Diaporthe limonicola Causing Leaf Spot Disease on Areca catechu in China

Abstract

The areca palm (Areca catechu L.), belonging to the Palmaceae family, is an important economic crop in Hainan. In June 2018, symptoms of leaf spot were observed on nearly 20% of A. catechu in Qionghai, Hainan (19°40′N; 110°33′E). At first, leaves exhibited small yellow spots; as symptoms progressed, the middle of the lesions appeared black with distinct yellow halos. Lesions were long oval, sometimes irregular with black to brown, small black spots and distinct yellow halos. As the lesions continued to expand, necrotic spots enlarged, gradually gray, and then combined to form larger necrotic areas. Ten leaves with typical symptoms were randomly collected. Small tissue sections (5 × 5 mm) were excised from the margins of lesions, disinfected with 75% ethanol for 10 s and 1% sodium hypochlorite for 2 min, followed by a triple wash with sterile water, plated on PDA, and incubated at 28°C. Three days after incubation, fast-growing fungal colonies with white mycelia appeared. Incubation continued for 3 weeks and few pycnidia developed. Alpha conidia were 5.9 to 8.5 × 2.3 to 3.0 µm (avg. 7.3 × 2.7 µm, n = 100), aseptate, hyaline, fusiform, and acute at both ends. Beta conidia were 14.8 to 29.6 × 1.3 to 2.1 µm (avg. 22.5 × 1.7 µm, n = 100), hyaline, filiform, curved, and tapering toward both ends. These morphological characteristics were similar to Diaporthe spp. (Guarnaccia and Crous 2017). To further confirm the identity of the isolate, single-spore isolates were cultured on PDA and selected for DNA extraction. The internal transcribed spacer (ITS) gene was amplified using primers ITS1 and ITS4 (White et al. 1990), a partial sequence of β-tubulin (TUB) gene by T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995), translation elongation factor 1-α (TEF1) gene by EF1-728F/EF1-986R, and calmodulin (CAL) by CAL-228F/CAL-737R (Carbone and Kohn 1999); sequence data were deposited in GenBank (MN424525, MN424539, MN424567, MN424581). BLAST analysis demonstrated that these sequences were 99% similar to the ITS (MF418423), TUB (MF418583), TEF1 (MF418502), and CAL (MF418257) of D. limonicola. Moreover, a phylogenetic tree was constructed using the method of maximum likelihood (MEGA7.0 (Kumar et al. 2016)) with a combined dataset of ITS, TUB, TEF1, and CAL sequences, which clustered the isolate with D. limonicola (CPC 31137) with high bootstrap support (100%). Based on the morphology, sequence data, and phylogenetic analysis, these isolates were determined as D. limonicola. To validate the results, a pathogenicity test was performed. Ten healthy leaves were washed with sterile water, and each leaf was slightly punctured eight times by a sterile pin and divided into two groups. The first group was placed on a mycelial disk taken from the edges of 4-day-old PDA cultures on each wounded leaf, whereas the other group was placed on PDA agar as a control. All plants were incubated at 28 ± 2°C and 100% relative humidity. Seven days after inoculation, leaves placed on the mycelial disk exhibited the typical symptoms (i.e., small black spots and gray to brown lesions surrounded by yellow halos), whereas controls showed no symptoms. Further, D. limonicola was reisolated from the leaf spot. Pathogenicity tests were repeated thrice with the same results. Koch’s postulate was achieved by the reisolation of D. limonicola from the inoculated leaves. D. limonicola has previously been reported to cause trunk canker of Citrus limon and C. sinensis in Italy (Guarnaccia and Crous 2017). To our knowledge, this is the first report of D. limonicola causing leaf spot on A. catechu in China.