Resistance Level And Mechanism Of Penicillium Digitatum From Quzhou, Zhejiang To Imazalil And Carbendazim

Penicillium digitatum is the most devastating pathogen of citrus fruit, being responsible for about 90% of production losses during post-harvest handling. Fungieide treatment is the main measure for postharvest disease control of citrus. However. the emergence and prevalence of fungicide-resistant biotypes followed by the successively and intensively using of fungieides often results in the decrease of control effect. In this study,70 isolates of P digitatum from 12 citrus storage houses in Quzhou city (included Kecheng and Qujiang districts, as well as Kaihua county), Zhejiang province, were assayed for their resistance to imazalil and carbendazim, and thier resistance machnisms were studied further. The results demonstrated that the frequency of imazalil-resistant isolates (minimum inhibitory concentration (MIC)≥0.5μg/mL) in Kecheng and Qujiang districts were 77.1% and 62.5%, respectively. The mean EC50 values of imazalil-resistant isolates from Kecheng and Qujiang distracts were 2.07±1.04μg/mLand 2.35±0.73μg/mL, respectively,41.4 and 47 times higher than those of imazalil-sensitive isolates (MIC≤0.1μg/mL). In Kaihua, however, none imazalil-resistant isolates was detected, and the mean EC50 values of P. digitatum was 0.04±0.02μg/mL. The frequency of carbendazim-resistant isolates (MIC>10μg/mL) in Kecheng and Qujiang ware 54.3% and 54.2% whereas 9.1% in Kaihua. These results indicated that the frequencies of imazalil and carbendazim resistance of P. digitatum were much higher in main citrus producing areas (Kecheng and Qujiang districts) than that in non-main citrus producing areas (Kaihua).To understand the resistance mechanism of P. digitatum to carbendaim, the sequences ofβ-tubulin gene (PdTUB2) of 8 Cab-S isolates and 10 Cab-R isolates were amplified and sequenced. Alignment of these sequences showed that the nucleotide at 1255 of PdTUB2 was thymine (T) in Cab-S isolates, whereas it was adenine (A) in Cab-R isolates, thus resulted in the change of amino acid at 200 from phenylalanine (F) in Cab-S isolates to tyrosine(Y) in Car-R isolates. No any other mutations correlated to Cab-R were found, thus indicated that the point mutatuion at 200aa from phenylalanine (F) to tyrosine (Y) inβ-tubulin gene must be responsive for resistance mechanism to carbendazim of P. digitatum from Quzhou.Our previous study indicated that the insertion of a four-times of extra tandem repeat of the 126-bp transcriptional enhancer (termed IMZ-R1) and the insertion of a 199bp unique sequence within the 126bp transcriptional enhancer unit (termed IMZ-R2) in the promoter region of imazalil target gene CYP51 were associated with imazalil resistance of P.digitatum. Based on these machinsms, a pair of primers CYP51A1/CYP51A2 were designed and a PCR detection method was developped for distinguishing the IMZ-R1 and IMZ-R2 P. digitatum isolates. However, there was no expected amplicons were amplified from the imazalil-resistant isolates from Quzhou, and any point mutations in CYP51 coding regions or promoter regions correlated to the imazalil resistance were detected, suggesting that an unknown imazalil resistance machnisms (termed as IMZ-R3) must be presented in these imazalil resistant P. digitatum.Two EST sequences, which were designated as CYP51B and CYP51C, were found in our annotated P. digitatum transcriptome database (unpublished). To obtain the full, as well as the partial sequences upstream and downstream of the PdCYP51B and PdCYP51C genes, primer pairs of CYP51B-R/CYP51B-F and CYP51C-R/CYP51C-F were designed according to the corresponding sequences in P. chrysogenum (NS₀00201). Results indicated that the cloned fragment with CYP51B-R/CYP51B-F from IMZ-S isolate PdKH8 contained 2919bp, encompassing a complete coding region (1751bp) and its upstream (742bp) and downstream (425bp) sequences.This gene contained an opening reading frame (ORF) of 1751bp, with 3 introns of 73,51, and 52bp, located between positions 989bp to 1061bp,1260bp to 1310bp, and 2376bp to 2427bp, respectively.PdCYP51C and partial sequences of its upstream and downstream were obtained using the same method. The cloned fragment with CYP51C-R/CYP51C-F has 2295nt, containing an ORF of 1497bp, with 5 introns located between 541bp to 596bp,682bp to 733bp,823bp to 885bp,1177bp to 1242bp, and 1292bp to 1348bp, respectively (data not shown). The nucleotide sequences of PdCYP51B and PdCYP51C genes were deposited in the GenBank as accession number HQ724322 and HQ724324.Phylogenetic analysis showed that the deduced amino acid sequence of PdCYP51B was 78% and 61% identical to Aspergillus fumigatus (AfCYP51B, XP₇49134.1) and Fusarium graminearum (FgCYP51B, FGSG₀1000), respectively. The PdCYP51C showed 19% identity with Fusarium graminearum (FgCYP51C, FGSG₁1024) and F. oxysporum (FoCYP51C, FOXG₁3138). Moreover, PdCYP51B was 59% and 22% identical to PdCYP51A and PdCYP51C, respectively, while PdCYP51C was 23% identical to PdCYP51A.PdCYP51B and PdCYP51C were amplified from eight IMZ-S isolates, one IMZ-R1, one IMZ-R2 and six IMZ-R3 isolates and sequenced. Alignment of nucleotide sequences of these PdCYP51B genes showed that an extra of 199bp fragment insertion was present of in promoter region in all 6 IMZ-R3 isolates, but was absent in both IMZ-R1 and IMZ-R2, as well as in all IMZ-S isolates. Apart from this difference, no any point mutation that related to imazalil resistance were detected either in the encoding or in the promoter regions of PdCYP51B of P. digitatum We speculate that this 199bp insertion is corelated to the observed resistance of IMZ-R3. So primer pairs of CYP51B-R/CYP51B-F were designed and used for PCR detected of IMZ-R3 P. digitatum. With this primer pair, the diagnostic band for IMZ-R3 were amplified from the imazalil resistant P. digitatum from Quzhou, indicating that the resistance molecular mechanism to imazalil of P. digitatum from Quzhou is the 199bp nucleotide-insertion mutation occurred in the promotor region of PdCYP51B.