First Report of Botryosphaeria dothidea Causing Leaf Blight on Parrotia subaequalis in China.

ABSTRACT

Parrotia subaequalis is of great ornamental value due to its unique bark, featuring interesting textures and colors, and its large, striking galls. These characteristics make it a popular choice for bonsai cultivation. (Yan et al. 2022) . In July 2023, an outbreak of leaf blight was observed on 40, six-month-old P. subaequalis seedlings in Anqing, Anhui, China, with an incidence rate of 80%. In the early stages of infection, small brown spots appear on the leaf surface, which gradually become round or irregular and darken to a deep brown color. As the disease progresses, the affected areas expand from the leaf margins towards the center, causing the leaf surface to become concave, wilt, and necrotize. This resulted in restricted plant growth, and in severe cases, partial or complete plant death. For isolation, 30 tissue slices (5 × 5 mm) were taken from the leaves of 10 symptomatic seedlings and surface sterilized with 75% ethanol for 5 seconds, followed by five rinses with sterilized distilled water. After two days of dark incubation at 28°C, hyphal tips of fungi were transferred onto new potato dextrose agar (PDA) plates and incubated until conidia production. Six unidentified isolates with similar morphological characteristics were obtained. The colonies, initially white, darken to black after 7 to 10 days of incubation. They produced colorless, aseptate conidia that were oblong or fusiform, measuring 18-26 µm in length and 5-8 µm in width (n=50). The morphological characteristics of the isolates resembled those of Botryosphaeria (Udayanga et al. 2015) . Isolate IS2116-1 was further confirmed through molecular methods. The rDNA internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α), and beta-tubulin (TUB2) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R, and Bt2a/Bt2b (Ferreira et al., 2021; Carbone et al., 1999), respectively. BLAST analysis revealed that the ITS (OR958722) sequence was 100% similar to the B. dothidea isolate HZ5(MH329650.1), TEF1-a (PP214058) sequence was 100% similar to the B. dothidea strain JZB310220(ON890458.1), and strain TUB2 (PP214057) sequence was 99.78% similar to the B. dothidea strain L14 (KR260833.1). Maximum likelihood analyses were performed for the combined ITS、TUB2、TEF datasets using PhyloSuite v1.2.2, the resulting phylogenetic tree indicated that isolate IS2116-1 clustered together with Botryosphaeria dothidea in a clade with 97% bootstrap support(Zheng et al. 2020) . Pathogenicity tests were conducted on 3-6 month-old P. subaequalis seedlings (n = 5) grown in a greenhouse. A conidial suspension (106 spores/ml) collected from the isolates was sprayed onto P. subaequalis seedlings, while the control was treated with distilled water. All plants were maintained in a growth chamber at 28°C with a 12-h photoperiod. The experiment was conducted twice independently . After 20 days of inoculation, brownish lesions similar to those observed in the field appeared on the treated plants, while the noninoculated control plants remained symptomless. The pathogen was reisolated from the leaves of the obviously diseased seedlings and confirmed as B. dothidea through morphological and sequence analysis. No isolates were obtained from uninoculated control plants, thus fulfilling Koch's hypothesis. This report marks the first record of B. dothidea causing leaf blight in P. subaequalis. In light of the rarity of natural P. subaequalis populations, it is imperative to assess both the extent of disease spread and its economic impact. These insights are crucial for devising strategies to protect this endangered species from disease threats and to preserve its ecological significance.