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 Fungi Lecanicillium spp.

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

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

Diserahkan: 6 Mac 2019/Diterima: 13 Oktober 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 entomopatogen Lecanicillium Gams dan Zare (dahulunya dikelaskan sebagai spesies Verticillium lecanii) merangkumi spesies yang sangat patogen ke atas banyak genera serangga. Dalam kajian ini, kami mengenal pasti enam strain pencilan Lecanicillium spp. (dinamakan sebagai V1-V6) yang merupakan L. 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 mutan nit 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 hibrid Lecanicillium 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

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