), 744–750.
Baumann, P. 2005. Biology of bacteriocyte-associated endosymbionts of plant sap-sucking insects. Annu. Rev. Microbiol., 59(1), 155–189.
Bi, J. and Wang, Y.F. 2020. The effect of the endosymbiont Wolbachia on the behavior of insect hosts. Insect Sci., 27(5), 846–858.
Ceballos, H., Hershey, C. and Becerra-López-Lavalle, L.A. 2012. New approaches to cassava breeding. Plant Breed. Rev., 36(1), 427–504.
Downie, D.A. and Gullan, P.J. 2005. Phylogenetic congruence of mealybugs and their primary endosymbionts. J. Evol. Biol., 18(2), 315–324.
Engel, P. and Moran, N.A. 2013. The gut microbiota of insects–diversity in structure and function. FEMS Microbiol. Rev., 37(5), 699–735.
Fukatsu, T. and Nikoh, N. 2000. Endosymbiotic microbiota of the bamboo pseudococcid Antonina crawii (Insecta, Homoptera). Appl. Environ. Microbiol., 66(2), 643–650.
García, M., Denno, B., Miller, D.R., Miller, G.L. and Ben-Dov, Y. 2016. ScaleNet: A literature-based model of scale insect biology and systematics. Available from: http://scalenet.info
Gupta, A., Sinha, D.K. and Nair, S. 2022. Shifts in Pseudomonas species diversity influence adaptation of brown planthopper to changing climates and geographical locations. Iscience, 25(7), 104550.
Harish, E.R., Jayaprakas, C.A., Jaganathan, D., KesavaKumar, H. and Muthuraj, R. 2022. Integrated management of mealybugs in cassava. Technical leaflet, ICAR-central Tuber Crops Research Institute, Thiruvananthapuram, Kerala.
Hashim, H.S., Al-Janabi, A.O.H. and Al Shammari, A.O.S. 2022. Efficiency of Pseudomonas aeruginosa in bioremediation of chlorpyrifos toxicity. Nat. Volatiles Essent. Oils, 9(1), 915–921.
Hedges, L.M., Brownlie, J.C., O'Neill, S.L. and Johnson, K.N. 2008. Wolbachia and virus protection in insects. Science, 322(5902), 702.
Hodges, A. 2017. Striped Mealybug Ferrisia virgata Cockerell (Insecta: Hemiptera: Pseudococcidae): EENY-674/IN1164, 2/2017. EDIS 2017(1), 4–4.
Hosokawa, T., Koga, R., Kikuchi, Y., Meng, X.Y. and Fukatsu, T. 2010. Wolbachia as a bacteriocyte-associated nutritional mutualist. Proc. Natl. Acad. Sci. U.S.A., 107(2), 769–774.
Husnik and Filip. 2016. Wolbachia from Maconellicoccus hirsutus mealybugs. figshare. Dataset. Available from: https://doi.org/10.6084/m9.figshare.2010390.v1.
Ibrahim, S., Gupta, R.K., War, A.R., Hussain, B., Kumar, A., Sofi, T., Noureldeen, A. and Darwish, H. 2021. Degradation of chlorpyriphos and polyethylene by endosymbiotic bacteria from citrus mealybug. Saudi J. Biol. Sci., 28(6), 3214–3224.
Indiragandhi, P., Yoon, C., Yang, J.O., Cho, S., Sa, T.M. and Kim, G.H. 2010. Microbial communities in the developmental stages of B and Q biotypes of sweet potato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae). J. Korean Soc. Appl. Biol. Chem., 53(5), 605–617.
Jose, P.A., Krishnamoorthy, R., Gandhi, P.I., Senthilkumar, M., Jamahiraman, V., Kumutha, K., Choudhury, A.R., Samaddar, S., Anandham, R. and Sa, T. 2020. Endomicrobial community profiles of two different mealybugs: Paracoccus marginatus and Ferrisia virgata. J. Microbiol. Biotechnol., 30(7), 1013–1017.
Karthikeyan, C., Patil, B.L., Borah, B.K., Resmi, T.R., Turco, S., Pooggin, M.M., Hohn, T. and Veluthambi, K. 2016. Emergence of a Latent Indian Cassava Mosaic Virus from Cassava which recovered from infection by a non- persistent Sri Lankan Cassava Mosaic Virus. Viruses, 8(10), 264.
Kikuchi, Y., Hayatsu, M., Hosokawa, T., Nagayama, A., Tago, K. and Fukatsu, T. 2012. Symbiont-mediated insecticide resistance. Proc. Natl. Acad. Sci. U.S.A., 109(22), 8618–8622.
Leontopoulos, S.V., Gowen, S.R., Topalidou, E., Vagelas, I.K. and Gravanis, F.T. 2011. Pseudomonas oryzihabitans suppresses damage caused by root-knot nematode Meloidogyne javanica on tomato. J. Agric. Sci. Technol. U.S.A., 5(4), 502–507.
Lin, D., Zhang, L., Shao, W., Li, X., Liu, X., Wu, H. and Rao, Q. 2019. Phylogenetic analyses and characteristics of the microbiomes from five mealybugs (Hemiptera: Pseudococcidae). Ecol. Evol., 9(4), 1972–1984.
Liu, X.D. and Guo, H.F. 2019. Importance of endosymbionts Wolbachia and Rickettsia in insect resistance development. Curr. Opin. Insect Sci., 33(1), 84–90.
Mamoon-ur-Rasheed., Bushra, S. and Tariq, M. 2014. Use and impact of insecticides in mealybug control. Int. J. Adv. Biol., 1(2), 1–11.
Megaladevi, P., Kennedy, J.S., Jeyarani, S., Nakkeeran, S. and Balachandar, D. 2020. Metagenomic exploration of the bacterial endosymbiotic microbiome diversity of papaya mealybug Paracoccus marginatus from different host plants. J. Entomol. Zool. Stud., 8(1), 429–439.
Menpara, D. and Chanda, S. 2013. Endophytic bacteria-unexplored reservoir of antimicrobials for combating microbial pathogens. Microbial pathogens and strategies for combating them: Science, Technology and Education, 1095–1103.
Mergaert, P. 2018. Role of antimicrobial peptides in controlling symbiotic bacterial populations. Nat. Prod. Rep., 35(4), 336–356.
Meyerdirk, D.E., Muniappan, R., Warkentin, R., Bamba, J. and Reddy, G.V.P. 2004. Biological control of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae) in Guam. Plant Prot. Q., 19(3), 110–114.
Muniappan, R., Meyerdirk, D.E., Sengebau, F.M., Berringer, D.D. and Reddy, G.V.P. 2006. Classical biological control of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae) in the Republic of Palau. Fla. Entomol., 89(2), 212–217.
Muniappan, R., Shepard, B.M., Watson, G.W., Carner, G.R., Sartiami, D., Rauf, A. and Hammig, M.D. 2008. First report of the papaya mealybug, Paracoccus marginatus (Hemiptera: Pseudococcidae), in Indonesia and India. J. Agric. Urban Entomol., 25(1), 37–40.
Nikoh, N., Hosokawa, T., Moriyama, M., Oshima, K., Hattori, M. and Fukatsu, T. 2014. Evolutionary origin of insect–Wolbachia nutritional mutualism. Proc. Natl. Acad. Sci., 111(28), 10257–10262.
Oliveira, M.D., Barbosa, P.R.R., Silva-Torres, C.S.A. and Torres, J.B. 2014. Performance of the striped mealybug Ferrisia virgata Cockerell (Hemiptera: Pseudococcidae) under variable conditions of temperature and mating. Neotrop. Entomol., 43(1), 1–8.
Popova, T.P., Trencheva, K.G. and Tomov, R.I. 2016. Investigation on the microflora of the longtailed mealybug Pseudococcus longispinus (Targioni-Tozzetti) (Hemiptera: Pseudococcidae) in order to assess its importance as a carrier of pathogenic microorganisms. Bulg. J. Agric. Sci., 22(1), 103–107.
Rogowska-van der Molen, M.A., Nagornîi, D., Coolen, S., de Graaf, R.M., Berben, T., van Alen, T., Janssen, M.A., Rutjes, F.P., Jansen, R.S. and Welte, C.U. 2022. Insect gut isolate Pseudomonas sp. strain Nvir degrades the toxic plant metabolite nitropropionic acid. Appl. Environ. Microbiol., 88(19), 719–722.
Saati-Santamaría, Z., Rivas, R., Kolařik, M. and García-Fraile, P. 2021. A new perspective of Pseudomonas—host interactions: distribution and potential ecological functions of the genus Pseudomonas within the Bark Beetle Holobiont. Biology, 10(2), 164.
Sambrook, J., Fritsch, E.R. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Singh, S.T., Priya, N.G., Kumar, J., Rana, V.S., Ellango, R., Joshi, A., Priyadarshini, G., Asokan, R. and Rajagopal, R. 2012. Diversity and Phylogenetic analysis of endosymbiotic bacteria from field caught Bemisia tabaci from different locations of North India based on 16SrDNA library screening. Infect. Genet. Evol., 12(2), 411–419.
Sousa, A.M., Machado, I., Nicolau, A. and Pereira, M.O. 2013. Improvements on colony morphology identification towards bacterial profiling. J. Microbiol. Methods, 95(3), 327–335.
Sreerag, R.S., Jayaprakas, C.A., Ragesh, L. and Kumar, S.N. 2014. Endosymbiotic bacteria associated with the mealy bug, Rhizoecus amorphophalli (Hemiptera: Pseudococcidae). Int. Sch. Res. Notices, 2014(2), 8.
Subramanian, S., Sagar, D. and Rajna, S. 2019, Genotyping of whitefly species complex and its associated Endosymbionts- A workshop Manual, ICAR-Indian Agricultural Research Institute, New Delhi, 22.
Teixeira, L., Ferreira, Á. and Ashburner, M. 2008. The bacterial symbiont Wolbachia induces resistance to RNA viral infections in Drosophila melanogaster. PLoS Biol., 6(12), e1000002.
Thao, M.L., Gullan, P.J. and Baumann, P. 2002. Secondary (gamma-Proteobacteria) endosymbionts infect the primary (beta-Proteobacteria) endosymbionts of mealybugs multiple times and coevolve with their hosts. Appl. Environ. Microbiol., 68(7), 3190–3197.
Qamer, S., Sandoe, J.A. and Kerr, K.G. 2003. Use of colony morphology to distinguish different enterococcal strains and species in mixed culture from clinical specimens. J. Clin. Microbiol., 41(6), 2644–2646.
Von Dohlen, C.D., Kohler, S., Alsop, S.T. and McManus, W.R. 2001. Mealybug beta-proteobacterial endosymbionts contain gamma-proteobacterial symbionts. Nature, 412(6845), 433–436.
White, J.A., Kelly, S.E., Perlman, S.J. and Hunter, M.S. 2009. Cytoplasmic incompatibility in the parasitic wasp Encarsia inaron: Disentangling the roles of Cardinium and Wolbachia symbionts. Heredity, 102(5), 483–489.
Xue, X., Li, S.J., Ahmed, M.Z., De Barro, P.J., Ren, S.X. and Qiu, B.L. 2012. Inactivation of Wolbachia reveals its biological roles in whitefly host. PLoS One, 7(10), e48148.
Downloads
Published
How to Cite
Issue
Section
License
Copyright © 2001-11 Kerala Agricultural University. Some rights reserved. This journal and the individual contributions contained in it are protected under copyright by the Kerala Agricultural University and the following terms and conditions apply to their use; Photocopying Single photocopies of single articles may be made for personal use as allowed by national copyright laws. Permission of the publisher and payment of a fee is required for all other photocopying including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Permission may be sought directly from the Editor, Journal of Tropical Agriculture, Department of Agronomy, College of Horticulture,, Kerala Agricultural University, KAU PO, Thrissur 680 656, Kerala, India. (Phone: +91-487-2438325; Fax +91-487-2371040; E-mail: editor.jta@kau.in. Derivative works Subscribers may reproduce tables of contents or prepare lists of articles including abstracts for internal circulation within their institution. Permission of the Publisher is required for resale or distribution outside the institution. Permission of the Publisher is required for all other derivative works, including compilations and translations. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products, liability, negligence, or otherwise, or from any use of any methods, products, instructions or ideas contained in the material herein.