【作者】 臧睿；

【导师】 黄丽丽；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2012， 博士

【摘要】 苹果树腐烂病是影响我国苹果生产最重要的枝干性病害之一，其主要危害果树的主枝、主干和小枝的皮层部分，造成主枝、主干枯死，从而严重的影响苹果的产量和品质。目前，由于对引起苹果树腐烂病的病原菌种类还存在一定的争议，导致对病原菌生物学特性和流行规律缺乏足够的认识，严重的影响到了病害防治策略的制定。由于苹果树腐烂病菌具有潜伏侵染的现象，潜伏期会因为寄主自身的生理条件的不同而长短不一。因此，在病害发展的早期阶段，果树是否被病原菌侵染不易察觉。当果树表现出明显的症状时，再进行防治不仅费时费力，而且往往因为错过了最佳的防治时间而收效甚微，造成了人力和财力的浪费。合理的选用抗病品种是被公认为防治腐烂病最为经济有效的方法，但由于对苹果树腐烂病菌群体遗传特性的研究一直以来都是空白，严重的阻碍了抗病育种工作的开展。本研究针对这些问题，主要采用分子生物学的方法，逐一进行了深入的研究，取得了以下主要结果：1.以rDNA-ITS，EF1α和β-tubulin三个基因序列和其形成的联合基因序列数据集为基础，通过MP、ML和BI三种系统分析法，明确了我国苹果树腐烂病菌的三个种类组成，即Valsa mali Miyabe et Yamada，V. malicola Z. Urb和V. persoonii（=Leucostompersoonii）。而V. mali种下又可以划分为两个不同的变种，即V. mali var. mali（Vmm）和V. mali var. pyri（Vmp），其中Vmm是我国苹果树腐烂病最主要的致病菌。来自于苹果或野苹果的V. cerastosperma与V. mali是同物异名，但其与来自其它阔叶树的V.cerastosperma有比较大的遗传差异。2.分别以Vmm，V. malicola和V. persoonii的rDNA-ITS的特异序列为基础，设计出针对每种病原菌的种特异性引物VmF/R，VmcF/R，VpF/R；并以其共有序列为基础，设计出针对壳囊孢属（Valsa）病菌的属专化性引物VF/R，以这两者相结合，建立起了针对这三种病原菌的巢氏PCR检测体系。检测体系的灵敏度检测结果表明，本实验所建立的巢氏PCR检测体系，对这三种病原菌的最低检测剂量分别达到了100fg/μl，1fg/μl和1fg/μl，表明其具有非常高的灵敏度，可以满足分子检测的需要。体系特异性的检测结果表明，这些引物只能从其要检测的靶标菌样品中扩增到目的条带，表明其具有高度的特异性。通过真菌分离法和巢氏PCR检测方法的比较，发现凡是通过真菌分离法发现病原菌的样品同时也能通过巢氏PCR检测到病原菌的存在。试验中不存在通过真菌分离法可以获得病原菌，但巢氏PCR却检测不到病原菌的现象。巢氏PCR和真菌分离法对有明显症状样品的检测率都达到了100%，对无明显症状样品的检测比率分别达到了64.7%和20.6%，表明巢氏PCR具有比传统真菌分离法更高的检测率和准确性。3.通过巢式PCR的方法，对采自陕西省苹果产区的279份节间组织样品进行了检测。检测结果表明在其中的150份样品中检测到了V. mali var. mali，占所检测样品的53.76%；在64份样品中检测到了V. malicola，占所检测样品的22.94%；在24份样品中，检测到了病原菌V. persoonii，占所检测样品的8.60%。检测结果说明，外表无明显腐烂症状的苹果树节间组织中，也普遍带有苹果树腐烂病菌，其中以V. mali var. mali最为普遍，V. malicola居中，V. persoonii检测到的频率最低。在检测的279份外表无症状的节间组织样品中，共有56份样品中检测到了两种或两种以上的病原菌，占所检测样品的20%，说明两种或两种以上病菌共同侵染的寄主的情况，在田间条件下广泛存在。4.采用巢氏PCR的方法，对苹果树不同组织样品的带菌率进行了检测，结果表明，不同种类的病原菌在不同的组织中的分布情况不同。V. mali var. mali在各种组织中的分布差异明显，顶芽的平均带菌率为89%，节间组织平均带菌率为71%，芽鳞痕组织平均带菌率为48%。而V. malicola在各种组织中的分布则没有明显差异。在病株率不同的两类果园内，病株率高的果园中，样品中Vmm的带菌率明显较病株率低的果园高，但样品中V. malicola的带菌率则没有明显差异。表明Vmm和V. malicola这两种病原菌在田间具有不同的分布规律，Vmm趋向于聚集分布，而V. malicola则更趋向于均匀分布。5.采用正交设计实验，对影响苹果树腐烂病菌ISSR-PCR的四种主要因素进行了三个不同水平的研究，确定出最优的PCR扩增体系，即：25μL的PCR反应体系中含：0.20μmol/L dNTP、0.1μmol/L ISSR引物、3.00mmol/L Mg2+和0.05U的Taq酶。ISSR-PCR最佳扩增体系的获得，保证了实验可以获得稳定、可靠的实验结果。通过对47条ISSR引物退火温度的逐一筛选，获得了各引物最佳的退火温度，并在此基础上，筛选到了11条对苹果树腐烂病菌具有较高多态性的引物。11条引物共从129个位点扩增到稳定的条带，在供试的87个陕西分离株中，119个位点为多态性位点，多态性位点百分率为92.25%；在126个中国分离株中，129个位点全为多态性位点，多态性位点百分率为100%。6.通过对陕西和全国不同地理区域种群遗传多样性的分析表明，不同地理区域种群的Nei’s（1973）基因多样性指数（H）和Shannon信息指数（I）均达到了H>0.2，I>0.3的水平，表明我国的苹果树腐烂病菌具有丰富的遗传多样性。对不同地理区域种群遗传结构的分析表明，不同地理区域种群间的遗传变异，只占总变异很小的一部分，各自然种群内的不同分离株之间的遗传变异是腐烂病菌最主要的变异来源。对陕西省苹果树腐烂病菌21自然种群遗传关系的UPGMA聚类分析结果显示，在相似系数为0.88时，可将供试自然种群聚成9个类群中，其中属于第二（Ⅱ）类群的分离株，数量多（60株）、分布广（10县区），表明该类群是陕西省苹果树腐烂病菌的优势种群。同时，聚类分析也发现来自于同一地理区域种群的不同自然种群可分布于不同的聚类群中，说明各个自然种群之间的遗传亲缘关系与其地理来源之间并没有明显的相关性。对中国不同地理区域种群内不同自然种群的UPGMA聚类分析发现，苹果树腐烂病菌种群的遗传分布与其所处的纬度位置之间具有一定的关系，但是这种关系却非常弱。更多还原

【Abstract】 Apple tree Valsa canker caused by species of Valsa, is one of the most destructive stemdiseases of apple tree (Malus domestica Borkh.) in China.The phloem of the twigs, limbs andbranches can be invaded by the pathogens, which can cause these tissues dead. Under thissituation, the production and quality of apple would be deeply affected. At present, it was stillcontroversial on the causal agents caused this disease. It was the partly reasons that limitedknowledgement was known on the physiological characteristics and epidemic rules, under thiscircustance, the effective disease control strategies have not been established by now. It wasreported that the pathogens were characteristical of latent infection.The latent period wasdependent on the physiological condition of the host. Because of latent infection, it wasdifficult in finding whether the apple trees were infected by the pathogens or not at the earlystage of disease development. Until the visible cankers can be seen on the trunks that controlmeasures will be applied. Thus, the optimal stage for controlling this disease will be missed,and the control efficiency was always not obtained after more labour and financial resourceswere spent. It was said that disease-resistant cultivar was the most effective and cost-optimalways for farmers to control this disease. However, it was still unknown on the populationgenetic diversity of the pathogens, which seriously impedes the breeding for diseaseresistance development. In order to solve these problems gradually, the molecular technologywas employed in this study, and the main results were obtained as follows.1. On the basis of rDNA-ITS, EF1α, β-tubulin and their combined genes sequences datasets, MP, ML and BI phylogenetic trees were repectively established. The results showed thatthere were three species of Valsa can caused this disease. They are repectively Valsa maliMiyabe et Yamada，V. malicola Z. Urb and V. persoonii (syn: Leucostom persoonii). Thecausal agent, V. mali Miyabe et Yamada, can be also divided into two varieties below the rankof species level, V. mali var. mali (Vmm) and V. mali var. pyri (Vmp). The variety of Vmm wasthe predominant pathogen of apple tree valsa canker in China. V. cerastosperma isolated from apple and Chinese crabapple was a synonym with V. mali, however, which was different fromV. cerastosperma isolated from other broad-leaf trees.2. Based on the rDNA-ITS conservative sequence of the Valsa genus, one pair ofgenus-specific primers was designed. Similarly, three pairs of species-specific primers weredesigned based on the specific sequence of Vmm, V. malicola and V. persoonii. Combinedthese two kinds of primers, a reliable and precise nested-PCR protocol was established. Thesensitivity of this assay was evaluated by serial dilutions of DNA extracted from Vmm, V.malicola and V. persoonii pure cultures, the results showed that the it was able to detect as lowas100fg of DNA in Vmm mycelial samples,1fg of DNA in V.malicola and V. persooniimycelial samples. The specificity of the three species-specific primers were evaluated againstVmm, V. malicola and V. persoonii isolates respectively, and four isolates from closely relatedValsa species and eight isolates from fungal species that are commonly isolated from naturallyinfected tissue. The results indicated that a distinct band of348bp in length was detected inall Vmm isolates, but not in other tested species. Similarly, a distinct band of312bp wasdetected in all V. malicola isolates and a354bp band generated in all V. persoonii isolates, butnot in other tested species. These data indicated that the protocol developed in this study hashigher specificity and sensitive, which can be applied in the detection of pathogens in theorchards. The efficiency of the nested PCR assay was compared to that of fungal isolationassays. All samples from which the pathogen was successfully isolated yielded a PCR productof the expected size.There was no incidence in which the pathogens were isolated, but failedto be detected by the nested PCR. For visible canker samples, the detection rate of the twomethods are both100%, while the detection rate of nested PCR for symptomless samples was64.7%, which was much higher than the detection rate of20.6%by fungal isolation. Thesedata showed that the nested-PCR had higher precision and detection rate than fungal isolation.3. A total number of279internodes tissues samples without symptoms from Shaanxiapple-producing area was detected by nested-PCR, the results showed that the species of Vmmwas detected in150samples, which had a proportion of53.76%. There were64samples inwhich V. malicola was detected and the proportion was22.94%. The species of V. persooniiwas detected in24samples, which had a proportion of8.60%. These data indicated that thecausal agents existed in the internodes tissues without visible symptoms. The detection rate ofVmm was the maximum, which indicated that this spcies was dominated. V. malicola was thesecond and V. persoonii was the minmum. In addition, there were56samples in which two orthree species were detected simultaneously by nested-PCR, this result showed that thecoinfection by two or three species was existed extensively in the orchards. 4. The nested-PCR detection results in different symptomless tissues showed that theincidence of different pathogens has their own features in different tissues of apple trees. Theincidence of Vmm in different tissues was significantly different. The average incidence ofVmm was89%in terminal buds,71%in internodes, and48%in bud scale scars. While theincidence of V. malicola in different tissues was almost the same. In the two kinds of orchardswith different diseased tree rate, Vmm incidence in the orchards with higher diseased tree ratewas significantly higher than the orchards with lower diseased tree rate. However, V. malicolaincidence in these two kinds of orchards was not significantly different. The distributionpatterns of the pathogens were different, Vmm trends to aggregation distribution, while V.malicola trends to uniform distribution pattern.5. The orthogonal designed experiments were employed to screen the optimal ISSRamplification protocol at three levels of four factors including Taq DNA polymerse, primer,dNTP, and Mg2+concentration. The optimized ISSR-PCR amplification protocol wasobtained, the25μL reaction mixture included0.20μmol/L dNTP,0.1μmol/L ISSR primer,3.00mmol/L Mg2+, and0.05U Taq DNA polymerase. The optimized amplification protocolpaved the way for getting reliable, steady and clear amplification bands. The annealingtemperatures of47ISSR primers were screened one by one, and the optimized annealingtemperatures of these primers were obtained. Regard these as foundation,11ISSRpolymorphic primers was screened. These primers were used to study the genetic diversity of87Vmm isolates isolated from Shaanxi province and126isolates isolated from differentprovinces of China. A total number of129loci were detected of which119loci werepolymorphic loci in Shaanxi isolates, the polymorphic loci percentage was92.25%. However,the129loci were all polymorphic loci in the isolates from different provinces of China, thepolymorphic loci percentage was100%.6. The results of genetic diversity of different geographical groups showed that Nei’s(1973) gene diversity index (H) and Shannon’s information index (I) were both greater than0.2and0.3respectively, which indicated that that the genetic diversity of Vmm geographicalgroups not only in Shaanxi but also in all over China was considerably abundant. The resultsof population genetic structure of different geographical groups showed that genetic variationamong geographical groups accounted for small part of the total genetic variation. The geneticvariation within the populations was the main source of genetic variation. The dendrogrambased on ISSR markers revealed that the21natural populations were clustered into nineclusters at the threshold of genetic similar coefficient0.88by UPGMA. The number ofisolates belongs to cluster Ⅱwas60, which occupied the great majourity of tested isolatesand distributed in a wider range, which indicated that this cluster was dominant. The populations belonged to the same geographical groups can be clusted into different clusters,which indicated that there was no significant correlation between the genetic relationships andtheir geographical originals. Moreover, the dendrogram of different populations from differentprovinces of China showed that the distribution of the population was associated with latitude,but this relationship was very weak.更多还原