First Report of Neopestalotiopsis piceana Causing Gray Blight in Camellia sinensis L. in China.

Abstract

Tea plants (Camellia sinensis L.) are an important cash crop and are cultivated worldwide for their commercial value (Palanisamy et al. 2014). Tea gray blight is an important tea plant disease as it can cause a decline in tea quality and reduce yields by 20-30% (Sanjay et al. 2008). In August 2018, a disease survey was conducted on 400 ha of organic tea plantations in the Pu'er area of Yunnan Province (22.48° N, 100.58° E). The survey found that widespread disease was causing damage to 40% of the tea plantations and that the most seriously affected tea variety was Yunkang No. 10, which had an average disease incidence of 30-35%. The affected leaves grew small yellow-green spots on their tips or margins in the early stage that expanded into round or irregular brown spots with distinct concentric whorls and black conidial disks arranged in whorls when the humidity was high (Fig. 1A-B), which is consistent with tea gray blight disease (Zheng et al. 2021). Twenty-four diseased leaf samples were collected from four different tea plantations and transported to the Pu-Erh Tea Research Laboratory. Leaves with disease spots were cut into 4 mm ×4 mm square pieces, surface-sterilized with 75% alcohol for 1 min, disinfected with 1% sodium hypochlorite for 3 min, and washed thrice with sterile water. The tissue pieces were placed on potato dextrose agar (PDA) plates containing 100 µg ml-1 of chloramphenicol (Wang et al. 2021). After 3 d of culturing in the dark at 28 C, twenty pure cultures with similar morphology were obtained, and two representative isolates were selected and transferred into new PDA media. After 7 d, circular fungal colonies with dense aerial mycelium produced black, wet spore masses that grew on the PDA media (Fig. 1C-D). The conidia were spindle-shaped with four septa, measuring 25.0 (21.0-26.0) × 6.0 (4.5-7.0) µm (n=15). The conidia had three median cells, two of which were dark brown in color with unclear separations, with a single basal hyaline appendage 3.8 (3.5-4.5) µm (n=30) in length and 2-3 apical hyaline appendages 31 (27-35) µm in length (n=30) (Fig. 1E), similar to the conidial characteristics of Neopestalotiopsis piceana (Maharachchikumbura et al. 2014). Two isolates were selected for DNA extraction. The internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (tef1-α) gene, and partial β-tubulin (tub2) gene were amplified using the ITS1F-ITS4 primer set (White et al 1990), the EF-1α-F and EF-1α-R primer sets (Li et al. 2018), and the tub1 and tub2 primers, respectively (Chauhan et al. 2007). The ITS (OP535632 to OP535632), tef1-α (OP589285,OP589287), and tub2 (OP589286,OP589288) sequences were submitted to NCBI GenBank. Basic Local Alignment Search Tool analysis demonstrated that these sequences were 100% similar to those of N. piceana isolates available in GenBank. The sequences were compared using the Mafft software package, and sequences with the same ID were concatenated using scripts. A maximum likelihood phylogenetic tree was constructed using the MEGA (ver. 5.1) software package based on the concatenated sequences (ITS, tef1-α, and tub2). Phylogenetic analysis revealed that C-5 and B-3 showed 95% bootstrap support with N. piceana isolates in references (Fig. 2). According to the morphology and molecular characterization, C-5 and B-3 were identified as N. piceana. Pathogenicity tests on these two isolates were conducted using 36 healthy tea plants. The leaves were scratched slightly with sterile toothpick tips, after which pathogen cakes (6 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain a moist environment. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). Seven days later, the inoculated leaves exhibited similar symptoms observed under natural conditions, whereas the control leaves exhibited no symptoms. The same isolates as the introduced strains were isolated from the diseased tea leaves, completing Koch's postulates. To our knowledge, this is the first report of N. piceana causing gray blight on tea leaves in China. These results provide valuable information for the prevention and management of gray blight on tea leaves.