Occurrence of Neopestalotiopsis clavispora Causing Leaf Blight on Acer oblongum in China.

Abstract

Acer oblongum is native to Southwest China and is also distributed in Nepal and Northern India. It is an excellent garden ornamental tree species, suitable for solitary planting in courtyards and parks. From June to August 2022, severe leaf blight occurred on A. oblongum in Baihe Wetland Park (32°5'42" N, 112°28'13" E) in Nanyang City, China. The foliar disease rate reached 59% (n=100). Early signs were yellow spots on the leaves, mainly on the middle and edge parts. Then, the lesions gradually expanded, became amorphous, and turned yellowish brown, eventually led to necrosis on leaves and branches. Twenty diseased leaves were collected and the junction areas between infected and healthy tissues were cut into 5 x 5 mm2 pieces. The collected plant materials were sterilized in 75% ethanol and 1% NaClO for 30 s and 1 minute, respectively, followed by rinsing in sterile water, and placing on a potato dextrose agar (PDA) plate supplemented with 50 µg ml-1 streptomycin at 25 °C for 3 days. Colony edges were cut and transferred to new PDA plates for purification culture. A total of 18 purified fungal strains were obtained, which showed similar phenotypes in morphological characteristics. All colonies had spread radially with wavy surfaces, and dense cream to white aerial hyphae. After 14 days in culture, black fruiting bodies appeared. Conidia were fusiform to slightly clavate, with five cells and two or three setae, 4.2 to 7.9 μm × 17.5 to 25.4 μm in diameter (n = 100). The apical and basal cells and setae were colorless, three median cells were brown, and the middle cell was dark brown. Morphological characteristics of all 18 strains were consistent with the genus description of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). Further molecular identification showed that the ITS region sequences of all strains have extremely high homology with Neopestalotiopsis spp. The β-tubulin gene (TUB), and the translation elongation factor 1-alpha gene (TEF1) were amplified for molecular identification (Shu et al. 2020). The sequences of three representative strains (FE-05, 09, 16) from different regions were deposited in GenBank with accession Nos. OQ867279, OQ867288, OQ867289 (ITS), OQ870207, OQ870208, OQ870209 (TUB), and OQ870204, OQ870205, OQ870206 (TEF1). BLASTn analysis of these sequences showed 99 to 100% identity to Neopestalotiopsis clavispora strains (OK655673, MZ648263 for ITS, ON000362, MZ286974 fr TUB, MH423941, MK512481 for TEF1). These morphological features and molecular identification indicated that the pathogen has the same characteristics as N. clavispora. Pathogenicity was tested on ten healthy 3-month-old seedlings using the three representative strains through in vivo experiments. For each strain, the conidial suspension (106 conidia ml-1) in absorbent cotton balls (50 µl of inoculum) were inoculated onto the healthy leaves of two seedlings, while a total of two other plants were served with sterile water as a blank control. The plants were potted in a climate incubator at 28°C and a relative humidity of approximately 90%. Symptoms consistent with natural lesions were observed on the inoculated leaves after 5 days while the control plants remained healthy. The strains of N. clavispora were reisolated from the symptomatic inoculated leaves, fulfilling Koch's postulates. N. clavispora is known to cause disease in a variety of plants in China, such as Dendrobium officinale (Cao et al., 2022), Fragaria ananassa (Shi et al., 2022), and Garcinia mangostana (Qiu et al., 2019). To the best of our knowledge, this is the first report of N. clavispora causing leaf blight on A. oblongum in China. The yellowing and falling off of leaves would seriously affects the garden landscape. It is necessary to further clarify the host range of the pathogen to select appropriate landscape matching plants in future planning.