Fusarium wilt is one of the most devastating diseases of cotton (Gossypium spp.). It is caused by the soil-borne pathogen Fusarium oxysporum Schlechtend. f. sp. vasinfectum (Atk.) Snyd. & Hans (Atkinson, 1892). To date, eight races of F. oxysporum f. sp. vasinfectum have been reported worldwide based on their diverse genetics and reactions to the host (Cianchetta, et al. 2015). Fusarium wilt symptoms appear at all developing stages. The fungus enters through the roots and colonizes the vascular system, leading to discoloration and wilting (Davis et al. 2006). During a survey in the July 2021 growing season, cotton plants showing typical wilting symptoms, stem discoloration, and root rot (Fig. 1A and B) were observed in two cotton fields in Sumner County, Kansas. In order to confirm the causal agent, root tissues and lower parts of the stems were collected from 25 diseased cotton plants and cleaned as previously described by Larren et al, (2001). Small segments (1-2 cm) of roots and stems were incubated on potato dextrose agar (PDA) at room temperature. The isolates were purified with the single spore method and 23 of 25 isolates (92%) showed typical morphology of F. oxysporum, with a white-peach pigmentation as reported previously (Leslie and Summerell 2008). Two isolates, CK13B and CK18A, one from each of the two fields were selected, and genomic DNA was extracted with E.Z.N.A Fungal DNA Mini kit (Omega Bio-tek, Norcross, GA). Polymerase chain reaction (PCR) assays were performed as reported previously (Ortiz, et al. 2017) to amplify and sequence a portion of three nuclear genes: translation elongation factor (EF-1α), phosphate: H* symporter (PHO), and β-tubulin (BT). BLASTn analysis of CK13B sequences showed 93-98% identity with F. oxysporum f. sp. vasinfectum isolates originating from Australia, with 93.43% (1706/1826 pb) identity of EF-1α to isolate AuSeed14 (KT323873), 95.11% (1849/1944 bp) of PHO to isolate AuK24232 (KT323909), and 97.56% (1840/1886 bp) identity of BT to isolate AusSeed14 (KT323833). Sequences of CK13B for EF1-α, PHO, and BT genes were submitted to GenBank, accession no. of ON754247, ON754248, and ON754249, respectively. The CK18A isolate showed high identity with F. oxysporum f. sp. vasinfectum races 1, 2, and 6 lineage isolates (Ortiz et al., 2017), with 99.89% (1822/1824 bp) identity of the EF1-α gene to isolate CDR238 (KT323838) from Arkansas, 98.36% (1916/1948 bp) identity of the PHO gene to isolate CDR1131 (KT323887) from Louisiana and 98.78% (1864/1887 bp) identity of the BT gene to isolate ATCC36198 (KT323799) from Brazil. Sequences of the CK18A isolate were submitted to GenBank, accession no. ON651444, ON725043, and ON725044, respectively. Pathogenicity of both isolates was tested on G. hirsutum as previously described (Kim et al. 2005) with three replicates per isolate and a control treatment in a growth chamber. Briefly, cotton seedlings were maintained with 12 h light/dark cycle at 25-20 °C. At the first true leaf stage seedlings were uprooted, roots were cleaned by rinsing with sterile water then placed in a conidial suspension (1×106 conidia/ml) for 2 h while the healthy control seedlings were dipped in water only. Plants were put into sterile soil, and the results were observed and recorded every seven days for a period of 30 days. The inoculated plants showed the same symptoms as those observed in the fields, wilting, leaf chlorosis, and necrosis. Both isolates were re-isolated from the roots of the inoculated plants. DNA was extracted and used for PCR with the specific gene primers as listed above. The sequences matched those of the original isolates, completing the Koch's pustulates. To our knowledge, this is the first report of F. oxysporum f. sp. vasinfectum causing Fusarium wilt disease of cotton in Kansas. These results will help growers select cultivars and design disease control strategies accordingly (Davis, et al., 2006). Further work is needed to determine the specific races in the area.
Keywords: Causal Agent; Disease management; Fungi; Pathogen detection; Subject Areas.