First report of leaf blight caused by Alternaria alternata on Lactuca indica in China.

Lactuca indica, an annual or biennial herbaceous plant, is widespread in valleys, shrubland, ditches, hillside meadows or fields (Wang et al. 2003). In China, it is widely used as medicine and high protein feed for herbivorous animal husbandry. In July 2022, leaf blight on L. indica was observed at Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. 70% of the 87 plants investigated were infected. Small brown spots with a yellow halos first appeared on the leaves, then became irregular necrotic spots until the entire leaf wilted and fell off. To identify the pathogen, four symptomatic leaves were collected and disinfected according to Wang et al. 2023. Then they were transferred onto potato dextrose agar (PDA), and incubated at 28°C for 7 days. To obtain the pure culture, the marginal mycelium was transferred to a new PDA plate. The colony of the isolated LPB-1 was light gray and regularly round at the early stage, and then changed to dark gray and villous. The back of the culture plate appeared sooty black. The conidia of the isolated fungi (n=50) were in chains, brown, obclavate, ovoid or ellipsoid, with an average size of 29.09 µm long and 6.41 µm wide, with 0 to 3 longitudinal and 1 to 7 transverse septa. These cultural and morphological characteristics were consistent with those of Alternaria alternata (Simmons 2007). To identify the strain, internal transcribed spacer (ITS) region, RNA polymerase Ⅱ second largest subunit (RPB2), and translation elongation factor 1-alpha (TEF1-α) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), RPB2-5F/RPB2-7cR (Liu et al. 1999) and EF1-728F/EF1-986R (Carbone & Kohn 1999). The RPB2 (OP909715), TEF-1α (OP909714), and ITS (OP776880) were 99 to 100% identical to those of A. alternata (GenBank accession nos. MZ170963.1, MK605900.1, and MK605895.1 for RPB2 sequences; ON951981.1, KJ008702.1, and MK672900.1 for TEF-1α sequences; OP850817.1, OP811328.1, and OP740510.1 for ITS sequences). In addition, the phylogenetic analysis also showed that the stain LPB-1 was A. alternata. To complete Koch's postulates, the conidial suspension (1×108 conidia/mL) were spray-inoculated on healthy leaves of three mature L. indica plants with sterile water as a control. All plants were incubated at 28 ℃ in a greenhouse with 12-h-light/12-h-dark photoperiod and approximately 70% humidity (Li et al. 2019). Fourteen days after incubation, the inoculated leaves showed symptoms similar to those of naturally infected leaves, while the controls remained asymptomatic. The pathogen reisolated from the inoculated leaves had the same morphological characteristics and molecular identification results as the original isolate. All the results shown above indicated that A. alternata was responsible for the leaf blight of L. indica. As far as we know, this is the first report of leaf blight caused by Alternaria alternata on Lactuca indica in China. The identification of the pathogen could provide relevant information for the establishment of methods to control the disease.