Kiwifruit (Actinidia) is a common fruit cultivated in many countries. Actinidia deliciosa and A. chinensis are two commercially important kiwifruit species. Over 70,000 ha are grown annually in China. In 2012, a leaf spot disease of A. chinensis was observed in several orchards in Leye County (106°34′ E, 24°47′ N), Guangxi Zhuang Autonomous Region, China. The disease mainly damaged the leaves during the fruit development stage through to the maturity stage. Initially reddish-brown small lesions appeared on the leaves; later, typical symptoms were tan to taupe lesions surrounded by purple brown margins, nearly circular to irregular, 2 to 10 × 2.2 to 15.5 mm in diameter. Some lesions exhibited a concentric pattern. The lesions eventually coalesced, causing extensive leaf necrosis and defoliation. The fungus that sporulated from lesions had the following morphological characteristics: light brown conidiophores with slightly swollen apexes, light brown conidia formed singly or in acropetal chains, straight or curved, cylindrical to oblavate, 52.9 to 240.5 μm long (avg. 138.9 μm) and 5.3 to 13.6 μm wide (avg. 8.4 μm), 5 to 12 distoseptate, with a flat, darkened, and thickened hilum. These morphological characteristics corresponded with that of Corynespora cassiicola (Berk. & Curt.) Wei (1). To isolate the pathogen of the disease, small pieces of symptomatic foliar tissues, including young lesions, typical older lesions, and atypical older lesions with concentric pattern were surface sterilized with 75% ethanol for 30 to 60 s, disinfected in 0.1% HgCl2 for 1 min followed by washing with sterile water, plated on PDA, and incubated at 28°C for 7 to 10 days. Gray to dark gray colonies and conidia of C. cassiicola were observed. To validate the identity of the fungus, the sequence of the ITS region of one of the purified strains, LYCc-1, was determined. DNA was extracted from the isolate that was grown on PDA at 28°C for 4 days, and the ITS region was amplified using the universal primer pair ITS4/ITS5 (2). The double strand consensus sequence was submitted to GenBank (KJ747095) and had 99% nt identity with published sequences of C. cassiicola in GenBank (JN853778, FJ852574, and FJ852587). Pathogenicity tests were carried out on detached leaves in petri dishes in an incubator at 28°C and on whole plants in a glasshouse at 25 ± 3°C. The isolations did not produce enough conidia in pure culture, so mycelial discs were used in pathogenicity tests. For both assays, 60-day-old healthy kiwifruit leaves were inoculated with a 5-mm mycelial disc obtained from the periphery of a 5-day-old C. cassiicola strain (LYCc-1) grown on PDA. The PDA discs were placed on the leaf surface with their mycelial surface down and secured with sterile wet cotton. Controls consisted of leaves that were inoculated with sterile PDA discs. For the detached leaf assay, the leaves were placed on filter paper reaching water saturation in petri dishes, and for the whole plant assays the inoculated leaves were kept moist with intermittent water sprays for 48 h. Four leaves of each plant were inoculated with the isolate in both assays, and experiment was repeated twice. Eight inoculated leaves of the detached leaf assay all showed the first water soaked lesions 36 h after inoculation, followed by extensive leaf rot 72 h after inoculation, and yielded abundant conidia of C. cassiicola. Six out of eight leaves inoculated on whole plants showed the first lesions 5 days after inoculation, whereas control leaves remained healthy. Only C. cassiicola was re-isolated from the lesions in both assays, fulfilling Koch's postulates. This is the first report of leaf spot caused by C. cassiicola on kiwifruit in China. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) T. J. White et al. In: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
An Improved Detached-Leaf Assay for Phenotyping Net Blotch of Barley Caused by Pyrenophora teres