Baseline Sensitivity, Molecular Mechanism And Detection Of Resistance Of Penicillium Digitatum To Fungicide Azoxystrobin

Green mold caused by Penicillium digitatum Sacc. is the most important postharvest disease of citrus fruits. Fungicide treatment is still a main measure for postharvest disease control. However, the emergence and prevalence of fungicide-resistant biotype followed by the successively and intensively using of fungicides often results in the decrease of control effect. Thus, screening the new fungicides with different mode would be a necessary step for sustainable postharvest disease control of citrus. Azoxystrobin is one of respiration inhibitors, belong to QoIs. It was registed for using in postharvest citrus recently in USA. However, the resistant risk, molecular mechanism and molecular detection are remained unknown so far. In this dissertation, these issues were explored, and the resultes are presented as follows.1. Baseline sensitivity for P. digitatum to azoxystrobin: Sixty-five isolates of P. digitatum were collected from Quzhou, Hangzhou, Jinhua and Lishui in Zhejiang province during 2000 to 2006, where strobilurin fungicide azoxystrobin had never been used for green mold control of postharvest citrus. The sensitivities to azoxystrobin were assayed by the methods of conidial germination rate and mycelial growth rate. The results demonstrated that the effective concentrations to reduce germination/growth by 50% (EC₅₀) values ranged from 0.0201 to 0.2600μg/ml and 0.0053 to 0.0794μg/ml, and averaged 0.0426±0.0304μg/ml and 0.0250±0.0129μg/ml for conidial germination and mycelial growth, respectively. The distributions of frequency of EC₅₀ values of 65 isolates were unimodal for both conidial germination and mycelial growth. Thus, the mean EC₅₀ values 0.0426±0.0304μg/ml and 0.0250±0.0129μg/ml of 65 isolates could be used as the baseline sensitivities for conidial germination and mycelial growth of P. digitatum to azoxystrobin, respectively.2. The resistant risk of P. digitatum to azoxystrobin: Five azoxystrobin-resistant spontaneous mutants and 23 UV-induced azoxystrobin-resistant mutants were screened in the azoxystrobin-amended PDA. The frequencies for spontaneous mutation and UV-induced mutation were 2.50×10^-7 and 1.91×10^-7 (20W UV, 30cm, 60～90s) respectively. All the azoxystrobin-resistant mutants were stable for azoxystrobin resistance. The MIC (minimum inhibition concentration) to these mutants was higher than 1000μg/ml, 1000 times higher of parental biotype of P. digitatum. In vitro test of adaptability indicated by mycelial growth and conidial production demonstrated that 2 of 5 spontaneous mutants and 3 of 23 UV-induced mutants had the equal adaptability of those of parental biotype. Comparison the products of PCR and RT-PCR of the first mutation hot spot of cytochrome b in P. digitatum (Pdcytb) demonstrated that there is no intron presence in this region. Thus, we suggest that P. digitatum has a high risk to form a population of azoxystrobin-resistance.3. Molecular mechanism of azoxystrobin-resistance: Partial sequences of Pdcytb gene covering the first hot mutation spot (aa120-160) were amplified and sequenced for all azoxystrobin-resistant mutants as well as the parental isolate. Alignment of these sequence indicated that a single nucleotide in the regions of Pdcytb for parental isolate is G, while it is C for all azoxytrobin-resistant mutants, suggesting that the nucleotide change from G to C, which resulting in aa143 from Glycine to Alanine (G143A), is response for azoxystrobin-resistant formation of P. digitatum.4. Molecular detection of azoxystobin-resistant P. digitatum: Based on the resistant molecular mechanism of P. digitatum against azoxystrobin, 3 forward primers were designed for allele specific-PCR (AS-PCR) detection of azoxystrobin-resistant P. digitatum. Among them, primer cytbMF2 (5'-CAAATGAGTTTATGACCA-3') was demonstrated to be the best one. By using this primer, an AS-PCR system was optimized. This AS-PCR system contained (20μl reactive volume): Buffer (10×) 2μl, dNTP (2mM) 2μl, MgCl₂(2.0mM) 1.6μl, Taq (5u/μl) 0.2μl, cytbR1 (5μM) 2μl, cytbMF2 (5μM) 2μl, 1μl template DNA (above 0.0610 ng/μl) and ddH₂O supplied to final volume of 20μl, followed by the PCR reactive program of 95℃5min, 95℃30s, 56℃30s, 72℃20s, 35 cycles, then, 72℃10min.