| Gummy stem blight( GSB) caused by Didymella bryoniae is one of the worldwide diseases, which caused a serious loss to various areas in our country. Currently, this disease was controlled mainly with fungicide, such as Difenoconazole and Carbendazim. However, the generation of fungicide resistance will lead reduce even or lose the control effect. Up to now, it is rarely report on Didymella bryoniae in China. So in this paper, we studied the development of resistance to Difenoconazole and Carbendazim, fungicides sensitivity, resistance mechanism of Carbendazim and biological characteristics of their resistant strains in Chongqing and Guizhou. All these did to provide a theoretical reference for Carbendazim resistance management on the watermelon gummy stem blight,so as to provide a theoretical basis for the Difenoconazole medication prospects and strategies. The results are as follows:1.Sensitivity baseline and distribution of field resistance of D. bryoniae to Carbendazim.A number of 106 wild D. bryoniae isolates were collected and their sensitivity to Carbendazim was measured by the mycelial growth diameter method. It turns out to be the maximum EC50 values of these isolates was 0.9482μg/m L, the minimum EC50 values was 0.5104μg/m L. The average value of EC50 was 0.7125±0.0842μg/m L, which was used as the sensitivity baseline to evaluate the resistance of D. bryoniae to carbendazim. The minimum inhibitory concentration(MIC) values of D. bryoniae to carbendazim were measured and the values were less than or equal to 4.5 μg/m L. So, 0.7125±0.0842μg/m L was used as the sensitivity baseline. 10 、 50 and 100μg/m L carbendazim was contained, seperatly as discrimination dosages of different carbendazim-resistant. 591 isloates of Didymella bryoniae were obtained in Beibei, Wulong, Yong chuan, Hechuan of Chongqing and Xifeng, Xiuwen of Guizhou province had monitored. And the most of strains are resistant, resistance frequency is 69.2%. In 2014, Wulong with 94.7% resistance frequency is the highest region, Hechuan with 38.5% resistance frequency was the lowest. In 2015, Wulong with 98.5% resistance frequency remain the highest, Xiuwen with 2.4% changed to the lowest. The fastest growing area of resistant is Hechuan, which has 36.5% resistance frequency in 2014 but increased to 86.9% in 2015. All the 19 isolates of D. bryoniae are high-resistant and the average resistance ratio is 358. So, Carbendazim should be stopped in this field.2. Main biological characteristics of D. bryoniae to CarbendazimThe mutants were cultured on PDA medium for 8 generations and measure their EC50 values every generation. Comparing the EC50 values found the resistance can inherited stable. One of the standard to judge suitability is the viability, fecundity difference between mutants and wild strains. The study showed that resistant strains’ all the ability are stay with sensitive strains. At the same temperature, resistant strains and sensitive strains have no significant differences in viability. With the development of resistance, tolerance to temperature without change, therefore, the resistant strains adaptation to the environment has not declined.Cross-resistance always occurs in the same type fungicide, repeat using of one fungicide should be avoid. This study showed there was a positive cross-resistance between carbendzim and Thiophanate-Methyl that average resistance ratio is 14.62. However, there were no cross-resistance found among difenoconazole, tebuconazloe, diethofencarb and kresoxim-methy 、 fludioxonil 、 boscalid 、 tolclofos-methyl for which can replace to use in fields.4. The mechanism of Carbendzim to D. bryoniae.The study was going to learn mechanism of Carbendzim to D. bryoniae. And whether β-tubulin gene mutation lead to resistance. Making use of primers GK-F1(5’-TGGTGCTGGTAACAACTG-3’)and GK-R1(5’-GTCCTCGACCTCCTTCAT-3’)with40 resistance strains and 12 sensitive strains’ amplification of β- tubulin gene, we got the partial nucleotide sequences of β-tubulin. Resistance strains with sensitivity strains were compared, there have 8 mutation of nucleotide, and other 7 mutation of nucleotide without led amino acids mutation. This mutation led to a related change of amino acid from Glu to Ala at site 593 in the translated protein.5. Sensitivity baseline and distribution of field resistance of D. bryoniae to DifenoconazoleIn order to master resistance dynamics of difenoconazole to D. bryoniae in Chongqing and Guizhou, this study monitoring the resistance of all the isolated strains. The sensitivity of baseline is the basis of resistance monitoring.A number of 101 D. bryoniae isolates were collected and their sensitivity to Difenoconazole was measured using the mycelial growth diameter method. The EC50 values of these isolates were between 0.01348 to 0.17061μg/m L and the highest EC50 for the least sensitive isolates was 12.7 times higher than that for the most sensitive isolates. The average value of EC50 was 0.05691±0.002890μg/m L, which was used as the sensitivity baseline to evaluate the resistance of D. bryoniae to Difenoconazole. 15 、 50 and 100μg/m L seperatly was assigned as the dose of different Difenoconazole-resistant. It’s turned out to be most strains are sensitive, and where also have a small amount of low resistance strains and secondary resistance strains. Frequency are 0.67% and 2.03%, and have no high resistance strains.6. Main biological characteristics of D. bryoniae to DifenoconazoleOne of the standard to judge suitability is the viability, fecundity difference between mutants and normal strains. The study showed that resistant strains have significant difference with sensitive strains in viability, fecundity, virulence and so on. All the biological characteristics of resistance strains are lower than sensitive strains. At the same temperature, resistant strains and sensitive strains in viability is serious different. With the development of resistance, tolerance to temperature has great change, therefore resistant strain adaptation to the environment has declined, this also means their risk to become resistance declined.In order to study Didymella bryoniae resistant strains whether genetically stable in the absence of selective pressure, the mutants were cultured 8 generations on PDA medium and measured their EC50 values every generation. Comparing the EC50 values found the resistance can inherited stable. Cross-resistance always occurs in the same type fungicide, repeat using of one fungicide should be avoid. We tested the sensitivity of diferent fungicides to resistance strains of difenoconazole. The results of tests showed that cross-resistance of D. bryoniae to diferent fungicides existed. There was a positive cross-resistance in diethofencarb tebuconazole, and kresoxim and their average resistance ratio are 15.54, 79.4 and 63.7. However, there were no cross-resistance found among Diethofencarb, Kresoxim-methy 、 Fludioxonil 、 Boscalid 、 Tolclofos-methyl for which can replace to use in fields.Through this study, we found that resistance of Carbendazm to Didymella bryoniae is wide and serious. Carbendazm can not effectively control the occurrence of the disease any more. Therefore we should have new fungicides to replace it. For difenoconazole, most strains are sensitive and there also have little decreased sensitivity of sub-populations which resistance are not high. So, it is wise to use difenoconazole with other types of fungicides instead of itself only. In the last, the mutation of β-tubulin nucleotide led to a related change of amino acid from Glu to Ala at site 593 in the translated protein is the mechanism of Carbendzim to D. bryoniae. |
PDF Full Text Request