| Hebei is one of the largest apple production provinces in China. Valsa canker is prevalent in all of the apple production areas in China and often lead to serious economic losses. At present, the species of valsa canker pathogen in hebei is not clear, and lack accurate, sensitive detection method. In this study, ITS gene was sequenced and analyzed to clarify the species of valsa canker and system evolution situation in Hebei. We also established a nested PCR detection system and preliminarily revealed the distribution of the pathogen in symptomless trees and symptomatic trees with different kinds of disease scars. Our study provided a reliable detection methods for valsa canker and laid foundation for the disease control. The main results are as follows: 1. The species of apple valsa canker pathogen in Hebei province were determined. Thiry-one isolates of valsa canker pathogen from different regions of Hebei(nine cities, twelve counties) were used as material. ITS gene of these isolates were amplified and sequenced using fungi universal primers ITS1/ITS4 and the phylogenetic tree was constructed according to the sequences. The results showed that the r DNA-ITS sequence length of 31 isolates was 542~563 bp. Blast analysis indicated that 20 isolates belonged to Valsa ceratosperma and 11 isolates belonged to Valsa mali var. mali. All of the 31 isolates were clustered to one branch with Valsa mali var. mali, Valsa mali and Valsa cerastosperma by analysis of the phylogenetic tree, which showed they belonged to the same species. These results proved the tested pathogens of valsa canker from Hebei belonged to Valsa mali. 2. We established a nested PCR detection assay of Valsa mali. The DNA extraction method suitable for diseased apple tree tissues was determined. The effect of the improved CTAB method, rapid salt formulation method and commercial kit was compared to extract DNA from apple tree tissues, including DNA concentration, purity, electrophoresis and PCR amplification effect. Through designing primers, determination of its specificity, optimizing amplification system, determination of the sensitivity and reliability, we builded a nested PCR detection assay of Valsa mali. The results showed that the improved CTAB method was suitable for xylem and phloem tissues with DNA concentration of 102.1~734.5 ng/μL, A260/A280 of 2.06~2.29, bands size of more than 2000 bp, and the amplification product was clear and stable by random primers. In nested PCR assay, fungi universal primers ITS1/ITS4 were used in the first amplification round and the specific primers V-F/V-R of the pathogen were used in the second amplification round, and the detection sensitivity reached 1 fg/μL. This nested PCR detection assay can be used for samples from orchards, which provided a railable method for the diagnosis of valsa canker on apple and laid a foundation for disease control. 3. The existence of Valsa mali in different kinds of tissues on symptomless apple tree was revealed. The extension of the pathogen around different types of disease scars was also made clear. We detect the pathogen on trunk of the tree, 3 years branches, 2 years branches and phloem and xylem tissues of 1 year branches by the nested PCR detection system. The results showed that the positive rate was relative higher(60%) for the phloem tissue. However, positive rate for xylem tissues was only 5%. The growth time of the branches showed no obvious relevance with the pathogen incidence. We detected the pathogen for the tissues locating 0-6 cm surrounding new disease scars, cured disease scars and recurrent disease scars. The results suggested phloem tissues around new disease scars and recurrent disease scars had larger range of pathogen carrying than that around cured disease scars. And phloem tissues around new disease scars and recurrent disease scars could reach 3 cm at least, the farthest could reach 6 cm, while phloem tissues around cure disease scars could only be detected in 0~1 cm, to 4 cm at most. |
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