Identification of the pathogenic fungi associated with apple valsa canker in Inner Mongolia, China

Abstract：【Objective】Apple valsa canker, also known as“apple cancer”, is one of the most important diseases of apple trees. Inner Mongolia Autonomous Region, located at the northeast district of apple growing area in China, is famous for its high-quality fruits owing to the abundant sunshine and larger temperature difference between day and night. The climate in Inner Mongolia is dry and cold, which is conducive for the occurrence of apple valsa canker diseases. Little information, however, has been known about the species of the pathogenic fungi associated with apple canker disease in this area. This study aimed to isolate and identify the pathogenic strains associated with apple canker in Inner Mongolia based on morphological and molecular characteristics.【Methods】Symptomatic branches were collected from‘Jinhong’trees in the Qianhe apple orchard around Hohhot, Inner Mongolia, China. Bark pieces at the border of healthy and diseased tissue were surface-disinfected with 1% sodium hypochlorite for 3 min and 75% ethyl alcohol for 30 s, and washed three times in sterile distilled water. The surface-disinfected pieces were placed on Potato Dextrose Agar (PDA) plates containing rifampicin, and incubated at 25℃. The hypha at the colony frontier was repeatedly transferred into the new PDA plates, until the pure colonies were received. Isolated fungi were tested for pathogenicity on sterilized healthy apple branches. Specifically, healthy branches on 2-year-old‘Jinhong’trees were cut into 20-cm-long segments, surface- disinfected in 1% sodium hypochlorite for 5 min and 75% ethyl alcohol for 30 s, washed three times with sterile distilled water, dried at the room temperature, and both ends were thensealed with wax. Subsequently, the sterilized branch segments were scalded by the head of nails, inoculated with the 6 mm diameter fungal mycelial plugs, and wrapped with wet degreasing cotton and parafilm. Inoculated branches were placed in the plastic tray, covered with parafilm, and incubated at 25 ℃, 16 h light/8 h dark cycle and 70% humidity. The degreasing cotton was removed after 3 days. The strain Cytospora Mali YL1, isolated from apple in Yangling, Shaanxi, was used as positive control, and PDA disk was used as negative control. The obtained pathogenic isolates were then identified based on their morphological and molecular characteristics. For the morphological characteristics, isolates were cultured on PDA plates at 25 ℃, and their colonial morphology was observed after 3, 10 and 60 days. The mycelium and conidia on the PDA plates were examined under a microscope, and the conidia and pycnidium on the inoculated branches were observed under the microscope or stereoscope. For the molecular characteristics, isolates were cultured at 25 ℃ for 7 days, their mycelium was collected by the sterilized toothpicks, and DNA was extracted from the obtained isolates. The genes of the internal transcriptional spacer (ITS), the ribosomal large subunit (LSU), the transcription prolongation factor (EF) and the beta- tubulin (BTU2) of the obtained isolates were amplified by the ITS1/ITS4, NL1/NL4, EF1- 688F/EF1-1251R, and Bt2a/Bt2b primer pairs. The PCR products were purified using the gel purification kit, and cloned into a PMD19-T vector. The reaction mixture was transformed into Escherichia coli DH5α competent cells. Three positive colonies from each transformation were selected and sequenced by Sangon Biotech. Obtained sequences were modified by the Vector NTI Advance11, and submitted to the NCBI. The received sequences were aligned with those published in NCBI by Clustal X software, edited manually by the BioEdit software, and connected as the ITS-LSU-EF-BTU2 sequences. The sequence identities based on ITS-LSU-EF-BTU2 sequences between the isolates and those published in NCBI were analyzed using SDT software with the Clustal W method, and phylogenetic trees based on the multi-locus sequences were built by MEGA 6.0 with the neighbor-joining method.【Results】Two strains, named QH1 and QH2, were isolated from the branches. Sterilized healthy‘Jinhong’branches inoculated with the QH1, QH2 or YL1 developed typical symptoms of apple canker disease, i.e., black fruiting bodies (pycnidium) formed, and yellow mucilaginous mass of conidia oozed out from the fruiting bodies under humid conditions. Conidia were hyaline, allantoid, and aseptate. The transverse microtome section of the pycnidium showed labyrinthine chambers. The colonies of QH1 and QH2 grown on PDA were white and villiform initially, and then changed moderately to kelly or brown separately after 10 days. Conidiomata began to form abundantly after 60 days grown on PDA plates. The morphological characteristics of QH1 and QH2 coincided with the Cytospora species. The sequence identity based on the ITS-LSU-EF-BTU2 genes of the QH1, QH2 and the reported Cytospora species was 76.1%-84.5%. Phylogenetic analysis based on the ITS-LSU-EF-BTU2 genes showed that QH1 clustered with C. schulzer, and QH2 clustered with C. mali.【Conclusion】Two strains of apple valsa canker associated pathogenic fungi were isolated in Inner Mongolia, China, and they were identified as C. schulzer and C. Mali. This is the first study on identifying the pathogenic fungi of apple valsa canker in Inner Mongolia, via both morphological characteristics and molecular marker genes.