First Report of Causing Branch Blight of Walnut in Chongqing, China.

Walnut (Juglans regia L.), a key economic tree species, plays an essential role in China's forestry sector. Our previous surveys indicated that the incidence of walnut branch blight reaches up to 60% in Chengkou County and Beibei District, Chongqing City, China. Over the past two years, branch blight has been frequently observed from July to December on both young and old branches, with a high incidence leading to fruit drop rates of up to 60% or even total crop failure, highlighting this disease as one of significant threats to the sustainable development of the walnut industry. In the field, symptoms on infected stems progressed from reddish-brown to dark brown, and longitudinal cracking. Four infected stems were collected from the walnut variety Yucheng No. 1 in Chengkou County and Beibei District. Twenty-six pathogenic fungi were isolated by tissue isolation method, including fifteen fungi with similar culture morphology. The pathogen was identified through microscopic examination, morphological characterization, pathogenicity tests, and the cloning of polygenic genes. Microscopic examination of the pure culture after 7 days of growth revealed that the pycnidia were ellipsoidal, with colorless, transparent conidia that were either spindle-shaped or oval, containing 1 to 2 oil droplets. 7-day-old conidia (n = 20) measured 4.3 to 8.5 μm in length and 1.5 to 2.8 μm in width. The culture was subsequently purified by single-spore separation. The strain CKZ4-3 was successfully reisolated from symptomatic plants, and display white, sparse mycelia on potato dextrose agar (PDA) medium. Following, the isolate CKZ4-3 was selected for further investigation. The pathogenicity test was conducted by using the needle pricking method to create small wounds on six healthy Yucheng No. 1 trees planted in an experimental field, and then inoculating the bacterial pellets and blank PDA on these wounds. Results showed that isolate CKZ4-3 induced necrotic lesions on the stems, causing them to turn dark brown and spread vertically, with necrotic spots ranging from 5 to 12 mm. The strain CKZ4-3 was successfully reisolated from symptomatic plants by single-spore separation, fulfilling Koch's postulates. Genomic DNA was extracted from cultures aged seven days. DNA was extracted and the ITS, TUB, GAPDH, Apn and EF1-α genes were amplified using primers ITS1/ITS4 (White et al. 1990), T1/T2 (Glass et al. 1995), GDF/GDR (Templeton et al. 1992), Apn2-F/R(Udayanga et al. 2014), and EF1-728F/EF1-986R (Udayanga et al. 2014) respectively. Sequence analysis using BLASTn in GenBank revealed that the ITS-rDNA, TUB2, Apn and EF1-α sequences of CKZ4-3 showed 99% (579/584 nt), 99% (533/539 nt), 99% (533/539 nt), 98% (750/768 nt) and 92% (320/349 nt)similarity, respectively, to Diaporthe eres (accession GQ281804, MZ724024, KJ380958 and KJ210550). The ITS (PP785566), TUB2 (PV052969), GAPDH (PV052972), Apn2 and EF1-α sequences of isolate CKZ4-3 clustered with Diaporthe eres using "Construct/Test Neighbor-joining Tree" in MEGA11. Previous studies have recognized Diaporthe as a major pathogenic group responsible for walnut branch blight (Chen et al. 2014; Wu et al. 2016; Liu et al. 2020; López-Moral et al. 2020). However, this study provides the first report of Diaporthe eres causing necrotic lesions on walnut branches in Chongqing city, China. The findings of this study will contribute to the theoretical framework for the comprehensive management of walnut branch blight, underscoring the need for effective disease control strategies in walnut-producing regions worldwide.