Cloning Of Carbendazim-Resistant Gene Tub2 From Mango Anthracnose And Its Transfer To Metarhizium Anisopliae Mediated By Agrobacterium Tumefaciens

Mango anthracnose is a serious disease caused by Colletotrichum gloeosporioides which is prevalent in tropical regions of China. The disease can be well controlled by benzimidazoles fungicides such as carbendazim, thiophanate-methyl, benomyl, thiabendazole, etc. However, single-site mutation occurred commonly leading to resistance to the fungicide, especially when the fungicide was applied indiscriminately.During the period 2003 to 2006 more than 300 isolates of C. gloeosporioides from mango-growing regions in China were tested for resistance to carbendazim. The results showed that resistance to the fungicide had developed in the field. The resistant isolates were able to grow normally on PDA medium with carbendazim at a high level of 500μg/mL.The resistance to carbendazim was stable and inherited unchanged through 12 consecutive generations on carbendazim-free PDA medium. There was no apparent correlation between biological characteristics of the fungal isolates with carbendazim-resistance .Numerous studies showed that single base-pair mutation of ?-tubulin gene especially at 198 amino acid of the tub2 was closely correlated with carbendazim-resistance. The genes of tub1 and tub2 of resistant(ZR46,ZR43,ZR51) and sensitive(ZS19,ZS29) strains were cloned, sequenced and aligned. The results showed a few base-pair mutations in tub1 and tub2 genes. No definite correlation between mutation and fungicide resistance could be detected in tub1 gene from resistant isolates. However, for tub2 gene, mutation of the amino acid in codon 198 resulted in the formation of Ala-A in all resistant isolates and Glu-A in all sensitive isolates. The mutation of amino acid 198 was detected by allele-specific PCR and enzyme assays and similar results were obtained in other resistant isolates. The results strongly suggested that amino acid change in codon 198 of tub2 played a leading role in conferring carbendazim-resistance to mango anthracnose in South China. Furthermore, the resistant gene of tub2 was cloned and used to construct fungicide-resistant strains of Metarhizium anisopliae, an entomopathogenic fungus.Species of Metarhizium have been proved effective in controlling more than 200 species of insect pests, and was widely used due to its harmless effect on human, animals, plants and other insects. In our study, 21 strains of Metarhizium were isolated from more than 200 cadavers of the host, Brontispa longissima Gestro adults and lavae in Wenchang and Haikou of Hainan. These isolates were identified initially as M. anisopliae based on the colony color, mycelial texture, and the characteristics of hyphae, conidiophores, conidia and conidial fructification. Two different groups of isolates (MA and MB) could be distinguished only based on their colony characters. The ITS1-5.8S-ITS2 of rDNA from MA and MB strains were cloned and sequenced for identification at the species level. The maximum parsimony tree and neighbor-joining tree were established using the software of MEGA3.1.The results showed that both MA and MB strains should be assigned to Metarhizium anisopliae var. anisopliae. These strains were highly pathogenic to adults and larvae of coconut hispid beetles.However, there were some environmental factors diminishing the effectiveness of Metarhizium in the field, such as temperature, UV light, insecticides and especially fungicides. So it was essential to construct fungicide-resistant strains of the fungus.The sensitivity of all 21 isolates of M. anisoplieae var. anisopliae to the fungicide, carbendazim was tested. The results showed that these isolates were highly sensitive (MIC<5μg/mL) to carbendazim. It would not be practical trying to screen for resistant strains from the field because of the scarcity of cadavers of hispid beetles. Only a few were collected from millions of thousands of coconut palms on Hainan island.Alteratively, UV irradiation was considered a good method to improve the resistance of the isolates. In our study several carbendazim-resistant stains were obtained by UV- induced mutation. Some strains could grow slowly on PDA agar medium even with a high concentration of carbendazim (600μg/mL) .Thus the resistant level had increased 120 times when compared with the parental strains. However, reverse-mutation occurred when these"resistant strains"were illuminated again with UV light. So these strains with unstable resistance could not be used in the field due to the presence of UV in the sunlight.Another approach was to transform the sensitive strains of Metarhizium with the transfer of a carbendazim-resistant gene(tub2) mediated by a bacterium, Agrobacterium tumefaciens. The binary vector pTUB2 was constructed based on the vector pCAMBIA1300, promotor TrpC cloned from vector pBHt2 and carbendazim-resistant gene tub2 cloned from resistant isolates of Colletotrichum gloeosporioides. TrpC-tub2 cassette was amplified by Gene Splicing by Overlap Extension(gene SOEing) of PCR. The hph gene cassette of pCAMBIA1300 was deleted by Bst XI/Xho I digestion,and its large line fragment was ligated with TrpC-tub2 into vector of pTUB2 using the blunt-end because it contained Bst XI and Xho I in the tub2 sequence.An enzyme assay was employed to detect the ligation direction. Then the pTUB2 was mobilized into A. tumefaciens (strain GV3103).In our study, a highly efficient and stable transformation system of Metarhizium was established by optimizing the A. tumefaciens-mediated procedure. Firstly, the effects of conidial concentration on transformation efficiency was tested, using a 2 day co-cultivation in the presence of 200μmol/L AS. Secondly, A. tumefaciens incubated for 6h in broth IM medium with 450μmol/L AS before co-cultivation resulted in highest transformation efficiency. Thirdly, A. tumefaciens (OD600:0.60-0.75) broth culture and Metarhizium spore suspension (1×10~6~-1×10~8conidial/mL) were mixed in equal volumes, and 0.01% Triton X-100 was added to dissolve the conidia. Fourthly, maximum efficiency was observed with the carbendazim at 4μg/mL. The transformation efficiency decreased with the increase of the concentration of carbendazim and there was no transformation when the fungicide used was at 10μg/mL.More than 200 transformants were compared with the parental strains. Differences in the colony color, production and morphology of the spores as well as decreased virulence were found. It is quite feasible that T-DNA was inserted into the functional DNA region of Metarhizium. The transformation was detected by PCR and the fragments were sequenced. The results proved that the tub2 cassette gene had been inserted into the genome of Metarhizium. Thus, the transformation of Metarhizium by the insertion of T-DNA could be readily performed on a large scale using the optimized procedure.The sensitivity of the transformants to carbendazim was tested. The results showed that the MIC was <10μg/mL and the resistant level increased only 2.8 times when compared with the parental strains. Nevertheless, the acquired resistance was stable and inherited unaltered through 12 consecutive generations on PDA medium. It is very likely that tub2 gene expressed only a low-level resistance after being inserted into the genome of Metarhizium. However, with the transformation vector established successfully and the transformation system optimized, a library of T-DNA mediated mutants can be readily obtained, providing a firm foundation for the investigation of fungicide-resistance and development in M. anisopliae.