First Report of Alternaria alternata Causing Alternaria Leaf Spot of Black Nightshade in China.

Black nightshade (Solanum nigrum L.) belongs to Solanaceae family, which is distributed all over China and has become one of the malignant weeds in cotton field in Xinjiang (Zhang et al., 2020; Zhang et al., 2021). During July 2016 to August 2023, leaf spot was observed on black nightshade in Hami and Shihezi city, Xinjiang, China. The spots appeared as brown circular or irregular, with some small black dots surrounded with yellow halos. Twenty five black nightshade plants were collected from a cotton field (100 m2) for pathogen isolation in Shihezi, July 2023. Leaf samples (2×2 mm) from the boundary between diseased tissue and healthy tissue, sterilized in 70% ethanol for 45 s, dipped in 2% NaClO for 30 s, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA) after dried and cultivated at 25°C in the dark for 4 days. Twenty-five fungal isolates were obtained and purified by using the single-spore isolation method, and all isolates were found to be similar in morphology, appeared as dark brown and produced dark brown pigmentation on PDA. Conidiophores were black brown, 8 to 58 × 10 to 20 μm, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, the results similar to those of Alternaria alternata isolated from buckwheat (Li et al., 2021). To confirm this identification, total genomic DNA of the isolates were extracted by CTAB method (Doyle et al., 1987). The internal transcribed spacer (rDNA-ITS), Alternaria major allergen gene (Alt α1) and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) of the A. alternata 26 were amplified using the primers ITS1/ITS4 (Glass et al., 1995), Altα1-F/Altα1-R (Li et al., 2024) and gpd-F/gpd-R (5'-CAACGGCTTCGGTCGCAT- TG-3' / 5'-GCCAAGCAGTTGGTTGTG-3'), respectively. The rDNA-ITS, Altα1 and GAPDH gene sequences were deposited in GenBank (KX904867, PP263361, PP263360), and showed 100% identity (rDNA-ITS: 572 out of 609 bp; Alt α1: 514 out of 516 bp; GAPDH: 619 out of 619 bp) to A. alternata (KU179665; MW522975; MK451977), respectively. The multi-gene phylogenetic tree showed that the representative isolate was grouped with A. alternata and identified as A. alternata. Black nightshade was used for pathogenicity assay. Fifteen plants at 3 to 4-leaf stage were selected and sprayed with the conidial suspension (1.0×106 spores/mL) of A. alternata 26 after wound the underside of leaves (2 mm length) with inoculating needle. Five plants were sprayed with sterilized water as control. After inoculation, placed the plants in a plastic box and covered with plastic wrap to keep 80% relative humidity for 3 days at 25℃ in the greenhouse, then removed the plastic box. Symptoms of leaf spots appeared within 12 - 15 days that were similar to the symptoms observed in the field, and the fungal pathogen that was re-isolated from symptomatic leaves was identical to original pathogen on the basis of morphological and molecular analysis to fulfill Koch's postulates. This experiment was repeated three times, and the disease incidence was above 80% after inoculation at each time. To our knowledge, this is the first report of A. alternata causing leaf spot of black nightshade in China. This report will help us to monitoring distribution of the disease and providing theoretical basis for disease prevention and control.