First Report of Alternaria tenuissima Causing Leaf Spot on Luffa cylindrica in China

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.