First Report of <i>Didymella bellidis</i> Causing Branch Blight of Walnut in Chongqing, China

Walnut (Juglans regia L.) is an economically important tree species widely cultivated in China. Over the past two years, branch blight has been frequently observed from July to December, with a high incidence leading to fruit drop rates of up to 60% or even total crop failure. Walnut branch dieback disease affected 30% to 40% of the observed walnut trees, culminating in significant financial losses for walnut growers in five major walnut planting towns (Gaoguan, Dong’an, Houping, Zhiping and Mingzhong) in Chengkou County and two towns (Tianfu and Jindaoxia) in Beibei District, Chongqing City, China. In the field, the phloem of the infected stems progressed from reddish-brown lesions to black-brown and developed longitudinal cracks. Diseased stems of the variety Yucheng No. 1 from Chengkou County and Beibei District were collected for organizational separation. A representative isolate, CKZ1-4, was obtained, purified, and identified based on morphological characteristics. Microscopic examination revealed that the pycnidium of CKZ1-4 was spherical, containing scattered conidia. The conidia were colorless, elliptical to circular, single-celled, measuring 4.2 to 8.6 μm in length and 1.4 to 2.9 μm in width (n = 20). The colony of CKZ1-4 appeared dark green and off-white, displaying dense and flat mycelia on potato dextrose agar (PDA) medium. Pathogenicity tests were conducted on Yucheng No. 1 trees planted in an experimental field using the needle penetration to create small wounds on six differen healthy branches from different trees and then inoculating with methods of inoculum cake in vitro on these wounds. Ten days after the inoculation, we observed that isolate CKZ1-4 induced necrotic spots on the stems, leading to dark brown stems with necrotic lesions measuring 7 to 11 mm. Upon cross-sectioning the lesions, it was observed that the lesions appeared sunken. Didymella bellidis was reisolated from the infected branches by organism isolation and single-spore separation, satisfying Koch’s postulates. Genomic DNA was extracted from 7-day-old cultures, and ribosomal DNA internal transcribed spacer (ITS-rDNA), β-tubulin (TUB2), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using the primers ITS1/ITS4 (White et al. 1990), T1/T2 (Glass et al. 1995), and GDF/GDR (Templeton et al. 1992), respectively. CKZ1-4 ITS-rDNA, TUB2, and GAPDH sequences showed a similarity of 99% (542/544 nt), 99% (336/337 nt), and 99% (152/154 nt) to Diaporthe bellidis (accession OQ190215, MZ546166, and KT287042, respectively). The sequences were submitted to GenBank: ITS (PQ779080), TUB2 (PV052970), and GAPDH (PV052971). The sequences of isolate CKZ1-4 were classified under Didymella bellidis. Previous research has identified Didymella bellidis as a significant pathogen responsible for leaf spot disease (Sun et al. 2022; Tang et al. 2023; Indermaur et al. 2023). This is the first report of Didymella bellidis causing necrotic spots on walnut stems in Chongqing, China. This discovery expands the known host range of the pathogenic Didymella bellidis and will help provide essential information for effective measures to manage this disease.