African Journal of Biotechnology Vol. 6 (2), pp. 143-147, 18 January 2007   

ISSN 1684–5315 © 2007 Academic Journals        

Full Length Research Paper

Cloning of a carbendazim-resistant gene from Colletotrichum gloeosporioides of mango in South China

ZHAN Ru-lin^1,2 and HUANG Jun-sheng¹*

¹State Key Laboratory of Tropical Crop Biotechnology, Institute of Environment and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China.

²Southern Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Science, Zhanjiang 524091, China.

*Corresponding Author. E-mail: H888111@126.com; Tel: 0898-66890763.

Accepted 29 September, 2006

Mango anthracnose caused by Colletotrichum gloeosporioides is an important disease and prevalent in tropical regions of China. High carbendazim (MBC)-resistant field strains were tested and collected. The fragments of tub2 were cloned, sequenced, and alignments were carried out between MBC-resistant and wild-type strains of C. gloeosporioides. The results showed that the amino acids were altered at residues 181,198, 237 and 363. All of the mutant positions were detected by allele-specific PCR. The allele-specific fragments were amplified in MBC-resistant strains by the positive primers but not in wild-type strains. On the contrary, the allele-specific fragments were amplified in wild-type strains by the negative primers but not in MBC-resistant strains. The preliminary findings proved that the point mutation occurred at amino acid codon 198 causing a change from glutamic acid (GAG) to alanine (GCG), which is closely associated with conferring MBC-resistance in the field. An enzyme assay was employed to further test the above results. It involved an Acc restriction site (CGCG) at the positions of the amino acid residues at 197 and 198 (GACGAG→GACGCG) in MBC-resistant strains, in which Acc digested a 329 bp fragment into 107 and 222 bp, while the fragments from wild-type strains remained undigested. Based on the above assays, all of the MBC-resistant and wild-type strains were detected successfully. It strongly suggested that the altered amino acid residue at position 198 played the leading role in conferring MBC-resistance in Mango anthracnose in south China.

Key words: Colletotrichum gloeosporioides, Mango, MBC-resistant gene, allele-specific PCR, enzyme assay, detection.