Isolation and identification of the pathogen causing acute blossom blight of sand pear (Pyrus pyrifolia) in Fujian

Abstract:【Objective】In recent years, a devastating fungal disease that damages flowers of sand pear (Pyrus pyrifolia Nakai), known locally as acute blossom blight of pear, has occurred in sudden out- breaks in Fujian province of China, causing severe wilting of petals and peduncle, rotting of young fruit, and resulting in economic loss. This study was carried out on identifying the species of pathogen causing acute blossom blight in Pyrus pyrifolia in Fujian province of China.【Methods】The occurrence and damage of the disease in the field was investigated, and damaged flower cluster samples from the diseased trees showing symptoms of acute blossom blight were collected. Diseased tissues (neighboring the asymptomatic regions) were cut into small pieces (4-5 mm2) after surface sterilization (75% ethanol for 45 s, washed three times in sterile water and dried on sterilized filter paper). Four single colonies of each isolate were picked up with a sterilized needle (insect pin, 0.5 mm diam.) and transferred onto po- tato dextrose agar (PDA, 20% diced potato, 2% glucose, 1.5% agar, and distilled water) plates and incu- bated at 28 °C in the dark. Pure cultures were stored in 25% glycerol at –80 °C. Representative isolates were chosen based on colony cultivation traits and morphological characteristics. Colony diameters were measured daily to calculate their mycelial growth rates (mm·d-1). The shape, color, and density of colonies were recorded after ten days. Moreover, the shape and color of conidia were observed under light microscopy (Olympus BX63, Japan) after 15 days. Multiple sequences of concatenated ACT, TUB2, CHS-1, GAPDH, and ITS sequences were aligned using MAFFT v.7 with default settings, and if necessary, manually adjusted in MEGA v.5.2.2. Maximum-likelihood (ML) was used to construct phy- logenies using IQ-TREE. The 14 representative isolates of Colletotrichum sp. were selected for pathogenicity tests with a spore suspension on detached pear flowers (approx. 1-wk-old) of P. ussuriensis‘Nan- guoli’, P. pyrifolia‘Hohsui’, and P. communis‘Guyot’. Briefly, the conidiospore suspension with a concentration of 1×106 conidia per mL was sprayed on the surface of flower clusters without being un- wounded, and sterile water was used in parallel as a control. Each treatment was performed with three independent repeats. After inoculation, the preservative film was wrapped to keep moisture and cultured in the greenhouse at 25 °C with a 12/12 h light/dark photoperiod. The humidity was maintained at 85%. Then the symptoms were observed continuously.【Results】The petals showed pale yellow wet lesions at first, and then expanded to the whole petals. The color of the lesions turned dark yellow, and then they became black-brown rotted. The peduncles were dark brown and large sunken rot lesions on fruits in the process of time. The occurrence of the disease was unlimited, and the lesions were large and irregu- lar, which can extend to the whole leaf, causing the whole leaf to fall black and brown. Pear acute blos- som blight could lead to substantial economic losses due to excessive fruit rot, or the severe wilting of blossom. A total of 31 Colletotrichum strains were isolated from the damaged flower cluster samples according to colony morphological characteristics. Colonies were light red in the center and pale white on the margin cultured for ten days. Conidia with mass orange color produced on the PDA plate after 15 days. Conidia were hyaline, smooth-walled, aseptate with both ends slightly acute under the light mi- croscopy. But no sexual organ was found. The 14 representative strains together with 17 reference strains from previously described species were subjected to multi-locus phylogenetic analyses with con- catenated ACT, TUB2, CHS-1, GAPDH, and ITS sequences. The results showed that the strains isolated from the damaged flower cluster samples belonged to C. fioriniae. The conidiospore suspensions of rep- resentative strains (CFZH1, CFZH6, CFZH15) were sprayed on the surface of the clusters of three spe- cies of pears (P. ussuriensis, P. pyrifolia and P. communis), which could cause the symptoms of acute blossom blight within 3 days. And the average rates of incidence were 81.3%, 95.8%, and 63.9%, re- spectively, of which P. pyrifolia had the highest incidence rate compared with other two species of pears (P. ussuriensis and P. communis), and no lesions developed on the flower clusters as control. Moreover, the symptoms caused by inoculation in the three species of pears were consistent with those of P. pyrifolia in the field. These results showed that the acute blossom blight in P. pyrifolia in Fujian province was caused by C. fioriniae.【Conclusion】In this study, we employed morphological and multi-locus phyloge- netic analyses, along with pathogenicity tests according to Koch’s postulates to identify the species ofColletotrichum causing acute blossom blight of sand pear. It is the first report that C. fioriniae is the pathogen of pear acute blossom blight, which may supply new useful information for understanding the pathogenic diversity of Colletotrichum. This study provides a theoretical basis for the effective control of the acute blossom blight in Fujian province, China.