Distribution and Frequency of Fusarium Species Associated with Soybean Roots in Iowa.

A 3-year survey was conducted in Iowa to characterize the distribution and frequency of species of Fusarium associated with soybean roots. Ten plants were collected from each of 40 to 57 fields each year at V2 to V5 and R3 to R4 soybean growth stages. Fusarium colonies were isolated from symptomatic and symptomless roots and identified to species based on cultural and morphological characteristics. Species identification was confirmed by amplification and sequencing of the translation elongation factor (EF1-α) gene. Fifteen species were identified; Fusarium oxysporum was isolated most frequently, accounting for more than 30% of all isolates. F. acuminatum, F. graminearum, and F. solani were also among the most frequent and widespread species. Eleven other species were recovered from few fields, accounting for less than 10% of all isolates in a given year. No consistent trends were observed in geographic distribution of species. Variability in species frequency was found between soybean growth stages. Fusarium oxysporum was recovered at higher frequency during vegetative stages (40%) than reproductive stages (22%). Conversely, species such as F. acuminatum, F. graminearum, and F. solani were recovered more often from reproductive-stage plants. No significant differences in species composition were observed among fields differing in tillage practices and row spacing.

First Report of Fusarium commune Causing Damping-off, Seed Rot, and Seedling Root Rot on Soybean (Glycine max) in the United States.

During 2007 to 2009, symptomatic and asymptomatic soybean plants were collected from fields in 18 Iowa counties. Fusarium isolates were recovered from surface-sterilized root tissue on peptone PCNB agar (2). Single-spore isolates were transferred to synthetic low nutrient agar (SNA) overlain with pieces (1 × 2 cm) of sterile filter paper, and to potato dextrose agar (PDA), and placed in the dark for 10 to 14 days for morphological identification (4). Twenty-three isolates were identified as Fusarium commune K. Skovg., O'Donnell & Nirenberg, previously in the F. oxysporum species complex (4). Colonies on PDA had white, fluffy, aerial mycelium with magenta to violet pigmentation in the medium. On SNA, macroconidia, chlamydospores, and microconidia on monophialides and polyphialides were consistent with the species description (4). Identification of all 23 isolates was confirmed by DNA sequencing of the translation elongation factor (EF1-α) gene, using ef1 and ef2 primers, and the mitochondrial small subunit (mtSSU), using primers MS1 and MS2 (4) [GenBank accessions for two representative isolates: EF1-α (JX289892 and JX289893), and mtSSU (JX289894 and, JX289895)]. Pathogenicity of two representative isolates of F. commune was tested on soybean (cv. AG2403) in a greenhouse, in water baths set at 18°C, using autoclaved soil mixed with infested sand-cornmeal inoculum (3). The experiment entailed a completely randomized design (CRD) with five replications (single plant/150 ml cone) per treatment, and was conducted three times. Dry root and shoot weights, and root rot severity (visual estimate of percent root rot on the entire root system) were evaluated after 6 weeks. Mean seedling emergence in soil infested with F. commune was 47 and 40% for the two isolates; in contrast, non-inoculated control plants had 100% emergence. There were significant differences in root (P < 0.0001) and shoot (P < 0.0001) weights, and root rot severity (P < 0.0001), between inoculated and non-inoculated plants. Seedlings that emerged were severely stunted and had dark brown lesions. F. commune was reisolated from infected roots of inoculated plants, but not from non-inoculated plants. Pathogenicity of both isolates to soybean (cv. MN1805) was also tested using a petri dish assay, in which eight seeds were placed on a plate with a 4-day-old culture growing on 2% water agar (1). Plates were rated 7 days later for seed germination, seed rot, and lesion development, using an ordinal scale (1). The experiment entailed a CRD with three replicate plates/treatment, and was conducted three times. Germination of inoculated seeds ranged from 37.5 to 75.0%, and germinated seedlings had dark brown lesions on the taproots. There was a significant difference between isolates in the petri dish assay (P = 0.0030); one isolate was less aggressive, but both isolates resulted in significantly more disease than on the non-inoculated control plants, which had 100% germination and no symptoms (P < 0.0001). F. oxysporum is a known soybean pathogen (1), but isolates of F. commune may have been misidentified as F. oxysporum in previous studies. To our knowledge, this is the first report of F. commune as a pathogen on soybean in the U.S.A. References: (1) K. E. Broders et al. Plant Dis. 91:727, 2007. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (3) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002. (4) K. Skovgaard et al. Mycologia. 94:630, 2003.

First Report of Fusarium armeniacum Causing Seed Rot and Root Rot on Soybean (Glycine max) in the United States.

In a survey for Fusarium root rot, soybean plants were sampled from eight counties across Iowa in 2008 to 2009. Fusarium isolates were recovered from surface-sterilized symptomatic and asymptomatic root tissue by culturing on peptone PCNB agar (2). Single-spore isolates were transferred to carnation leaf agar (CLA) and potato dextrose agar (PDA) for morphological identification; 11 isolates were identified as F. armeniacum (Forbes, Windels, and Burgess) Burgess and Summerell (previously F. acuminatum ssp. armeniacum) (2). Colonies on PDA produced white aerial mycelium, red to apricot pigment in agar, and bright orange sporodochia in the center of the culture. Some isolates produced a pionnotal form of slow-growing colonies with little aerial mycelium and abundant orange sporodochia. On CLA, macroconidia in orange sporodochia on carnation leaves and chlamydospores formed abundantly, but microconidia were absent (2). Species identity for the 11 isolates was confirmed by sequencing of the elongation factor gene (EF1-α) using ef1 and ef2 primers (4) (reference sequences deposited in GenBank JX101763 and JX101764). Pathogenicity of seven F. armeniacum isolates was tested using surface-sterilized soybean seed, cv. AG2403, in a petri dish assay with 3-day-old cultures on 2% water agar (1). Germination, seed rot, and lesion development were scored 7 dai using an ordinal scale (1). The experiment was a completely randomized design (CRD), had three replicate plates per isolate, and was conducted twice. All seven isolates were pathogenic on soybean, though variation in aggressiveness was observed among isolates (P < 0.0001) related to colony morphology on PDA. Seed germination was 0 to 40% when inoculated with four isolates showing white fluffy aerial mycelium on PDA. Seedlings were severely stunted with dark brown lesions covering a majority of the root system. When inoculated with three isolates showing the pionnotal form of slow-growing mycelium, germination was 70 to 100%, with few small brown lesions (~5 to 10 mm) on the roots. Noninoculated controls showed 100% germination and no symptoms. Pathogenicity was also tested in a growth chamber assay at 18°C using autoclaved soil mixed with an infested sand-cornmeal inoculum (3). Data for dry root and shoot weights and root rot severity (visually scored on a % scale) were collected at 6 weeks. The CRD experiment had five replications (single plant in a cone containing 150 ml infested soil), and was conducted twice. Root symptoms and similar variation in aggressiveness among isolates (based on colony morphology) was observed in inoculated plants. Isolates differed significantly for effects on root weight (P = 0.0125), shoot weight (P = 0.0035), and root rot severity (P = 0.0158). F. armeniacum was reisolated from infected root tissue, but not from noninoculated controls. Recovered isolates maintained their original colony morphology. F. armeniacum was previously reported in Minnesota on symptomless corn (2), but it has not been reported on soybean and its pathogenicity has not been established on any crop. To our knowledge, this is the first report of F. armeniacum as a pathogen on soybean in the United States. References: (1) K. E. Broders et al. Plant Dis. 91:727, 2007. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (3) G. P. Munkvold and J. K. O'Mara. Plant Dis. 86:143, 2002. (4) K. O'Donnell et al. Proc. Natl. Acad. Sci. 95:2044, 1998.