Camellia azalea Wei (Theaceae) is a critically endangered species with high ornamental value in China. Its wild individual plants, less than 1,000, are only found in Yangchun, Guangdong Province, China. Since 2010, a severe dieback on C. azalea has been observed in several commercial plantations in Foshan, Guangdong Province, during the process of artificial propagation. The infection started from the middle portion of the new shoots, where necrosis spots developed and expanded to girdle the stems. Consequently, the shoots died and became brown in color. Later, the necrotic spots turned pale gray, and many small, black fruiting bodies emerged. In the end, more than half of the dead shoots broke off from the necrotic spots. Generally, about 10 to 20% new shoots were infected for one individual plant. Although the older branches with leaves were not infected and showed no symptoms, the dieback of crown outer layer greatly reduced the ornamental value of the plants and the sale price went down. Another part of the plants that is often infected is the stalk, resulting in the drop of fruits. By using routine isolation methods and single-spore purification technique, 18 single-conidial isolates with similar colony morphology were obtained from five diseased plants. The cultures of single-conidial isolates grew at an average rate of 6.8 mm per day on PDA at 28°C. The central part of colony became gray-green with age, and acervuli formed on the medium after incubation for 7 to 10 days. Conidia, round at both ends, were 13.65 to 18.3 × 3.61 to 5.92 μm (avg. = 16.1 ± 1.6 × 4.8 ± 0.8 μm, n = 50) in size. After culturing for 50 to 60 days, perithecia matured. Ascopores were hyaline, straight, aseptate, and 10.02 to 13.77 × 3.27 to 4.45 μm (avg. = 12.2 ± 1.1 × 3.9 ± 0.4 μm, n = 50) in size. The cultural and morphological characteristics of these isolates are consistent with the description of Glomerella cingulata f. sp. camelliae (1). The sequences (GenBank Accession Nos. KJ668576, KJ668577, KJ676642, KJ689374, KJ689375, and KJ689376) of ITS, GPDH, GS, actin, β-tubulin, and CAL regions of three representative isolates are identical and share 99, 99, 100, 99, 100, and 100% identity with those of the type specimen of G. cingulata f. sp. camelliae ICMP 10643 (JX010224, JX009908, JX010119, JX009540, JX010436, and JX009630), respectively (2). Twenty randomly selected shoots with young leaves on the top of them, detached from different trees, were scratched in the middle part with a fine scalpel to generate a 5-mm-long wound, 50 μl conidial suspension (1 × 105 conidia ml−1) was then dropped onto the wound for inoculation. The control shoots were inoculated with the same volume of sterile distilled water. All inoculated shoots were placed into an intelligent artificial climate incubator with 12-h photoperiod and 100% relative humidity at 28 ± 1°C. Each treatment replicated on five shoots, and the tests were repeated twice. Symptoms resembling those in the field were observed on all conidia-inoculated shoots after 10 to 14 days, and control shoots were asymptomatic. The same fungus G. cingulata f. sp. camelliae was consistently re-isolated from the diseased shoots, fulfilling Koch's postulates. G. cingulata f. sp. camelliae has been reported on other species of Camellia outside China, but this is the first report in China where the species is endemic and endangered (1,2). References: (1) J. S. W. Dickens et al. Plant Pathol. 38:75, 1989. (2) B. Weir et al. Stud Mycol. 73:115, 2012.
Characterization of Alternaria isolates causing leaf spots in radish in Brazil
