First Report of Verticillium dahliae on Cotton in Alabama

Verticillium wilt of cotton (Gossypium hirsutum L.) in Alabama is estimated to reduce yields by an average of 1.5%, for over $3 million lost to this disease (Lawrence et al. 2015). It was believed that Verticillium albo-atrum was the causal agent of wilt in multiple crops statewide (Palmateer et al. 2004). However, Verticillium dahliae has recently been identified as the causal agent of Verticillium wilt in northern Alabama cotton, which may be why the disease is more economically devastating that region. Two cotton variety trials were conducted in Colbert and Madison counties in north Alabama in 2013 and 2014 in fields with a history of Verticillium wilt. Petioles were collected in early September during flowering and boll opening from symptomatic plants of multiple cotton varieties across replications. Petioles were diced into 1-mm pieces, surface sterilized, and aseptically placed onto water agar media with 10 diced petioles per petri dish. Cultures were incubated at 22.2 to 24.4°C for 5 to 14 days when aerial whorled conidiophores with conidia appeared, allowing for identification to a Verticillium species. Isolates were initially identified to species by transferring vacillated conidiophores onto Verticillium minimal medium (VMM) (Puhalla and Mayfield 1974). Verticillium dahliae was identified by observing morphological characteristics (Hawksworth and Talboys 1970). Phialides for the V. dahliae species ranged from 16 to 35 µm in length × 1.0 to 2.5 µm wide as found in the original descriptions. Conidia also extended between 2.5 to 6 × 1.4 to 3.2 µm, just 2 µm shorter the type species. Microsclerotia were abundant producing a colony with a black reverse. The microsclerotium presence separates V. dahliae from V. albo-atrum morphologically. DNA sequencing was also used to confirm V. dahliae. DNA was isolated using the DNeasy Plant Mini Kit from Qiagen Inc. (Germantown, MD). The primers ITS1 and ITS4 (White et al. 1990) were used to amplify the internal transcribed spacer (ITS) region (∼500 bp), which was sequenced at the Auburn University Genomics and Sequencing Lab. Sequence results were cross-referenced with NCBI GenBank. GenBank Megablast showed 99% shared identity between the sequence generated in this study (Accession No. KT318493) and V. dahliae VdLs.17 (Accession No. CP010980.1). Koch’s postulate were fulfilled using V. dahliae cultures in microplot tests. Microplots were located outside of the greenhouse. Plots were planted in late May and samples and incidence values were collected in early September. V. dahliae microsclerotia were increased on VMM plates, blended in water, filtered through cheesecloth, quantified; 250 ml added to 24 liters of Decatur silt loam soil and cv. Croplan 3787 B2RF was planted. The experiment was a RCBD, replicated 5 times, and the test was repeated once for a total of 130 experimental units. Wilt symptoms were initially observed at midseason with incidence recorded. Verticillium wilt incidence ranged from 5 to 43% of the plants with discolored vascular cylinders in the infested soil. The plants in the noninfested microplots did not develop foliar symptoms of Verticillium wilt or was vascular discoloration observed. Petioles collected from symptomatic plants confirmed the presence of V. dahliae by reisolation. This is a confirmation of V. dahliae causing wilt in cotton in northern Alabama. To our knowledge, this is the first report of V. dahliae causing Verticillium wilt in Alabama. © 2016, American Phytopathological Society. All rights reserved.