First Report of Leaf Spot Disease Caused By Colletotrichum gloeosporioides on Pouteria caimito in China

Pouteria caimito (Ruiz & Pav.) Radlk is a tropical fruit tree that is of high commercial value. It has been widely cultivated in Hainan Province of China since 2009, and planting has rapidly expanded. In March 2017, P. caimito seedlings in several nursery gardens of Haikou city were affected by a leaf spot disease with an incidence of up to 39%. The disease symptoms of irregular brown spots appeared on the edges of the leaves as small lesions, ranging from 0.2 to 0.3 × 0.3 to 0.4 cm in size, which later merged and gradually expanded to the middle of the leaves, resulting in coverage of more than 60% of the leaf surface area. Interestingly, no similar disease symptoms on leaves of mature plants were observed. White or grayish-green fungal colonies were consistently isolated from infected spots after surface sterilization and placed on potato dextrose agar for 3 to 7 days. Conidia were cylindrical with blunt ends, some slightly curved, ranging from 10.4 to 16.7 μm (mean 13.3 μm) × 4.2 to 5.8 μm (mean 4.8 μm) (n = 100). Similar colonies were isolated from different infected leaf samples from other nursery gardens. To further identify these isolates, the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase, chitin synthase (CHS-1), calmodulin (CAL) and manganese-superoxide dismutase genes were amplified and sequenced using primers developed by Weir et al. (2012). The nucleotide sequences were deposited in GenBank (accession nos. MF197434, MG196057, MG196058, MG196059, MG196060, and MG196061, respectively). The results of sequence analyses showed the isolate was highly similar to Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., about 99% homology with C. gloeosporioides by comparing with the sequences of ITS (KM513611.1), ACT (KM604707.1), CHS (MF554857.1), and CAL (KP145359.1). Pathogenicity experiments were carried out by spraying a conidial suspension (10⁵ CFU/ml) on three tender leaves of healthy P. caimito seedlings, and three seedlings sprayed with sterile water served as the control. The sprayed seedlings were incubated in a growth chamber at 20 to 25°C of 90% relative humidity under natural daylight. The test was repeated five times. The symptoms of leaf spot disease developed on seedlings after 6 days, whereas the negative control was asymptomatic. The same pathogen was reisolated from the infected leaves, fulfilling Koch’s postulates for proof of pathogenicity. According to morphological characteristics and molecular analysis, the fungal strain was identified as C. gloeosporioides (Penz.) Penz. & Sacc. (Bailey et al. 1992). C. gloeosporioides not only can cause leaf spot disease on P. caimito but also causes the same symptoms on citrus (Peres et al. 2004) and pepper (Manandhar et al. 1995), which are the important cash crops in Hainan. To our knowledge, this is the first report of leaf spot disease caused by C. gloeosporioides on P. caimito in China.