First Report of Pestalotiopsis chamaeropis Causing Leaf Spot on Eurya nitida in China.

Eurya nitida Korth. belonging to the family Theaceae is an evergreen shrub or small tree and is usually used as a very important ornamental tree and nectar source plant (Khan et al. 1992; Ma et al. 2013). It also has high medicinal values with the treatment of rheumatoid arthritis, diarrhea, innominate inflammatory of unknown origin, ulcer fester and traumatic hemorrhage (Park et al. 2004). In October 2020, symptoms of leaf spot were observed on E. nitida in Meiling Scenic Spot of Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). We surveyed about 300 m2 of the mountain area which holds about 100 trees of E. nitida scattered naturally near the waterside or regularly planted on either side of the mountain road. Most of the infected plants were observed from humid environments or waterside, with 15~20% disease incidence, and the disease severity on a plant basis was determined to be 25% to 30%, depending on the field. Sixty infected leaves were collected from 20 individual trees which have the same symptoms. The symptoms on infected leaves appeared as tiny circular spots that gradually enlarged into brown circular necrotic lesions and then became a light gray with brown borders and black acervuli at the later stages of the disease. Ten leaves of infected tissues randomly selected from collected sixty infected leaves were cut into 4 mm2 pieces, and surface disinfected with 75% ethanol for 30s and 1% hypochlorite for 1 min, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 to 7 days. Five isolates with similar morphological characteristics were obtained. Colonies developed copious white aerial mycelium covering the entire Petri dish area after 7 to 10 days. Conidiogenous cells were discrete, hyaline, and smooth. Conidia were fusiform, ellipsoid, 4-euseptate and ranged from 21.86 to 29.80 × 5.95 to 9.80 µm. Apical cells were hyaline with 2 to 3 unbranched, tubular apical appendages (mostly 3); basal cell was hyaline, obconic with a truncate base; three median cells doliiform to subcylindrical, brown. The morphological characteristics of all isolates matched features described for Pestalotiopsis chamaeropis Maharachch., K.D. Hyde & Crous (Maharachchikumbura et al. 2014). Two single representatives (JAUCC L001-1 and JAUCC L002) were used for molecular identification, which were verified based on the amplification of DNA sequences of internal transcribed spacer region (ITS) gene and translation elongation factor 1 alpha (TEF1-α) gene, using the primers ITS4/ITS5 (White et al. 1990) and EF1-526F/EF1-1567R (Rehner and Buckley 2005), respectively. The sequenced loci (GenBank accession nos. ITS: MW845761, MW828589 and TEF1-α: MW838967, MZ292464) exhibited over 99% homology with P. chamaeropis strain CBS 186.71 in GenBank (GenBank accession nos. KM199326 and KM199473), confirming the morphological identification. Phylogenetic reconstruction was generated by using the maximum likelihood (ML) method based on the Kimura 2-parameter model, with bootstrap nodal support for 1000 pseudoreplicates in MEGA software, version 7.0. The result showed that our isolates were clustered together with P. chamaeropis at 99% bootstrap values. Based on morphological characteristics and molecular phylogenetic analysis, the isolates were identified as P. chamaeropis. The pathogenicity of one representative isolate (JAUCC L001-1) was tested indoor by inoculating the top leaves of six healthy E. nitida plants. Three plants with three leaves were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and other three plants wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and non-infested PDA plugs on three leaves each. Treated plants were incubated in an artificial climate box with high relative humidity at 25 °C. After 10 days, symptoms on all wounded inoculated plants were similar to those previously observed with distinct tiny circular spots, whereas no symptoms appeared on inoculated plants. Pestalotiopsis chamaeropis was re-isolated from symptomatic tissues but not from the mock-inoculated plants, and its identity was confirmed by morphological characteristics and molecular data, which confirmed Koch's postulates. Pestalotiopsis chamaeropis was previously reported as the causal agent of leaf blight diseases on Camellia sinensis in China (Chen et al. 2020), Pieris japonica in Japan (Nozawa et al. 2019) and Prostanthera rotundifolia in Australia (Azin et al. 2015). To our knowledge, this is the first report of P. chamaeropis causing a leaf spot disease on E. nitida in China, and this disease may be more widespread than the sampled location. This finds is beneficial to the better protection of E. nitida, a widespread medicinal and nectar source plant with high economic value.