Molecular Detection Of Venturia Inaequalis Using Internal Transcribed Spacer

Apple scab, which is one of the most important diseases of apple in most producing countries all over the world, is caused by an ascomycete fungus, venturia inaequalis (Cooke)Wint. The fungus infected leaves and fruit, often causing an early apple tree leaves, the drop in fruit yield, quality became inferior, and thus reduce the amount of flower bud differentiation and the next year output. From studying the previous studies, we can get some informations about the spread of germs and development and popularity of the law. In this study, the gene of ribosomal DNA internal transcribed spacer rDNA (ITS) sequence specific sprimers inaequalis molecular detection, achieved the following results:1. Application of universal primers ITS1 and ITS4 of the apple scab pathogen,, Alternaria mali , Marssonina cororlar and Podospharea leucotricha of 26 virus strains have tested their DNA-ITS PCR amplification and sequencing, 26 ITS sequences obtained.2. 26 about the internal transcribed spacer (ITS) sequences, which is of the apple tested strains, were compared screening, a set specific primer 320A/320B, is 320 bp for the fragment size, was obtained. Its PCR reaction system optimized was performed with a total volume of 25μL reaction mixture containing 2μL MgCl2(25 mmol/L), 2.5μL 10*buffer, 3μL dNTP(2.5 mmol/L), 1.2μL forward and reverse primer 320A/320B (10μmol/L), 0.5μL Taq polymerase (5 U/μl), 1μLDNA template(30 ng/μL), and ddH2O. Besides, PCR was carried out in a thermocycler programmed for 35 cycles consisting of a denaturing step at 94℃for 30 s, and annealing step at 60℃for 30 s, and an extension step at 72℃for 30 s. These cycles were preceded by an initial denaturation step at 94℃for 3 min, and ended with a final extension at 72℃for10 min.3. The ITS-PCR molecular detection for the tested strains and the different stages of apple leaf tissue inoculated by V. inaequalis have detected by specific primers 320A/320B designed. The results showed that, DNA extracted from V. inaequalis strains provided a single 320bp amplicon, but DNA extracted from A.mali, M.cororlar, P. leucotricha, host tissue, and double distilled water did not amplify using these primers. At the same time, DNA was extracted from the apple leaf tissue inoculated by V. inaequalis during the different stages can be amplified 320bp amplicon.In the study, the sensitivity of specific primers 320A/320B have examined, indicating that the primers sensitivity for genomic DNA detection is on the level of 100 fp/μL.The results of the study provides a rapid, simple, sensitive molecular detection technology, which is an important theoretical and practical significance for integrated management of apple scab. And this method could detect the pathogen in the early time when V. inaequalis had been inoculated. It can be also a reference for the quarantine and field investigations of the plants and plant products in the national epidemic and non-infected. The method also has vital significance on the control of apple scab pathogen spreading in our country, while the establishment of the system also provides technical guidance and theoretical basis for the detection of other pathogens.