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First Report of Colletotrichum boninense Causing Leaf Anthracnose on Eucalyptus robusta (Smith) in the Upper Reaches of Yangtze River

Zhang, J., Zhu, T. H.
Plant disease 2018 v.102 no.7 pp. 1446
Colletotrichum, DNA primers, Eucalyptus robusta, anthracnose, branches, color, culture media, ethanol, forest diseases, forest trees, fungi, greenhouses, growth chambers, humid zones, internal transcribed spacers, islands, leaves, mycelium, pathogenicity, pathogens, photoperiod, planting, plastic bags, polymerase chain reaction, sodium hypochlorite, spores, surveys, tissues, tree diseases, China, Japan, South Africa, Yangtze River
Eucalyptus robusta (Smith) is a fast-growing and highly productive timber species mostly located in the upper reaches of Yangtze River in China (from 24 to 30°N, from 100 to 111°E). In 2014, anthracnose symptoms on E. robusta leaves were first observed in planting areas in Panzhi Hua (26°58′N, 101°51′E), Sichuan Province, in China. However, in March 2017, 69% of E. robusta individuals of the Yangtze River exhibited symptoms similar to those observed in 2014 according to our general survey on forest diseases. So far, the symptoms have only been reported as a causal agent of a disease on Eucalyptus in South Africa (Smith et al. 1998). Colletotrichum boninense was first described and collected in the Bonin Islands, Japan (Moriwaki et al. 2003). The color of symptomatic leaves gradually changed from light yellow to brown. Some of the leaves showed red round spots later, which was as a sign of the pathogen. Infected branches did not develop new growth. During the later stages of symptom development, the leaves wilted. Red or red brown spores on diseased leaves were found in humid climates. To isolate the responsible fungus, leaf and stem fragments from 100 plants were taken from the diseased tissues. Subsequently, the surface was sterilized for 30 s in 70% ethanol and 3 min 5% NaClO followed by rinsing in sterile water before placing on potato dextrose agar (Difco). Fifty-eight isolates were obtained from all leaf fragments, and these exhibited the typical morphology described for the so-called C. boninense taxa, such as varied from white to gray, to dark orange or pink-gray, and the reverse side of the colonies was white, dark gray, orange, or a mixture and with regular colony margins, hyphal branching pattern, growth rate, mycelium, and the presence of loose or compact growth. The size of spore is 8.0 to 18.0 × 4.0 to 6.5 μm, and the shape and color of the spores were the same as described in previous reports (Diao et al. 2013; Pileggi et al. 2009). The internal transcribed spacer (ITS) region of the fungus was amplified using the primers ITS1/ITS4, and eight isolates were selected randomly and sequenced based on the variation observed in colony morphology. It turned out the sequenced data were the same for all isolates (GenBank accession no. KJ619456). BLAST comparison of the 597-bp product showed a 100% similarity with several sequences of C. boninense (JQ926743, GU935881, and EU520087). We also identified the pathogen with the species-specific primers Col1/ITS4 (Pileggi et al. 2009), the sequence was deposited in GenBank (accession no. MG752909), and it also turned out that the fungus was C. boninense. To confirm pathogenicity and to fulfill Koch’s postulates, 30 1-year-old E. robusta (Smith) plants (20 inoculated with C. boninense and 10 negative controls) were grown in a greenhouse in 5.0-liter pots. Plants were covered with plastic bags and cultured in a growth chamber at 25°C in a 12-h photoperiod for 20 days. After 14 to 17 days, symptoms similar to those reported from diseased plants were observed in the positive group. Twenty isolates of C. boninense were reisolated from all symptomatic plants identified by polymerase chain reaction, sequenced, and analyzed. No C. boninense strain was isolated from control plants. To our knowledge this is the first report in an international journal of a leaf anthracnose disease on E. robusta (Smith) in the Upper Reaches of the Yangtze River, in China.