The Phylogentic Relationship, Molecular Detection Of Apple Tree Valsa Canker Causal Agents In China, And Population Structure Analysis Using Issr Markers

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.