First report of Diaporthe sojae causing stem canker on blueberry in China

Blueberry (Vaccinium corymbosum) plants are popular all over the world due to their high nutritional value and health benefits. In October 2020, blueberry stems (cv. O'Neal) displaying reddish brown necrotic lesions were observed from a blueberry field in Anqing (Anhui, China), with the incidence of approximately 90%. The affected plants were somewhat stunted that had smaller fruit, and in severe cases, partial or whole plant died. We randomly selected three sampling sites to collect stems with the symptoms. Samples at the margin between diseased and healthy tissues were taken out, cut into 5 mm pieces in length，and then mixed them together. Twenty small samples were surface-sterilized, and plated onto potato dextrose agar (PDA). The plates were incubated at 25°C in the dark until fungal colonies were observed. After subculturing single hyphal tips, 9 out of 12 fungal isolates with similar morphologies were obtained. The representative isolate, LMKY12 was selected for further identification. The colonies on PDA showed white, fluffy aerial mycelia with 7.9  0.2 mm (n=5) diameter after inoculation in darkness at 25°C for one week. The colony darkens in color with age, yellowish pigmentation in reverse were observed. After 15 days of incubation, dark brown, irregular hard particles (fruiting bodies in sexual stage) accumulated on the surface of the colonies. Asci were 8-spored, sessile, club-like, hyaline, and 35-46 x 6-9 μm (n=30) in size. The ascospores were oval or spindle shaped, two-celled, constricted at division, and containing four guttulates with larger guttules at centre and smaller one at ends, measured 9-11 x 2-4 um (n=50). No sporulation observed on blueberry stems after inoculated 30 days. In order to induce the production of conidiophores, mycelial plugs were placed on blueberry leaves and cultured in darkness at 25°C. There are two types of conidia observed after 20 days of inoculation. Alpha conidia were aseptate, hyaline, smooth, ovate to ellipsoidal, often biguttulate, measured 5.33-7.26 x 1.65-2.53 μm (n=50). Beta conidia were hyaline, linear, measured 12.60-17.91 x 0.81-1.38 μm (n=30). The morphological characteristics matched the previous description of D. sojae (Udayanga et al. 2015; Guo et al. 2020). To confirm the identification, the mycelial genomic DNA of LMKY12 was extracted as a template. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1-α), and calmodulin (CAL) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. BLAST analysis revealed that the ITS (ON545758), CAL (OP886852), and TEF1-α (OP886853) sequences were 100% (527/527 base pairs), 99.21% (504/508 base pairs), and 99.41% (336/338 base pairs) similar to the strain FAU636 of D. sojae (KJ590718, KJ612115, KJ590761), respectively. Phylogenetic analysis based on concatenated sequences of ITS, TEF1-α, and CAL using MEGA 7.0 by maximum likelihood attributed the isolate LMKY12 to the D. sojae clade. Pathogenicity tests were performed on blueberry cv. O'Neal using detached stems (n=8) in laboratory, one-year-old potted plants (n=4) in greenhouse. Inoculations were done by placing mycelial plugs (7 mm in diameter) taken from a 7-day-old PDA culture on wounded stems. Inoculations with uncolonized agar plugs served as negative controls. Reddish dark brown lesions similar to the symptoms were observed on all inoculated stems 7 days after inoculation. No symptoms developed on control stems. Reisolations were successfully made from all the inoculated stems, and the pathogen was confirmed by the presence of pycnidia, alpha conidia and beta conidia. To our knowledge, this is the first report of D. sojae causing blueberry stem canker in China.