Species of pathogens causing cherry brown rot and their resistance to common fungicides in Zhejiang province

Abstract:【Objective】The aim of the experiment was to completely identify the pathogen diversity of cherry brown rot (CBR) disease and to investigate the resistance of pathogenic fungi to boscalid [a nov- el SDHI (succinate ubiquinone reductase inhibitor)], difenoconazole [a SBI (ergosterol synthesis inhibi- tor)], thiophanate-methyl [a MBC (tubulin inhibitor)] and azoxystrobin [a new QoI (quinine outside in- hibitor)], so as to provide a scientific basis for the reasonable prevention and control of CBR disease in Zhejiang province, China.【Methods】According to Koch’s law, diseased fruit samples with typical symptoms of CBR were collected from different regions of Zhejiang province and candidate pathogenic fungi were isolated. Then each candidate isolate was re-inoculated onto healthy cherry fruits to deter- mine the pathogenicity. Then, the pathogenic fungi were systematically classified by combining analy- sis of both the morphological characteristics including growth colony, sporulation structures and conid- ia, and the molecular identification though amplifying the internal transcribed spacer (ITS) of ribosome gene using the universal primer pair ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’). The resistance status of each isolate to boscalid, difenoconazole, thiophanate-methyl and azoxystrobin was respectively determined by the method of differential dose. Isolates that were unable to grow on potato dextrose agar (PDA) plates amended with 5 mg · L- 1 fungi- cide were considered as sensitive (S); those that could grow on 5 mg·L-1 but not on 25 mg·L-1 were de- fined as low resistance (LR); those that could grow on 25 mg·L-1 but not on 50 mg·L-1 were defined as moderate resistance (MR); and those that could grow on 50 mg · L- 1 were considered high resistance (HR). Based on the resistance results, isolates resistant to azoxystrobin were selected to further analyze the molecular mechanism of resistance to the pathogen of CBR.【Results】Only one kind of fungus was isolated from all the diseased samples. A total of 32 isolates causing CBR symptoms were got at ran- dom. They were identified into three fungus species based on morphological and molecular characteris- tics, which were responsible for causing CBR: Monilinia fructicola accounting for 65.6%, M. laxa ac- counting for 18.8%, and M. fructigena accounting for 15.6%. Most of the isolates of M. fructicola, the dominant species causing CBR, were grayish brown or grayish yellow, with neat edges and no rose-petal structure on PDA plates. The conidia were colorless, single cell, elliptic or lemon-shaped, arranged in chains, and the conidial size was (7.128-12.534) μm×(6.157-9.336) μm; The colony morphology of M. laxa was significantly different from other species, showing a grayish yellow color, a darker color in the middle, irregular lobes on the edge, thin mycelium thickness. Its conidia were binomial branches, with clear conidia, monosporin and chain arrangement, and the conidial size was (9.785-12.708) μm× (6.867- 10.335) μm; M. fructigena was pale or beige. The conidia were colorless, elliptic or lemon, and conidi- um chain, obviously not bifurcate or binary-shaped branch, and the condial size was (8.549-13.675) μm× (3.388-9.762) μm. For pathogenicity, all isolates could cause disease after inoculation on cherry fruits, but there were significant differences in the size of disease spots, and the symptoms of inoculated cherry fruits were consistent with those of natural disease in the fields, among which M. laxa was the most pathogenic, which was the second frequently isolated fungi from CBR fruits. The following was M. fructicola, and then M. fructigena. All the isolates of CBR were sensitive to boscalid, thiophanate-methyl and difenoconazole except for two isolates, HF-4-5 and HF-5-4, which were resistant to azoxystrobin and were detected with resistance frequency of 6.25%. The study on resistance mechanism showed that no glycine to alanine substitution at amino acid 143 (G143A) in the mitochondrial cytochrome b (cyt b) gene of the pathogen of CBR occurred in the azoxystrobin HR isolates【. Conclusion】There was diversity in the pathogen of CBR in Zhejiang, China: M. fructicola (65.6%), M. laxa (18.8%) and M. fructigena (15.6%). The pathogenic ability to cherry fruits was M. laxa >M. fructicola >M. fructigena, which was not consistent with the responding frequency of isolation. The pathogens of CBR were sensitive to boscalid, thiophanate-methyl and difenoconazole and had the HR frequency of 6.25% to azoxystrobin with a different resistance mechanism from previously reported G143A point mutation in the Cyt b.