| Mango(Mangifera indica L.)is an important economic crop in the tropics,and Hainan Province is the main producing area of Mango in China.Benzimidazole fungicides are prone to resistance due to the single characteristics of the action sites and the high frequency of application of these drugs in Hainan.On the basis of previous studies,3 strains of MBC-S,4 strains of MBC-MR and 5 strains of MBC-HR were selected,and the genetic stability of strain resistance,sensitivity to temperature,sensitivity of hypertonic solution,mycelium conductivity and related enzyme activity were tested,with the aim of exploring the possible resistance characteristics of strains and the biochemical mechanism of resistance.Cloning and analysis of the beta-tubulin protein gene sequence of the strains of the resistant carbendazim Botryodiplodia theohromae,and the relationship between the expression of beta-tubulin protein gene and the sensitivity of carbendazim,and to explore the molecular mechanism of resistance;According to the point mutation of the 198th and 200th codons of the beta-tubulin protein gene in MBC-HR and MBC-MR strains,the rapid detection technique of resistance of Botryodiplodia theobromae to carbendazim by PCR Primer LYT-R/LYT-Z and YT-R1/YT-Z1 was designed according to ASO-PCR method.The results show:1.The resistance of 12 strains to carbendazim Botryodiplodia theobromae can stabilize heredity,high temperature and low temperature will inhibit the growth of strains,antimicrobial strains have low temperature sensitivity.The growth of mycelium of 12 strains was first promoted and then inhibited with the increase of glucose and NaC1 concentration,and the high salt environment of MBC-S strain was more sensitive than that of MBC.-HR and MBC-MR strains.2.The relative conductivity of 12 strains showed a tendency to rise first and then tend to flatten.After treatment of the carbendazim of 50 mg/L,the relative conductivity of sensitive strains increased obviously and was always greater than that of resistant strains,which also indicated that sensitive strains were sensitive to carbendazim and serious leakage of substances in cells damaged by niedicaments.The content of glycerin in the mycelium of 12 strains was determined,and the results showed that there was no difference in glycerin content in the mycelium of the 12 strains.3.There were significant differences in CAT enzyme activity in mycelium of 12 strains,and the enzyme activity in resistant strains was higher,and CAT enzyme activity in high resistant strains was about 4 times that of sensitive strains.The activity of GST enzyme in resistant strains was also higher than that of sensitive strains,and there was no significant difference in POD enzyme activity of resistant strains.4.Cloning the comparison of the gene sequences of beta-tubulin protein,the 198th amino acid of MBC-HR high resistant strains,the codon glutamate GAG(Glu)mutated into alanine GCG(Ala),and the base mutation occurred.The 200th amino acid codon 200th TTC(Phe)of the resistant strains in MBC-MR became tyrosine TAC(Tyr).The cDNA length of the beta-tubulin protein gene sequence was 447 amino acids,and the relative expression of beta-tubulin protein in MBC-HR high antibacterial plant was greater than that of MBC-S sensitive strain in MBC-MR.5.The ASO-PCR method designed the PCR primer LYT-R/LYT-Z and YT-R1/YT-Z1.Primers LYT-R and LYT-Z amplified a product ofthe expected size(700 bp)only from MBC-MR strains,Primers YT-R1 and YT-Z1 amplified a 700 bp fragment only from MBC-HR strains,.Conclusion:Resistant Botryodiplodia theobromae to carbendazim of beta-tubulin protein gene occurrence point mutation,resistance strains have stability and high suitabilityr high resistance risk.In addition to gene mutations,the permeability and enzyme activity of strains in cell membranes are related to the mechanism of resistance. |
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