First Report of Tomato Verticillium Wilt Caused by <i>Verticillium dahliae</i> in Mexico

Mexico is the seventh largest producer of tomatoes worldwide, with approximately 4.2 million tons reported for 2023. In November 2023, tomato cultivar Pai Pai with symptoms of wilting and yellowing of lower leaves, which rapidly affected young leaves, was observed in the Baja California State (Geographical coordinates 30°34'00.0"N 115°56'00.0" W). The disease incidence was 20%, with a severity index of 25% in a combined area of 10 ha corresponding to two greenhouses. Pieces of leaves were taken from the edge of the lesions from five plants and disinfested with 70% ethyl alcohol and washed with distilled water twice; subsequently, they were placed on plates with PDA (potato dextrose agar) culture medium and incubated at 25°C for 7 days. Pure cultures were obtained by transferring hyphal tips from the growth medium to new plates containing PDA. The colonies obtained changed from white to black, and preparations were mounted with lactophenol blue and analyzed under the microscope. Characteristic structures for the fungus Verticillium dahlie such as a large number of black microsclerotia of variable shape and size measure 49.8 (33.8-63.5) µm (n=30); as well as hyaline, and vertically branched conidiophores; and elliptical and hyaline conidia which measure 4.98 (3.6-5.9) x 1.52 (1.01-2.3) µm (n=100) were observed (Yu et al., 2016). For molecular identification, four isolates were selected to carry out the DNA extraction. First, the species-specific pair of pimers Df (5’-CGGTCCATCAGTCTCTCTG-3’) and Dr (5’- CTGTTGCCGCTTCACTCG-3’) reported by Inderbitzin et al. (2013) was used to amplify a fragment of 490 bp. To confirm this result, the amplification and sequencing of a partial fragment of the ITS region for one representative isolate were conducted with the primers ITS1 and ITS4 (White et al., 1990). The sequence was aligned in the GenBank Database, showing a match of 466/466 base pairs with 100% homology with Verticillium dahliae from TYPE material (GenBank Access number NR_126124), and the phylogenetic analysis placed the strain BC-COL (GenBank Access number PQ096068) in a monophyletic clade with several strains of V. dahliae. Koch's postulates were conducted. Thirty-day-old seedlings of cultivar Pai Pai were root-dipped into a spore suspension of the strain BC-COL containing 1 X 106 esp/mL for 10 min. Non-inoculated control seedlings were dipped in sterile distilled water (n=10). Seedlings were transplanted into coco peat grow bags and maintained under greenhouse conditions at 21°C-25°C. This test was repeated twice. Characteristic V-shaped inter-veinal yellowing was observed in all inoculated plants one month after inoculation. The fungal pathogen was re-isolated from damaged leaves on plates with PDA medium. For confirming colonization with V. dahliae, the Df-Dr primers were used in PCR to generate a fragment of 490 bp only from the DNA obtained from inoculated plants. Verticillium dahliae is a soilborne fungal pathogen distributed worldwide, which colonizes the vascular system and interferes with water and nutrient transport. In the tomato crop, V. dahliae has been reported in Canada, the United States, the Netherlands, and Uzbekistan. In Mexico, there are reports of this fungus affecting two wild potato species (Solanum cardiophyllum and Solanum ehrenbergii) (Rodríguez-Alvarado et al., 2000). This is the first report of V. dahliae causing Verticillium wilt symptoms in tomatoes in Mexico, and this information is relevant to contribute to the prevention of the spread of this fungus.

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