| In this study, peach brown rot fungi from China were characterized in details for the first time, which revealed the presence of three distinct Monilinia species, including a new species and a new record species on peach. Subsequently, mating type loci were sequenced and analysed from all of the Monilinia species, indicating that Monilinia spp. are heterothallic fungi. In consideration of M. fructicola was the most widely distributed one of the Monilinia species in China, the genetic diversity was investigated. In addition, risk of resistance development to QoI fungicides was assessed in M. fructicola. And a suitable medium was selected for sensitivity determination of M. fructicola mycelium to SDHI fungicides. Results achieved so far were summarized as following:1. Based on the biological characteristics, and phylogenetic analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (G3PDH), β-tubulin (TUB2) revealed the presence of three distinct Monilinia species. These species included M. fructicola, M. mumecola, and a previously undescribed species designated Monilia yunnanensis sp. nov. Phylogenetic analysis of ITS, G3PDH and TUB2sequences indicated that M. yunnanensis is most closely related to M. fructigena, a species widely prevalent in Europe. Interestingly, there were considerable differences in the exon/intron structure of the Cyt b gene between the two species. Among these three brown rot fungi, M. fructicola was the most widely distributed in China, the samples which came from Beijing (BJ), Shandong (SD), Hubei (HB), Zhejiang (ZJ), Fujian (FJ), Shanxi (SX), Gansu (GS), and Yunnan (YN) provinces. M. mumecola had primarily only been isolated from mume fruit in Japan, and were isolated from peach in HB for the first time. As a new species, M. yunnanensis had been only isolated from YN and SX so far. A new multiplex PCR method was developed to facilitate the detection of M. yunnanensis and differentiation of Monilinia spp. causing brown rot of peach in China.2. Mating type loci were cloned from all of the five Monilinia spp. on peach, the sequences indicated the typical MAT organization of heterothallism in M. fructicola, M. fructigena, M. laxa, M. mumecola and M. yunnanensis. All of the MAT1-1isolates contained MAT1-1idiomorph including a MAT1-1-1alpha-domain gene and a MAT1-1-5gene, whereas MAT1-2isolates contained a MAT1-2idiomorph including a MAT1-2-1HMG-domain gene and a MAT I-2-4. In addition, a930bp insertion was located on the noncoding downstream of MAT1-2-4in MAT1-2isolates of M. yunnanensis. Interestingly, the idiomorph of all the Monilinia MAT1-1isolates harbored a fragment of MAT1-2-1 gene, while MAT1-1-1gene of MAT1-1isolates showed77.4%to91.8%sequence similarity (coverage:7.6%to27.8%) with MAT1-2-4gene of MAT1-2isolates. Based on the differences in idiomorphs among the Monilinia species, three specific primers were designed to discriminate the mating type from different Monilinia isolates. Subsequently, MAT1-1and MAT1-2isolates of M. mumecola or M. yunnanensis were selected to inoculate peach fruit simultaneously, and apothecium was successfully induced from M. yunnanensis but not from M. mumecola.3. RAPD technique was applied to analyze the genetic diversity of M. fructicola isolates from different areas in China. The results showed that FJ isolates were genetically most distant from other populations. Nei’s locus diverisity and Shannon’s index indicated the highest diversity in isolates from BJ. These results showed that the M. fructicola should have long history in China even it has not been reported until2005.4. Cyt b gene, the target gene of QoIs was cloned and sequenced in M. fructicola isolates. The Cyt b gene of M. fructicola was11,927bp in size and contained seven introns located at cDNA positions (5□→3□)201,393,429,490,506,779and811with sizes of1592,1318,1166,1252,1065,2131and2227bp respectively. Sequence analysis revealed that the third1166bp intron, a self-splicing group I intron, was located just at the G143codon. The Cyt b gene region covering the G143location and the adjacent1166bp intron was PCR amplified and sequenced from Chinese and USA isolates, indicating that the intron was omnipresent in M. fructicola. Despite the fact that G143is the’hot spot’of point mutation G143A that can confer a high level of QoI resistance in plant pathogenic fungi, the G143A point mutation in M. fructicola isolates was unlikely occurred because that the1166bp intron is just located at the G143codon.5. When rich nutrient medium (such as PDA) was used to determine sensitivity of M. fructicola mycelium to SDHI fungicides, even at high doses the mycelium could not be completely arrested. In this study we determined the mycelial growth rate of M. fructicola isolates on various media. Minimal medium (MM) supported mycelial growth the best and yielded low EC50values for three SDHI fungicides. On MM, the EC50values for boscalid, fluopyram and penthiopyrad ranged from0.013to2.134μg/ml,0.059to15.557μg/ml, and0.0002to0.4582μg/ml, respectively. Strong cross resistance relationships were detected among three SDHIs. |
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