First Report of Leaf Spot Caused by Alternaria alternata on Sonchus asper in China

Sonchus asper (Spiny sowthistle), belonging to the Asteraceae, is a problematic weed in grain crops, orchards as well as turf (Cho et al., 2019). In April 2016, about 90% of the S. asper plants infesting a pear orchard had symptomatic with black spots densely distributed on the leaves and stems in Da Yang Town, Luyang District, Hefei, Anhui (N31°55'43″, E117°11'40″). The foliar lesions were mostly circular (3.0 mm to 1.5 cm in diameter) or irregular in shape; lesions on the stem were elliptical to irregular in shape running along the vascular bundle and about 3 to 6mm long × 2 to 3mm wide. Severely affected plants had their leaves become completely blighted and eventually the plants died. Fifteen tissue pieces from five symptomatic leaves and stems were surface sterilized with 0.5% NaClO for two min, rinsed three times with sterile water, and then plated on 1/4-strength potato dextrose agar (PDA) medium. Fourteen fungal isolated were obtained from the tissues, the isolation frequency from the plant tissue pieces was almost 93%. Fungal colonies were circular, initially white, and eventually turned to dark olive or black along with profuse sporulation. The conidia were tawny to brownish green, obclavate to obpyriform, with a cylindrical or coniform short beak, ranging from 16.8 to 39.8 μm long × 6.3 to 14.7 μm wide with two to five transverse and zero to two longitudinal septa (n = 50) and were borne on branched conidiophores. These morphological characteristics were similar to those described for Alternaria alternata (Simmons, 2007). For one representative isolate, the internal transcribed spacer (ITS) region of rDNA, partial coding sequence of the actin (ACT) gene, partial Alt a 1 major allergen (ALT) gene and partial Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene were amplified and sequenced with primers ITS1/ITS4 (White et al., 1990), ACT512F/ACT783R (Carbone and Kohn, 1999), Alt-4for/Alt-4rev and gpd1/gpd2 (Lawrence et al., 2013) respectively. Sequences were submitted to GenBank (ITS: MH886523; ACT: MH892480; ALT: MN655781; GAPDH: MN655782). Based on a BLAST analysis, ITS, ACT gene and GAPDH gene had 100%-99.8% identity with the existing sequences of the ex-type CBS 916.96 of A. alternata (Fries) Keissler (ITS: AF347031; ACT: JQ671702; GAPDH: AY278808, respectively) and ALT gene showed 100% identity with the A. alternata isolate SCWC10 (MG199093). Thus the pathogen was identified as A. alternata based on its morphological and molecular characteristics. Pathogenicity tests were conducted on six to seven-leaf stage potted S. asper plants (one plant per pot). Five mm diameter fungal disks, which were excised from the edge of a three-day old A. alternata colony, were placed on healthy leaves of five plants. Same size sterile PDA disks were used as controls. In addition, a conidial suspension (107 conidia/ml) was also inoculated on healthy leaves of five plants, sterile distilled water was used as control. Both inoculation experiments were conducted in the greenhouse at 25 ± 2°C, 90 % relative humidity, 12 h light/dark photoperiod and repeated at least three times. Lesions were observed on all the leaves within 12 h after fungal disk inoculation. All pathogen inoculated plants developed lesions, similar to those observed on the symptomatic leaves in the field 12 days after conidial inoculation. No symptoms were observed on both control treatments. A. alternata was re-isolated from the inoculated leaves and confirmed by both morphological and molecular techniques, confirming Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf spot on S. asper in China. The pathogen could cause severe disease in S. asper, and has the potential to be further studied as a fungal weed biocontrol.