Isolation and identification of endophytic fungus JK2 antagonistic against pear Valsa canker caused by Valsa pyri

Abstract:【Objective】Pear is one of the most important fruits in China, where both the growing area and production of pear rank first in the world. However, the pear tree is frequently infected with certain microbial pathogens during growth. Pear Valsa canker disease is one of the most destructive diseases in the main production area in China, and poses a great threat to pear production. Pear Valsa canker disease is caused by fungus Valsa pyri. The pathogen can infect host bark wounded by injury, making plants ex- hibit reddish-brown, water-soaked, softened, and decayed symptoms. In some cases, it even can cause the whole plant death, resulting in severe production and economic losses. In recent years, pear cankerdisease has become more common and serious in most of planting regions of China and the development of pear industry has been greatly affected by this disease. Chemical control of this disease is still one of the most common and effective methods. However, due to the long-term use of chemical fungicides, it could increase the selection pressure on the presence of fungicides resistant isolates. In addition, it could result in environmental pollution, which greatly threatens the food safety. Therefore, it is urgently need- ed for new methods or means to replace chemical control of plant diseases in production. Biocontrol of plant diseases with endophytes has the characteristics of safety, low toxicity and high efficiency, which has always been the direction and hot spot of plant diseases control research. The aim of this study is to isolate and identify endophytic fungi antagonistic against pear Valsa canker caused by V. pyri, thereby providing new biocontrol fungi resources.【Methods】The pear branch infected with V. pyri was collected from Korla city, Xinjiang Uygur Autonomous Regions and used to isolate entophytic fungi by tissue sep- aration method. The isolated entophytic fungi were screened by plate confrontation experiment culture  method, to select strains antagonistic against pear Valsa canker caused by V. pyri. The supernatant of an- tagonistic strain was further used to test the inhibition activity. The classification of antagonistic strain was first identified by morphology method. The morphology of colony was observed by placing it on PDA medium for culture and the morphology of conidia was observed under a microscope. Subsequent- ly, antagonistic strain was further identified by molecular biology method. Total genomic DNA of antag- onistic strain was extracted with a CTAB method and used as template for PCR amplification. Two loci including rDNA-ITS (ITS) region and Tubulin (Tub) were amplified with the indicated universal prim- ers. The PCR production was purified by using gel extraction kit. After sequencing, the obtained DNA sequences were used to blast the NCBI database. Similar sequences were used to construct phylogenetic trees using MEGA 7 software. Effect of antagonistic strain on pear canker was determined on detached branches of pear. After treatment with supernatant or conidia of JK2, the branches were inoculated with mycelia plugs of V. pyri.【Results】An entophytic fungus strain named JK2 was isolated from pear Valsacanker diseased branch. JK2 showed strong inhibitive effect on mycelia growth of V. pyri. Statistical re- sult indicated that the inhibition rate of JK2 against V. pyri reached to above 95%. Microscopic observa- tions revealed that JK2 resulted in abnormal mycelial morphology of V. pyri. After treated with JK2, the hypha of V. pyri became slender with less branches and part of hyphae were transparent. Statistical result showed that the treated hyphal diameter was about 3.43 μm, which was significantly reduced, comparing to CK. Different concentrations of JK2 supernatant (10%, 20%) also exhibited inhibition on mycelia growth of V. pyri. The inhibition rate of 10% and 20 % supernatant against V. pyri was 52.15% and 64.04%, respectively. This result indicated that supernatant of JK2 had the antifungal substances. Based on the result of colony and conidia morphology observation, strain JK2 was identified as Penicillium spe- cies. BLAST result showed that ITS sequence of JK2 was 100% similar to Penicillium funiculosum, Pen- icillium oxalicum and Penicillium purpurogenum, which could not identify the specific species of JK2. We further used Tubulin gene sequences to construct the phylogenetic tree, which indicated that JK2 was most closely related to Penicillium purpurogenum and the similarity rate was above 99.5%. Therefore, strain JK2 was identified as Penicillium purpurogenum. The result of pathogenicity test showed that both supernatant and conidia of JK2 could inhibit pear Valsa canker disease caused by V. pyri. Compar- ing to CK, supernatant treatment did not affect the disease incidence, and however the lesion length was greatly inhibited. Conidia suspension could both significantly reduce disease incidence and lesion length. These results indicated that strain JK2 was an effective endophytic fungus to control the pear Val- sa canker disease.【Conclusion】An endophytic fungus, Penicillium purpurogenum JK2, was isolated form diseased pear branch. JK2 showed strong antagonistic activity against pear Valsa canker caused byV. pyri, which indicated that strain JK2 was a potential biocontrol fungus resource of pear Valsa canker.