The Sensitivity Of B. Berengeriana F. Sp. Piricola To Tebuconazole And Molecular Detection

Apple ring rot is one of the important diseases of apple in China. The causal pathogen (Botryosphaeria berengeriana f.sp. piricola) can infect apple fruit, stem and branch. For its difficulty to control, and composes one of the major limiting factors of apple production.To confirm the sensitivity of Botryosphaeria berengeriana f.sp.piricola to triazole fungicides tebuconazole, 98 strains of Botryosphaeria berengeriana f.sp.piricola were isolated from typical and representative location of Henan, Shandong and Liaoning Province. The sensitivities of 98 strains to triazole fungicide tebuconazole were measured by colonial growth-rate. The result showed that the 50% effective concentration values(EC₅₀)to the 47 strains for tebuconazole ranged from 0.0054 to 3.3253 mg/L, and the mean EC₅₀ values( Se t 0.05)were 0.26670.2083 mg/L. Isolate XX2B(EC₅₀ 3.3253 mg/L)showed the lowest sensitivity to tebuconazole, strain QF2(EC₅₀ 0.0054 mg/L)was the most sensitive strain to tebuconazole, whose toxicity ratio was 620.38-fold compared to strain XX2B. Furthermore, the mean EC₅₀ values of Shandong, Shan xi, Henan, and Liaoning Province were 0.5715 0.4612 mg/L, 0.5348 0.6935 mg/L,0.4020 0.1006 mg/L and 0.2667 0.2083 mg/L, separately. This showed that the isolates from Liaoning were more sensitive to tebuconazole than the isolates from Shandong and Henan, the sensitivity of strain from Shandong was the lowest. The result of Duncan test and hierarchical cluster analysis showed that the sensitivities to tebuconazole of Botryosphaeria berengeriana f.sp.piricola from different regions were no evident difference in the strains.The pathogenicity of 33 B.berengriana f.sp.piricola strains coming from different regions and sensitivities was studied after inoculation. The results showed that there was the appearance of pathogenicity differentiation of B.berengriana f.sp.piricola strains. The average of the disease index of 33 isolates which were inoculated on apple branchs was 25.94±4.82, and different isolates showed the different disease index on apple branchs.Three extraction methods, urea, benzyl chloride and SDS-CTAB, were applied to extract the genomic DNA of Botryosphaeria berengriana f.sp.piricola. The results showed that all the three methods were capable of extracting the genomic DNA of Botryosphaeria berengriana f.sp.piricola. The SDS-CTAB method had a highest quality DNA extraction and was the most efficient method, but consumed longest extraction time. The urea method was the simplest and most convenient procedure, but had a lower quality extraction. The benzyl chloride method had a lowest quality and the extracted DNA contained a lot of protein impurities. The rDNA ITS analysis and electrophoresis analysis showed that the DNA extracted by all the three methods has fairly good quality for DNA amplification and satisfied the requirements for rDNA ITS analysis of the Botryosphaeria berengriana f.sp.piricola.The ITS andβ-tubulin gene sequence of apple ring rot pathogen was determined with primer ITS1/ITS4 and Bt2a/Bt2b. Sequence comparing of ITS andβ-tubulin gene of apple ring rot pathogen was conducted and a phylogenetic tree based on multiple alignment of their ITS andβ-tubulin gene nucleotide sequences was constructed. The results showed that the three isolates (QF8B, PL7, XX2B) in lower sensitivity ITS andβ-tubulin gene shared the highest sequence similarity (99%) with Botryosphaeria rhodina (Lasiodiplodia theobromae) and they could be grouped into one branch with Botryosphaeria rhodina. It showed that Botryosphaeria rhodina may infect apple and then cause apple ring rot.