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First Report of Leaf Spot Disease Caused by Alternaria alternata on Carya illinoensis in China

Pecan (Carya illinoensis) is an important tree for commercial nut production in North America and widely cultivated in China. In September 2019, leaf spot symptoms were observed on the leaves of C. illinoensis in an ecologic orchard in Chuzhou, Anhui, China (32°10′20″N, 118°20′12″E), with a disease incidence of 90%. The initial symptoms appeared as small circular to irregular dark brown or black spots on the leaves. The lesions enlarged and coalesced into large necrotic areas, which later resulted in leaf abscission and stunting. Disease symptoms were not observed on the fruits. To isolate the pathogen, leaf fragments (3 to 4 mm) from symptomatic leaves were surface sterilized with 75% ethanol for 30 s and 0.1% HgCl₂ solution for 30 s, rinsed three times in sterile distilled water, placed on potato dextrose agar (PDA) plates, and incubated at 25°C in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate, CZ-15, growing on PDA was olivaceous and circular, with abundant aerial mycelium, and light brown on the reverse with white borders on PDA. The conidia were ellipsoidal, subellipsoidal, to ovoid with a short conical beak at the tip, light brown to dark brown with one to six transverse and zero to three longitudinal septa and were in the range of 17.15 to 28.41 × 7.54 to 18.54 µm (n = 50). Based on observed cultural and morphological features, this fungus was tentatively identified as Alternaria alternata (Simmons 2007). Genomic DNA was extracted from single conidial cultures of representative isolate CZ-15, and the internal transcribed spacer (ITS), 18S ribosomal RNA (SSU), 28S ribosomal RNA (LSU), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and anonymous region (OPA10-2) genes were amplified with the primers described by Woudenberg et al. (2015). The obtained sequences showed 98 to 99% similarity with those from A. alternata accessions in GenBank. The sequences from this isolate were deposited in GenBank under the following accession numbers: ITS, MN636274; SSU, MN636283; LSU, MN636275; GAPDH, MN650588; TEF1, MN650589; and OPA10-2, MN650590. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate CZ-15 clustered in the A. alternata clade with 99% bootstrap support. To test pathogenicity, 10 detached healthy leaves and 10 1-year-old C. illinoensis plants were inoculated by excising 5-mm mycelial plugs from a 7-day-old colony grown on PDA and placing them on the adaxial surfaces of leaves. As a control treatment, 10 additional detached leaves and potted seedlings were inoculated with 5-mm PDA plugs without mycelia. All plants were covered with clear polyethylene bags and incubated in a growth chamber at 23 ± 5°C, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. Seven days after inoculation, the symptoms were similar to those on the original infected plants, whereas the control leaves remained symptomless. A. alternata was reisolated from the lesions and morphologically identified, confirming Koch’s postulates. To our knowledge, this is the first report of A. alternata associated with leaf spot disease on C. illinoensis. This study provides a foundation to further investigate the biology, epidemiology, and management of this disease.