First Report of Leaf Spot Caused by Colletotrichum fructicola on Kiwifruit in China.

Abstract

Kiwifruit (Actinidia spp.) is an important fruit with high nutritional and economic value, which is widely cultivated in China. In April 2021, leaf spots were observed on the leaves of 'Xuxiang' (A. deliciosa) in a kiwifruit plantation of Hefei city, Anhui province, China (117°26'E, 31°85'N). Disease incidence was about 10% of the observed plants. Small yellow spots initially developed on the leaves and gradually expanded into irregular dark brown spots, and eventually the diseased leaves curled and withered. Leaf tissues (n=10, 5×5 mm) were collected from five infected plants, sterilized in 75% ethanol solution for 30 s and 1% NaOCl for 5 min, washed, dried and plated on PDA at 25°C. In total, ten isolates were obtained, including two previously reported Botryosphaeria dothidea (Zhou et al. 2015) and Diaporthe actinidiae strains (Bai et al. 2017) and eight unknown isolates with similar morphology. All unknown isolates initially appeared white with many aerial hyphae, and at the later stage, the center of all colonies turned gray. Colonies were transferred to new PDA with 0.1% yeast extract for three days. Then, aerial hyphae were scraped with sterile cotton swabs, and continued to grow for four days. Orange conidial masses were produced. Conidia were hyaline, smooth-walled, single-celled, cylindrical with broadly rounded ends, with average size around 4.1-5.5×13.2-18.2 µm (n=100). Appressoria (n=50) were ovoid in shape with average size around 4.9-6.7×8.6-11.8 µm. Morphological features were similar to Colletotrichum. gloeosporioides species complex (Weir et al. 2012). To confirm their species identification, internal transcribed spacers (ITS), β-tubulin (TUB2), glyceraldehydec-3-phosphate dehydrogenase (GAPDH), actin (ACT) and chitin synthase (CHS) were amplified by PCR using the primer pairs ITS1/ITS4, Bt2a/Bt2b, GDF/GDR, ACT-512F/ACT-783R CHS-79F/CHS-234R, respectively (Weir et al. 2012). Based on alignment analysis, sequences of the eight unknown isolates were 100% homologous. The representative isolate LSD3-1 was selected for further study. BLAST analysis showed that the ITS (OM033371), TUB2 (OM044376), GAPDH (OM044377), ACT (OM044379) and CHS (OM044378) sequences of isolate LSD3-1 were 98.7%-100% identical with the collected sequences of C. fructicola strain ICMP:18581 (NR_144783, JX010405, JX010033, JX009866, JX009501). Phylogenetic analysis of multiple genes was conducted with the Maximum likelihood method using MEGA 7. Based on morphological and molecular characteristics, the LSD3-1 was identified as Colletotrichum fructicola (Prihastuti et al., 2009). Koch's postulates were performed on six one-year-old 'Xuxiang' plants, which were used to test pathogenicity in the greenhouse (at 28℃, relative humidity 80%, 16/8 h light/dark). Surface-sterilized leaves were sprayed with a conidial suspension (107 conidia/mL). Yellow and brown lesions were formed 14 to 21 days after inoculation, whereas the mock-inoculated controls remained asymptomatic. The experiment was performed three times. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Although C. fructicola has been reported as a leaf spot disease on many plants (Shi et al. 2018), this is the first report of leaf spot caused by C. fructicola on kiwifruit in China. This result is helpful to better understanding the pathogen of kiwifruit leaf spot diseases in China and formulate effective control strategies.