【作者】 胡勐郡；

【导师】 罗朝喜；

【作者基本信息】 华中农业大学 ， 植物病理学， 2013， 博士

【摘要】 本研究第一次系统地描述了我国的桃褐腐病菌,确定了包括一个新种,一个新新记录种在内的三种桃褐腐病菌。克隆并分析了不同桃褐腐病菌的交配型位点,揭示了桃褐腐病菌为异宗配合真菌。此外,对我国广泛分布的美澳型核果褐腐病菌Moniliniafructicola进行了遗传多样性分析,并评估了其对QoI类杀菌剂产生抗药性的风险,最后筛选了一种适合利用M. fructicola菌株菌丝测定对SDHI杀菌剂敏感性的培养基,具体结果如下：1.通过对我国桃褐腐病菌生物学性状和对其ITS序列,微管蛋白(TUB2)以及甘油醛-3-磷酸脱氢酶(G3PDH)基因片段的测序分析,明确了我国的桃褐腐病菌共有3个种,分别是M. fructicola,新记录种Monilia mumecola,和新种Monilia yunnanensis sp. nov.。对ITS, TUB2和G3PDH的系统发育分析表明M. yunnanensis与欧洲最为普遍的桃褐腐病菌M. fructigena最为相近。有趣的是,M. yunnanensis和M. fructigena的Cyt b基因在内含子/外显子结构分布上差异明显。三种桃褐腐病菌中,M. fructicola在我国分布最广,从北京,山东,湖北,浙江,福建,陕西,甘肃以及云南等地的病桃上都分离到了M. fructicola, M. mumecola之前仅在日本的梅上分离到,本研究首次从湖北省桃上分离到该种。M. yunnanensis则是我们新发现的一个种,目前为止仅从云南,陕西等省分离到。同时设计了一个多重PCR检测方法,可快速鉴定我国桃褐腐病菌M. fructicola, M. mumecola和M. yunnanensis。2.克隆了桃褐腐病菌全部五个种的交配型位点,经过测序分析表明M.fructicola, M. fructigena, M. laxa, M. mumecola和M. yunnanensis菌株都为异宗配合真菌。在所有桃褐腐病原菌MAT1-1型菌株交配型位点上,包含一个MAT1-1-5基因和含有alpha-domain的MAT1-1-1基因,而MAT1-2型菌株交配型位点上则存在一个含HMG-domain的基因MAT1-2-1与MAT1-2-4。此外,M. yunnanensis的MAT1-2型菌株MAT1-2-4下游的非编码区中存在一段930bp的插入。进一步分析发现,所有MAT1-1型菌株交配型位点中都含有MAT1-2-1的部分片段,而MAT1-1型菌株的MAT1-1-1与MAT1-2型菌株的MAT1-2-4基因之间具有77.4%～91.8%的序列相似性(覆盖率为7.6%～27.8%)。利用不同桃褐腐菌株间’idiomorph’区域的差异,设计了三个特异性引物鉴定桃褐腐病菌全部五个种的交配型。进而根据PCR结果,选取了不同交配型的M. mumecola菌株和M. yunnanensis菌株分别同时接种桃,最后成功诱导出M. yunnanensis的子囊盘。3.利用RAPD技术对我国不同地区的M. fructicola菌株进行多样性分析,结果表明福建菌株遗传距离与其它菌群最远。Nei氏多样性指数和香农多样性指数表明北京的菌株群体的多样性最高。这些结果揭示了M. fructicola菌株可能早在2005年,其被首次报道以前就已经存在于我国。4.克隆并测序分析了M. fructicola菌株QoI类杀菌剂的靶标Cyt b基因,基因全长11927bp,包含7个长度为1592,1318,1166,1252,1065,2131和2227bp的内含子,分别位于其cDNA序列的第201,393,429,490,506,779和811位。其中,上述1166bp的内含子为Ⅰ型自剪切内含子,位于Cytb基因cDNA序列429位(氨基酸序列143位)。PCR扩增测序表明美国和中国的M. fructicola菌株Cytb基因143位点后都存在此内含子,预示着143位点的1166bp内含子是广泛存在的。虽然143位点发生突变(G143A)是植物病原菌抗QoIs的突变热点,但由于M.fructicola菌株普遍存在143位点的1166bp内含子,其产生G143A突变从而导致对QoIs高抗性的可能性极小。5.当使用富营养培养基(如PDA)测定M. fruticola菌丝对SDHI类杀菌剂的敏感性时,即使在含高浓度SDHIs的培养基中也不能完全抑制菌丝生长。而在所测试的培养基中,MM培养基不仅能使菌丝最快生长,而且在低浓度的SDHIs存在时,就能很好地抑制M. fructicola菌丝生长。MM培养基上,M. fructicola菌株对啶酰菌胺(boscalid),氟吡菌酰胺(fluopyram)和吡噻菌胺(penthiopyrad)的ECso分别为0.013～2.134μg/ml,0.059～15.557μg/ml,和0.0002～0.4582μg/ml三种SDHI类杀菌剂之间存在显著的交互抗性。更多还原

【Abstract】 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.更多还原