Sequence analysis and identification of the pathogen causing white root rot disease in sand pear (Pyrus pyrifolia) in China

Abstract:【Objective】White root rot disease mainly damages root of pear and causes the death of the infected tree, and occurs commonly in the main producing areas of pear in China, resulting in serious economic losses. This study investigated the occurrence of the disease and clarified the pathogen species in the main producing areas of sand pear (Pyrus pyrifolia) in China.【Methods】The occurrence and damage of the disease in the main producing areas of P. pyrifolia, including Hubei, Fujian, Guizhou, Sichuan and Shandong provinces, was investigated, and root samples were collected from the infected trees. Diseased tissues (neighboring the asymptomatic regions) were cut into small pieces (4-5 mm² ), disinfested in 2% NaClO for 3 min, washed three times in sterile water, dried on sterilized filter papers and plated onto potato dextrose agar medium (PDA, 20% diced potato, 2% glucose, 1.5% agar, and distilled water). Cultures were incubated on PDA at 25 ℃ in darkness. Pure cultures were stored in 25% glycerol at -80 ℃. After incubation for 2 d at 25 ℃ in the dark, morphologically similar colonies were isolated from all samples and representative isolates chosen based on colony and morphological characteristics. Colony diameters were measured daily to calculate their mycelial growth rates (mm·d^-1). After incubation for 6 d, the shape, color and density of colonies were recorded. The mycelial characteristics were observed by light microscopy (Olympus BX63, Japan) on the 4th and 12th day. Strains were selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the unclear rDNA, beta-tubulin (TUB) and the second largest subunit of the RNA polymerase II (RPB2). Maximum-likelihood (ML) was used to construct phylogenies using IQ-TREE. The three strains including HB1-1-15, FJ1-2-2 and GZ1-2-3, were selected for pathogenicity tests and the inocula were prepared by inoculating wheat grains with the fungi. The grains were soaked in water for 12 h, autoclaved at 120 ℃ for 30 min at an atmospheric, and inoculated with an agar disk, 5 mm diameter, taken from the margin of 7- day- old culture. After flasks were incubated at 25 ℃ for 14 d, the grains were used as inocula. The seedling plants of P. betulifolia were planted in pots containing 1.7 g infected wheat grains per 60 g heavy soil, and sterile wheat grains were used in parallel as the control. Each treatment was performed with five independent repeats, and the test was repeated for three times. After inoculation, the plants were cultured in the greenhouse at 25 ℃ with a 12/12 h light/dark photoperiod. The humidity was maintained at 52%, then the symptoms of inoculated plants were observed continuously.【Results】The results of investigation and symptom observation of white root rot showed that this disease occurred in the main producing areas of P. pyrifolia, including Hubei, Fujian, Guizhou, Sichuan and Shandong in China and caused substantial loss of yield and quality. The disease mainly damages the roots of P. pyrifolia. At the initial stage, the diseased plants show leaf yellowing and weakened growth, at the later stage, wilting of all the leaves, withering of the branches and finally death of the whole plant. The surfaces of roots in infected plants were covered with white mycelia. A total of 128 Rosellinia strains were isolated from the infected root samples according to colony morphological characteristics. The colors of colonies of all strains were initially white and became afterwards brown- black on the PDA media. Microscopic examination showed that the hyphae branched and septum swelled like the shape of pear. The 36 representative strains obtained in this study together with 12 reference strains from previously described species were subjected to multi-locus phylogenetic analyses with concatenated ITS, LSU, RPB2 and TUB sequences. The results showed that the strains isolated from the infected root samples belonged to Rosellinia necatrix, and there were obvious differences in gene sequences among different strains, which were clustered in three sub branches. In the pathogenicity tests, five healthy seedling plants of P. betulifolia were inoculated respectively with 14-day-old cultures in wheat grains of three strains (HB1-1-15, FJ1-2-2 and GZ1-2-3) selected from three sub branches and another five plants inoculated with sterilized wheat grains served as the controls. After 9 days, all inoculated plants showed the symptoms that were similar with symptoms in the field. However, there was significantly difference between pathogenicities of three strains. At 9 days post inoculation, the incidence percentages of roots of inoculated plants with three strains were all 100%. Whereas control plants remained symptomless. Moreover, the same fungi were re-isolated and identified from symptomatic roots. These results showed that pear white root rot was caused by R. necatrix.【Conclusion】The results of this study indicated that white root rot disease, occurring in the main producing areas of sandy pear(P. pyrifolia) in China, was caused by R. necatrix, but there were obvious differences between gene sequences of strains from different sources, which were clustered in three sub branches. It is the first report on the pathogen identification of pear white root rot in China, which provides useful information about the molecular variation as well as pathogenic diversity of Rosellinia fungus.