China is the world's largest producer and exporter of carrot (Daucus carota L. var. sativa), a well-known and nutritious root vegetable. In the spring seasons of 2021-2023, dark brown lesions were observed on field-grown and cold-stored carrot roots in the Xiamen City, Fujian Province, China. Although just discovered in recent years, the disease has expanded from the initial point to the most planting area in there, and causing over 20% yield loss in the most severely affected fields. This disease symptom is consistent with black rot, a carrot disease found globally and caused by the fungal pathogen Alternaria radicina (Saude et al. 2006). Small pieces of symptomatic roots (3 to 5 mm) from diseased carrot roots were surface disinfested with 75% alcohol for 3 minutes and 10% sodium hypochlorite solution for 8 minutes, and then rinsed in sterile distilled water and cultured on potato dextrose agar (PDA) medium at 28°C with a 12-h dark/light photoperiod. By tissue isolation and single-spore culture, six isolates were obtained from the disease plants in the past three years, and used for morphological and molecular analyses. Unexpectedly, the morphological characteristics of conidia of the six isolates, shape (long and ellipsoid), size (27 to 60 × 17 to 21 μm, n =50), and color (dark olive-brown) were similar to those of A. radicina, but with more transverse septae (Figure 1, Trivedi et al. 2010). Meanwhile, compared to A. radicina, the colony's margin was highly uniform and smooth, without an accumulation of yellow pigment and with fewer dendritic rhizomycin crystals at the bottom of agar media. These characteristics were similar to those of Alternaria carotiincultae, but not to those of A. radicina (Park et al. 2008). Genomic DNA of the six isolates was extracted and further molecular identification was performed. The EF-1α gene was amplified using EF-1/EF-2 primer pairs and sequenced (O'Donnell et al. 1998.). The EF-1α gene sequences of the six isolates were compared using DNAMAN 5.2.9 software. They were found to be 100% identical, and the sequences has been deposited in GenBank under accession number OR449062. BLAST analysis of the amplicon revealed 100% nucleotide sequence identity with the A. carotiincultae strain YZU 151039 (GenBank accession No. MK279390), while 2bp difference between the sequences of A. radicina strains present in GenBank. Pathogenicity tests of the isolate were carried on the unwounded carrots (5 roots each, with three replications). These tap roots were surface disinfected with 75% alcohol and disinfected by immersion in 0.75% sodium hypochlorite solution for 10 minutes, then immediately rinsed with sterile water and dried with sterile filter paper. Mycelial plugs (6 mm diameter) were taken from the margin of a vigorously growing colony and inoculated into the sterilized carrot roots. Another group of sterilised tap roots inoculated with sterile agar plugs were used as negative control. All the roots were incubated in the dark at 25°C with 80% humidity. After 2 days, colonies were observed on the surface of the roots that had been inoculated with the mycelial plugs. After 7 days, the inoculated tap roots showed symptoms of black rot with dark brown sunken lesions as the diseased plants in the field. However, no disease was observed on the control roots. Following the previous method, a strain was re-isolated from the inoculated carrot roots and again identified as A. carotiincultae, thus fulfilling Koch's postulates, and confirming that A. carotiincultae is the pathogen causing dark brown lesions of carrots. To our knowledge, this is the first report of A. carotiincultae causing carrot black rot in China. Attention should be paid to the damage caused by this pathogen during the production and storage stages of carrots, and strategies should be developed to prevent its spread.
Keywords: Causal Agent; Crop Type; Fungi; Pathogen detection; Subject Areas; Vegetables.