First Report of Alternaria tenuissima Causing Leaf Black Spot on Pecan in China

China is one of the largest markets for pecan (Carya illinoinensis) consumption in the world, and pecan production in China has increased since 2008 (Zhang et al. 2015). From July to September 2019 and 2020, leaf black spot was observed on several pecan cultivars, including Pawnee, Burkett, Kiowa, and Western Schley, in the germplasm in Liuhe County, Nanjing, Jiangsu Province, China. Disease incidence was approximately 40% in 2019 and 50% in 2020. Small, dark-brown to black spots on leaves were observed initially, and spots expanded quickly into circular or irregular patches when spots coalesced. In severe cases, the disease can obviously weaken tree vigor, ultimately leading to losses in yield. Disease symptoms were not observed on the fruit. To determine the causal agent of black leaf spot, symptomatic leaves were collected and cut into pieces (approximately 3 by 3 cm²), surface sterilized with 1% sodium hypochlorite for 2 min and 75% ethanol for 30 s, and rinsed twice with sterile double-distilled (dd)H₂O. Dried tissues were placed on potato dextrose agar (PDA) amended with rifampin (Solarbio, Beijing, China) at a final concentration of 100 µg/ml and incubated at 25°C for 7 days in darkness. Five colonies were obtained and purified by single-spore culture for morphological characterization. Colonies were initially white, then turned to dark olivaceous with a white margin and moderate to abundant gray aerial hyphae. Conidiophores were linear, light brown in color, and appeared as individuals or in clusters. Conidia were pale brown to brown, typically obclavate or obpyriform (8 to 49 × 3 to 18 µm), with one to five transverse septa and zero to three longitudinal septa (n = 50). Conidia of all isolates produced no beaks or a short beak. Based on conidial morphological characteristics, isolates were tentatively identified as Alternaria tenuissima (Simmons 2007). To further confirm the five isolates, the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA), partial region of the histone 3 (H3) gene, translation elongation factor 1-α gene (TEF), and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with PCR primer sets ITS1/ITS4 (White et al. 1990), H3-1a/H3-1b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), and GDF1/GDR1 (Berbee et al. 1999) respectively. The sequences were deposited in GenBank (ITS, MN822659 to MN822661 and MZ182355 to MZ182356; H3, MN840997 to MN840999 and MZ202355 to MZ202356; TEF, MZ246595 to MZ246599; and GAPDH, MZ246590 to MZ246594). BLAST analysis of the resulting sequences showed 99 to 100% nucleotides identical to those of A. tenuissima isolates (KP278184 [ITS]; MH824352; MN046379 [TEF]; and MK683840 [GAPDH]). Therefore, based on morphological characteristics and DNA sequences data, the five isolates were identified as A. tenuissima. To determine the pathogenicity of the five isolates, 10 µl of a suspension of 10⁵ conidia/ml from each isolate was placed on three intact young leaves (Pawnee). Leaves inoculated with ddH₂O in the same manner served as the controls. Inoculated leaves were placed in a growth chamber at 28°C with 95% relatively humidity. Black spot symptoms appeared on all inoculated leaves by 7 days postinoculation, and A. tenuissima was reisolated from the inoculated leaves with isolates and reconfirmed by morphological characteristics, thus fulfilling Koch’s postulates. Controls remained symptomless. Pathogenicity tests were conducted twice. To our knowledge, this is the first report of A. tenuissima causing black leaf spot on pecan in China. Although we have not found any fruit infection, we think the disease is also a potential threat to pecan fruit and production. If more reports of this pathogen are found on pecan, then it will be necessary to study and develop effective control strategies.