First Report of <i>Neopestalotiopsis chrysea</i> Associated with Gray Blight of Tea (<i>Camellia sinensis</i>) in China

Tea plants (Camellia sinensis (L.) O. Kuntze) are commercial perennial woody crops, which has been widely cultivated in more than 60 countries all over the world (Hajiboland 2017). Tea gray blight is a foliar disease that reduces quality and yield by 20-30% (Horikawa 1986). During March of 2022, a disease survey was conducted on a 300 ha tea plantation (Liyang Tianmu Lake tea research Institute) in the Liyang area of Jiangsu province (31°19'37.64''N, 119°23'46.37''E). The disease damaged 30% of the tea plants, and 'Baiye No. 1' was the most impacted with an average disease incidence rate of 50-60%. In early stages of infection, leaves exhibited small yellow-green lesions at the tips and margins. These lesions gradually expanded into round or irregular brown spots characterized by distinct concentric rings and black conidial disks arranged in whorls. These symptoms are consistent with tea gray blight disease associated with Neopestalotiopsis piceana reported by the previous study (Wang et al. 2022). A total of 15 lesions were collected from different plants and sent to the Plant Protection Laboratory of Nanjing Agricultural University for analysis. To identify the pathogens, leaves with necrotic lesions were cut into 5 × 5 mm pieces at the junction between diseased and healthy tissues, surface sterilized with 75% ethanol for 1 min, disinfected with 2% sodium hypochlorite for 3 min, and then rinsed three times with sterile water. The tissues were placed on potato dextrose agar (PDA) plates containing 100 μg/ml of streptomycin sulfate (Wang et al. 2022) and incubated at 25°C in the dark. A total of 15 isolates were obtained from 15 different lesions with typical symptoms as described earlier and identified as Pestalotiopsis-like fungus by spore morphological identification (Maharachchikumbura et al. 2014), the conidia length and width of which were shorter than that of Pseudopestalotiopsis theae. Pure cultures were obtained by monosporic isolation, and the representative isolates J15 and F22 randomly selected were used for morphological studies and phylogenetic analyses. The cultures of the two isolates grown on PDA were white, cottony, and flocculent and contained undulate edges with dense aerial mycelium on the surface, black, wet conidial masses emerged after 7 days. Conidia were five celled, clavate to fusiform, smooth, and 17.9 to 25.3 × 5.3 to 9.1 μm (n = 50). The three median cells were dark brown to olivaceous, the central cell was darker than the other two cells, and the basal and apical cells were hyaline. Conidia developed filiform appendages: one basal appendage (3.5 to 9.3 μm long; n = 50) and two to three apical appendages (15 to 35 μm long; n = 50). Morphological features were similar to Neopestalotiopsis chrysea (Maharachchikumbura et al. 2012). To further identify, the partial internal transcribed spacer (ITS) regions and ß-tubulin 2 (TUB2) regions and translation elongation factor 1-alpha (TEF1-a) genes of the two isolates were amplified from genomic DNA using the primers ITS1/TS4, Bt2a /Bt2b, and EF1-728F/EF1-986R (Glass and Donaldson 1995; Carbone and Kohn 1999), respectively. The ITS, TUB2, and TEF1-a sequences of J15 and F22 were submitted to GenBank and the accession numbers were PQ489357, PQ489381, PQ505998, PQ505999, PQ539625 and PQ539626. The sequences were compared using the MEGA (ver. 11), and sequences with the same ID were concatenated using the PhyloSuite (ver.1.2.3) software package, and then, a maximum likelihood phylogenetic tree was constructed based on the concatenated sequences (ITS, TUB2, and TEF1-a). The phylogenetic analyses on the basis of multilocus gene sequences revealed that two isolates J15 and F22 were located in one clade with the known N. chrysea isolates (FZXM038, host: blueberry, China) in references (Fig 2). The other hosts of N. chrysea reported in China were Ligustrum lucidum, Liquidambar formosana or Carya illinoinensis. According to the morphology and molecular characterization, J15 and F22 were identified as N. chrysea. Pathogenicity tests were conducted on 18 healthy tea plants (1-year-old rooted cuttings of 'Baiye No. 1' purchased from Nanjing Yarun tea Co., LTD) which were cultivated in a Light incubator at 23°C, 16h light /8h dark, light intensity 200 μmol m-2s-1, 65% RH. Leaves were scratched slightly with a sterile needle, and pathogen plugs (5 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain moisture. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). The inoculated leaves showed symptoms similar to those observed in the field, whereas the control leaves were asymptomatic after 14 days inoculation. The fungi N. chrysea were consistently reisolated only from the inoculated and symptomatic leaves by morphological and molecular identification, fulfilling Koch's postulates. To our knowledge, this is the first report of N. chrysea infecting tea plants associated with gray blight in China. The sequences from this work have been submitted to GenBank, and this work provides crucial information for the prevention and management of gray blight on tea plants.