First Report of Colletotrichum lindemuthianum Causing Anthracnose on Pepper in China

Pepper (Capsicum annuum L.) is an important economic vegetable crop worldwide (Valadez-Bustos et al. 2009). China is currently the second largest exporter of pepper (De Silva et al. 2017). Anthracnose caused by Colletorichum spp. is one of the most destructive diseases in pepper production, resulting in 40% yield loss. Of the 15 Colletotrichum species reported to infect pepper (cvs. Chaotianjiao and Xianjiao), Colletotrichum fioriniae, C. fructicola, C. gloeosporioides, C. scovillei, and C. truncatum have been previously identified as the dominant pathogens causing anthracnose of pepper in China (Diao et al. 2017). In August of 2016, the typical anthracnose symptoms (circular, slightly sunken, necrotic, and dark lesions) were observed on pepper fruit in Tai’an, Shandong Province, China. Two orchards, separated by 20 km, were selected and three peppers were collected in each orchard. Diseased tissues were cut from one lesion on each fruit, disinfected with 0.5% NaOCl for 30 s, rinsed three times with sterile distilled water, and placed onto oatmeal agar (OA) plates. The OA plates were incubated at 25°C in the dark for 3 days. Six purified cultures were obtained by the single spore method. On OA, fungal colonies of the six isolates were gray to olivaceous black on the upper surface with sparse aerial mycelium and gray to olivaceous gray in the lower surface with an average colony diameter of 62 mm after 10 days. Conidia were hyaline, unicellular, guttulate, smooth-walled, cylindrical, straight or slight curved, both ends obtuse or with an acute base, and 8.69 to 16.45 × 3.11 to 6.27 μm. These morphological characteristics were consistent with descriptions of C. lindemuthianum (Liu et al. 2013). Because these isolates had similar morphology, one representative isolate was selected for multilocus phylogenetic analyses. DNA was extracted from the representative isolate using the CTAB method. The nucleotide sequences of the internal transcribed spacers (ITS), beta-tubulin (TUB2), actin (ACT), histone3 (HIS3), glyceraldehyde-3-phosphate (GADPH), and chitin synthase 1 (CHS-1) were amplified using specific primer sets (Diao et al. 2017), sequenced, and deposited in GenBank (MF511774–79). Based on the combined dataset of ITS, TUB2, ACT, HIS3, GAPDH, and CHS-1, alignments were made using MAFFT v. 7, and phylogenetic analyses were processed in MEGA v.7.0. The studied isolate (GY3) clustered together with C. lindemuthianum reference strain CBS151.28 (100% bootstrap). To confirm the pathogenicity, detached pepper cv. Xianjiao fruit were surface-sterilized and wounded using a sterilized needle to a depth of 1 mm. Fruits were inoculated by pipetting 15 μl conidial suspensions (1 × 10⁶ conidia ml⁻¹) onto each wound, and controls were inoculated with 15 μl sterile distilled water. Each treatment had 10 replicate fruit. These fruits were incubated in a growth chamber with 12 h photoperiod and 85% relative humidity at 28°C. The test was repeated three times. After 12 days, typical anthracnose symptoms (sunken, brown to dark lesions) appeared on inoculated fruit but not on controls. Reisolation of fungus was performed as previously described and identified as C. lindemuthianum, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. lindemuthianum causing anthracnose on pepper in China. Considering the economic value of pepper, this identification can help producers select appropriate field management measures for this disease.