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First Report of Pathogenicity of Fusarium sporotrichioides and Fusarium acuminatum on Sunflowers in the United States
F. Mathew and B. Kirkeide, Department of Plant Pathology, North Dakota State University, Fargo, ND 58102-6050; T. Gulya, USDA Northern Crop Science Laboratory, Fargo, ND 58105-5677; and S. Markell, Department of Plant Pathology, North Dakota State University, Fargo, ND 58102-6050
Corresponding author: S. Markell. email@example.com
Mathew, F., Kirkeide, B., Gulya, T., and, Markell, S. 2010. First report of pathogenicity of Fusarium sporotrichioides and Fusarium acuminatum on sunflowers in the United States. Online. Plant Health Progress doi:10.1094/PHP-2010-0915-02-BR.
A sunflower field (Helianthus annuus L. cv. Pioneer 63M82) with uneven maturation was observed in Todd County, MN, in September 2009 (2). Thirty seven percent of the plants were wilted and approximately 12% of the plants had lesions consistent with charcoal rot [Macrophomina phaseolina (Tassi) Goid] despite a sub-optimal environment for disease development (2,3). Stem sections of the basal portion of infected plants were harvested and dissected. In additional to spherical microsclerotia consistent with M. phaseolina (3), a pink discoloration of the pith was observed (Figs. 1 and 2).
Small pieces of pink pith were surface-disinfected in 0.5% sodium hypochlorite and 70% ethanol for 60 s each, rinsed in sterile distilled H5O, transferred to potato dextrose agar (PDA) in petri dishes and incubated for 7 days at 22° to 25°C under fluorescent lights with a 12-h photoperiod. Macroconidia from aerial hypha were harvested and streaked on 1.5% water agar and after 3 days, individual colonies were transferred to PDA to record colony morphology, density, and extent of mycelia growth. Cultures from single-spore isolates formed characteristic pale pink to purple colonies, floccose mycelium, and slow growth. In addition to colony morphology, Fusarium spp. were identified by morphological characteristics of macroconidia as Fusarium oxysporum Schlechtend: Fr. (three septate, 23-54 × 3-4.5 µm); F. sporotrichioides Ellis & Everhar. (primarily three septate, 39-51 × 4-5 µm); and F. acuminatum Sherb (primarily three septate, long tapering apical cell, 30-55 × 3-4 µm) (4). DNA was extracted from lyophilized mycelium of the latter two Fusarium spp. using the DNeasy Plant Mini Kit (Qiagen, Valencia, CA) and its translation elongation factor 1-alpha (TEF1-α) gene region was amplified using ef1 and ef2 primers (6). PCR amplicons of ~700 bp were directly sequenced in both directions using the same primers, and a BLASTN search against the NCBI nucleotide database was performed using the consensus sequence generated by alignment of the forward and reverse sequences for this region. BLASTN search results confirmed 99% identity with F. sporotrichioides (closest match was accession no. EU744849) and 100% identity with F. acuminatum (closest match was accession no. FJ154737).
Kochs postulates were performed in a greenhouse with air temperatures ranging from 20° to 25°C and a 14-h photoperiod. For each pathogen, ten 3-week-old sunflower plants (cv. Pioneer 63M82) at the six to eight leaf growth stage were inoculated by securing mycelia agar plugs of 7-day-old cultures (PDA) with parafilm on stems wounded with sterile pipette tips using a method modified from Zazzerini and Tosi 1987 (8). The experiment was repeated twice. Necrosis was observed 10-days after inoculation with each pathogen. Plants inoculated with F. sporotrichioides exhibited necrotic girdling lesions on 100% of plants at 14-day post-inoculation, and wilting occurred on 80% of the plants by 21-days post inoculation (Fig. 3). Girdling necrotic lesions developed on plants inoculated with F. acuminatum at 14-days post inoculation, but wilting was not observed by 21-days when experiments were terminated. No plants inoculated with sterile agar plugs had symptoms. Both Fusarium spp. were re-isolated from necrotic tissue fragments within 3-cm of the inoculation point of five randomly-selected inoculated-plants per pathogen, fulfilling Koch's postulates. Association of M. phaseolina and an unidentified Fusarium spp. was previously reported in the United States (7). Despite their co-occurrence, Orellana (7) found that the Fusarium (then unidentified spp.) caused wilt and was more aggressive than M. phaseolina. In this brief, the specific Fusarium species causing disease on sunflowers in Minnesota were identified and pathogenicity was demonstrated but the relationship among them and M. phaseolina was not investigated. The occurrence of M. phaseolina and pathogenic Fusarium spp. may have implications for accurate diagnoses of the primary pathogen of diseased sunflowers. Sunflower plants infected by M. phaseolina exhibit visually recognizable symptoms and are easily identified by the presence of microsclerotia, but the presence of pink tissue (caused by Fusarium spp.) in stalks should not be assumed to be a secondary infection. In a disease survey of sunflower fields conducted between 1999 and 2001 in the Krasnodar region in Russia, sunflowers with wilt symptoms were collected and F. oxysporum, F. solani, and F. sporotrichoidies were the most common species recovered (1). When the effect of F. oxysporum and F. sporotrichoidies isolates on sunflower emergence were compared, F. sporotrichoidies reduced emergence more than F. oxysporum (1). F. sporotrichoidies was commonly found on wheat-bioassays in North Dakota (5), the leading producer of sunflowers in the United States and a border state to Minnesota. Despite pathogen ubiquity in the region, current and future implications of Fusarium spp. on disease incidence, severity and yield loss are unclear. To our knowledge, this is the first report of F. sporotrichoides and F. acuminatum causing disease on Helianthus annuus in the United States.
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