A three-year survey from 2010 to 2012 was conducted in Kansas to investigate the identity and diversity of seedborne Fusarium spp. in soybean. A total of 408 soybean seed samples from 10 counties were tested. One hundred arbitrarily selected seeds from each sample were surface-sterilized for 10 min in a 1% sodium hypochlorite solution to avoid contaminants and promote the isolation of internal fusaria. Seeds were rinsed with sterile distilled water and dried overnight at room temperature (RT). Surface-sterilized seeds were plated on modified Nash-Snyder medium and incubated at 23 ± 2°C for 7 days. Fusarium isolates were single-spored and identified by morphological characteristics on carnation leaf agar (CLA) and potato dextrose agar (PDA) (3). From 276 seedborne Fusarium isolates, six were identified as F. thapsinum (2). On CLA, F. thapsinum isolates produced abundant mycelium and numerous chains of non-septate microconidia produced from monophialides. Microconidia were club-shaped and some were napiform. No chlamysdospores were found. On PDA, three of the isolates presented characteristic dark yellow pigmentation and three were light violet. Confirmation of the isolates to species was based on sequencing of an elongation factor gene (EF1-α) segment using primers EF1 and EF2 and the beta-tubulin gene using primers Beta1 and Beta2 (1). Sequence results (~680 bp, EF primers; ~600 bp, beta-tubulin primers) were confirmed by using the FUSARIUM-ID database (1). All isolates matched F. thapsinum for both genes sequenced (Accession No. FD01177) at 99% identity. Koch's postulates were completed for two isolates of F. thapsinum under greenhouse conditions. Soybean seeds (Asgrow AG3039) were imbibed with 2.5 × 105 conidia ml−1 for 48 h. After inoculation, seeds were dried for 48 h at RT. One isolate each of F. equiseti and F. oxysporum were used as the non-pathogenic and pathogenic inoculation controls, respectively. In addition, non-inoculated seeds and seeds imbibed in sterile distilled water (mock) were also used. Twenty-five seeds from each treatment were planted in pots (500 ml) with autoclaved soil and vermiculite (1:1). The experiment was a completely randomized design with three replicates (pots) per isolate. The entire experiment was repeated three times. After 21 days, aggressiveness of both F. thapsinum isolates was assessed using initial stand (%), final stand (%), and seed mortality (% of non-germinated seeds). Both seedborne F. thapsinum isolates caused reduced emergence and final stand, and increased seedling mortality when compared to the non-inoculated and F. equiseti controls (P< 0.0001). No significant difference was observed between F. thapsinum isolates and F. oxysporum. F. thapsinum isolates were re-isolated from wilted seedlings and non-germinated seeds, but not from the control treatments. Typically, F. thapsinum is considered a pathogen of sorghum, but it has also been recovered from bananas, peanuts, maize, and native grasses (3). However, its presence on soybean plant tissues and its pathogenicity has never been reported. To our knowledge, this is the first report of seedborne F. thapsinum and its pathogenicity on soybean in the United States. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) C. J. R. Klittich et al. Mycologia 89:644, 1997. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006.
Decolourization of congo red synthetic dyes by dark septate endophytes
