Sains Malaysiana 50(6)(2021): 1639-1650

http://doi.org/10.17576/jsm-2021-5006-11

New Hybrid Strains via Intraspecific Protoplast Fusion of the Entomopathogenic FungiLecanicillium spp.

(Strain Hibrid Baharu melalui Pelakuran Protoplas Intraspesies oleh Kulat Entomopatogen Lecanicilliumspp.)

LIANG LIU¹, CHUNLAI LIU², LILI YAN³, FANYANG², SHUANG WANG², XIFENG JIANG² & XINMIN LI²*

¹Heilongjiang Academy of Agricultural Sciences, Postdoctoral Programme Harbin, Heilongjiang, 150086, China

²Institute of Plant Protection (IPP), Heilongjiang Academy of Agricultural Sciences, Scientific Observing and Experimental Station of Crop Pest in Harbin, Ministry of Agriculture, Harbin, Heilongjiang, 150086, China

³School of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China

Received: 6 March 2019/Accepted: 13 October 2020

ABSTRACT

The entomopathogenic fungal genus Lecanicillium Gams and Zare (formerly classified as the species Verticillium lecanii) includes species that are highly pathogenic to many insect genera. In this study, we identified six Lecanicillium spp. isolated strains (designated as V1-V6) belonging to L. lecanii (V1, V3 and V5) and L. attenuatum (V2, V4 and V6). In addition, these strains were used to obtain new strains via protoplast fusion, and nit mutants were used for protoplast selection. Genetic recombination of the hybrid strains was determined using the random amplified polymorphic DNA (RAPD) technique. We obtained nine stable fusant strains from 176 new hybrid strains, which were termed V₁₂-10, V₁₄-3, V₁₆-4, V₂₃-6, V₂₅-8, V₃₄-14, V₃₆-5, V₄₅-16 and V₅₆-7. Morphological characteristics varied between the hybrid and parental strains. Genomic DNA analysis of the fusants also showed genetic recombination. The median lethal concentration (LC₅₀) for the fusants were lower than that for parental strains, and the median survival time (LT₅₀) for the fusants were reduced compared with that for parental strains. Thus, these results showed that we produced new, more virulent hybrid Lecanicillium spp. strains as biological control agents via intraspecific protoplast fusion.

Keywords: Hybrid strains; L. attenuatum; L. lecannii; pathogenicity; protoplast fusion

ABSTRAK

Genus kulat entomopatogenLecanicillium Gams dan Zare (dahulunya dikelaskan sebagai spesiesVerticillium lecanii) merangkumi spesies yang sangat patogen ke atas banyak genera serangga. Dalam kajian ini, kami mengenal pasti enam strain pencilanLecanicillium spp. (dinamakan sebagai V1-V6) yang merupakanL. lecanii (V1, V3 dan V5) dan L. attenuatum (V2, V4 dan V6). Selain itu, strain ini digunakan untuk memperoleh strain baharu melalui pelakuran protoplas dan mutannit digunakan untuk pemilihan protoplas. Gabungan semula genetik strain hibrid ditentukan menggunakan teknik DNA polimorfik rawak (RAPD). Sembilan strain fusant stabil diperoleh daripada 176 strain hibrid baharu yang disebut sebagai V₁₂-10, V₁₄-3, V₁₆-4, V₂₃-6, V₂₅-8, V₃₄-14, V₃₆-5, V₄₅-16 dan V₅₆-7. Ciri morfologi didapati berbeza antara strain hibrid dan induk. Analisis DNA genom fusant juga menunjukkan berlakunya gabungan semula genetik. Median kepekatan maut (LC₅₀) untuk fusant adalah lebih rendah daripada strain induk dan median masa kebolehhidupan (LT₅₀) untuk fusants berkurang berbanding strain induk. Oleh itu, keputusan ini menunjukkan bahawa kami menghasilkan strain hibridLecanicillium spp. baru yang lebih virulen serta mampu bertindak sebagai agen kawalan biologi melalui pelakuran protoplas intraspesies.

Kata kunci: Kepatogenan; L. lecannii; L. attenuatum; pelakuran protoplas; strain hibrid

REFERENCES

Abbott, R., Albach, D., Ansell, S., Arntzen, J.W., Baird, S.J., Bierne, N., Boughman, J., Brelsford, A., Buerkle, C.A., Buggs, R., Butlin, R.K., Dieckmann, U., Eroukhmanoff, F., Grill, A., Cahan, S.H., Hermansen, J.S., Hewitt, G., Hudson, A.G., Jiggins, C., Jones, J., Keller, B., Marczewski, T., Mallet, J., Martinez-Rodriguez, P., Most, M., Mullen, S., Nichols, R., Nolte, A.W., Parisod, C., Pfennig, K., Rice, A.M., Ritchie, M.G., Seifert, B., Smadja, C.M., Stelkens, R., Szymura, J.M., Vainola, R., Wolf, J.B.W. & Zinner, D. 2013. Hybridization and speciation. Journal of Evolutionary Biology 26(2): 229-246.

Aiuchi, D., Inami, K., Kuramochi, K., Koike, M., Sugimoto, M., Tani, M. & Shinya R. 2008. A new method for producing hybrid strains of the entomopathogenic fungus Verticillium lecanii (Lecanicillium spp.) through protoplast fusion by using nitrate non-utilizing (nit) mutants. Micologia Aplicada International 20(1): 1-16.

Akbari, S., Ali Safavi, S. & Ghosta, Y. 2014. Efficacy of Beauveria bassiana (Blas.) Vuill. against cabbage aphid Brevicoryne brassicae L. (Hem.: Aphididae) in laboratory condition. Archives of Phytopathology and Plant Protection 47(12): 1454-1458.

Almeida, J.E.M., Leite, L.G. & Batista Filho, A. 2019. Natural Enemies of Insect Pests in Neotropical Agroecosystems. New York: Springer. pp. 223-233.

Asi, M.R., Bashir, M.H., Afzal, M. & Imran, S. 2009. Effect of conidial concentration of entomopathogenic fungi on mortality of cabbage aphid, Brevicoryne brassicae L. Pakistan Journal of Life and Social Sciences 2(7): 175-180.

Correll, J.C., Klittich, C.R.J. & Leslie, J.F. 1987. Nitrate non-utilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology 77: 1640-1646.

Couteaudier, Y., Viaud, M. & Riba, G. 1996. Genetic nature, stability, and improved virulence of hybrids from protoplast fusion in Beauveria. Microbial Ecology 32(1): 1-10.

Dahlmann, T.A., Böhm, J., Becker, K. & Kück, U. 2015. Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains. Current Genetics 61(4): 679-683.

El-Fadly, G., Abou-Shoshah, A. & Rehan, M. 2018. Interspecific Streptomyces protoplast fusants as biological control agents. Catrina: The International Journal of Environmental Sciences 4(2): 37-44.

Enkerli, J. & Widmer, F. 2010. Molecular ecology of fungal entomopathogens: Molecular genetic tools and their applications in population and fate studies. BioControl 55(1): 17-37.

Fang, W., Lu, H.L., King, G.F. & Leger, R.J.S. 2014. Construction of a hypervirulent and specific mycoinsecticide for locust control. Scientific Reports 4(1): 7345.

Gams, W. & Zare, R. 2001. A revision of Verticillium Sect. Prostrata. III. Genetic classification. Nova Hedwigia72(3-4): 329-337.

Hasan, S., Singh, R.I. & Singh, S.S. 2011. Development of transformation system of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: hyphomycetes) based on nitrate reductase gene of Aspergillus nidulans. Indian Journal of Microbiology 51(3): 390-395.

Hassan, M.M. 2014. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.Biotechnology and Biotechnological Equipment 28(6): 1014-1023.

Hirpara, D.G. & Gajera, H.P. 2018. Molecular heterozygosity and genetic exploitations of Trichoderma inter-fusants enhancing tolerance to fungicides and mycoparasitism against Sclerotium rolfsiiSacc. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases 66: 26-36.

Inderbitzin, P., Davis, R.M., Bostock, R.M. & Subbarao, K.V. 2011. The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range. PloS ONE 6(3): e18260.

Kim, J.J., Goettel, M.S. & Gillespie, D.R. 2007. Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea. Biological Control 40(3): 327-332.

Kim, H.Y., Lee, H.B., Kim, Y.C. & Kim, I.S. 2008. Laboratory and field evaluations of entomopathogenic Lecanicillium attenuatum CNU-23 for control of green peach aphid (Myzus persicae). Journal of Microbiology and Biotechnology 18(12): 1915-1918.

Korolev, N. & Katan, T. 1997. Improved medium for selecting nitrate-nonutilizing (nit) mutants of Verticillium dahliae. Phytopathology 87(10): 1067-1070.

Kumari, N. & Thakur, S.K. 2014. Randomly amplified polymorphic DNA - A brief review. American Journal of Animal and Veterinary Sciences 9(1): 6-13.

Lalithakumari, D. 2019. Fungal Protoplast: A Biotechnological Tool. Boca Raton: CRC Press.

Lee, W.W., Shin, T.Y., Bae, S.M. & Woo, S.D. 2015. Screening and evaluation of entomopathogenic fungi against the green peach aphid, Myzus persicae, using multiple tools. Journal of Asia-Pacific Entomology 18(3): 607-615.

Leland, J. & Gore, J. 2017. Microbial Control of Insect and Mite Pests. Oxford: Academic Press. pp. 185-197.

Mitina, G.V., Kazartsev, I., Vasileva, A. & Yli Mattila, T. 2017. Multilocus genotyping based species identification of entomopathogenic fungi of the genus Lecanicillium (= Verticillium lecanii s.l.). Journal of Basic Microbiology 57(11): 950-961.

Mitina, G.V., Mikhailova, L.A. & Yli-Mattila, T. 2008. RAPD-PCR, UP-PCR and rDNA sequence analyses of the entomopathogenic fungus Verticillium lecaniiand its pathogenicity towards insects and phytopathogenic fungi. Archives of Phytopathology and Plant Protection 41(2): 113-128.

Modi, A., Gajera, B., Subhash, N. & Kumar, N. 2017. Evaluation of clonal fidelity of micropropagated date palm by random amplified polymorphic DNA (RAPD). In Date Palm Biotechnology Protocols Volume II. New York: Humana Press. pp. 81-89.

Muralidhar, R.V. & Panda, T. 2000. Fungal protoplast fusion: A revisit. Bioprocess and Biosystems Engineering 22(5): 429-431.

Nyasani, J.O., Subramanian, S., Poehling, H.M., Maniania, N.K., Ekesi, S. & Meyhöfer, R. 2015. Optimizing western flower thrips management on French beans by combined use of beneficials and imidacloprid. Insects 6(1): 279-296.

Oborník, M., Klíc, M. & Zizka, L. 2000. Genetic variability and phylogeny inferred from random amplified polymorphic DNA data reflect life strategy of entomopathogenic fungi. Canadian Journal of Botany 78(9): 1150-1155.

Parthiban, P., Baskaran, R.K.M., Chinniah, C., Ravikumar, A. & Thangavel, K. 2018. Genetic improvement of fungal pathogens. Advances in Plants and Agriculture Research 8(1): 4-9.

Patil, N.S., Patil, S.M., Govindwar, S.P. & Jadhav, J.P. 2015. Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion. Journal of Applied Microbiology 118(2): 390-398.

Peng, G. & Xia, Y. 2015. Integration of an insecticidal scorpion toxin (BjαIT) gene into Metarhizium acridum enhances fungal virulence towards Locusta migratoria manilensis. Pest Management Science 71(1): 58-64.

Reddy, S.G.E. & Sahotra, S. 2018. Multiplication of entomopathogenic fungus (Lecanicillium lecanii) on apple pomace and its toxicity against aphid (Aphis craccivora). Toxin Reviews 39(8): 1-6.

Ruiu, L. 2018. Microbial biopesticides in agroecosystems. Agronomy 8(11): 235.

Singh, H.B., Keswani, C., Ray, S., Yadav, S.K., Singh, S.P., Singh, S. & Sarma, B.K. 2015. Beauveria bassiana: Biocontrol beyond lepidopteran pests. In Biocontrol of Lepidopteran Pests. New York: Springer. pp. 219-235.

Solter, L.F., Hajek, A.E. & Lacey, L.A. 2017. Exploration for Entomopathogens. In Microbial Control of Insect and Mite Pests. New York: Elsevier Inc. pp. 13-23.

Strom, N.B. & Bushley, K.E. 2016. Two genomes are better than one: History, genetics, and biotechnological applications of fungal heterokaryons. Fungal Biology and Biotechnology 3: 4.

Vega, F.E., Goettel, M.S., Blackwell, M.S.A., Chandler, D., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzon, A., Ownley, B.H., Pell, J.K., Rangel, D.E.N. & Roy, H.E. 2009. Fungal entomopathogens: New insights on their ecology. Fungal Ecology 2(4): 149-159.

Wang, D., Deng, J., Pei, Y., Li, T., Jin, Z., Liang, L., Wang, W., Li, L. & Dong, X. 2017. Identification and virulence characterization of entomopathogenic fungus Lecanicillium attenuatumagainst the pea aphid Acyrthosiphon pisum(Hemiptera: Aphididae). Applied Entomology and Zoology 52(3): 511-518.

White, T.J., Bruns, T., Lee, S. & Taylor, J.W. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, edited by Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J. Oxford: Academic Press. pp. 315-322.

Wraight, S.P., Lopes, R.B. & Faria, M. 2017. Microbial control of mite and insect pests of greenhouse crops. In Microbial Control of Insect and Mite Pests. Oxford: Academic Press. pp. 237-252.

Wu, J.D. & Chou, J.C. 2019. Optimization of protoplast preparation and regeneration of a medicinal fungus Antrodia cinnamomea. Mycobiology 47(4): 483-493.

Xie, M., Zhang, Y.J., Zhai, X.M., Zhao, J.J., Peng, D.L. & Wu, G. 2015. Expression of a scorpion toxin gene BmKit enhances the virulence of Lecanicillium lecaniiagainst aphids. Journal of Pest Science 88(3): 637-644.

Zare, R., Gams, W. & Culham, A. 2000. A revision of Verticillium sect. Prostrata. I. Phylogenetic studies using ITS sequences. Nova Hedwigia 71: 465-480.

Zhang, Y.J., Xie, M., Zhang, X.L., Peng, D.L., Yu, W.B., Li, Q., Li, Q., Zhao, J.J. & Zhang, Z.R. 2016. Establishment of polyethylene-glycol-mediated protoplast transformation for Lecanicillium lecaniiand development of virulence-enhanced strains against Aphis gossypii. Pest Management Science 72(10): 1951-1958.

*Corresponding author; email: shengfangshi408@126.com