Trichothecene Genotype of Fusarium graminearum Isolates from Soybean (Glycine max) Seedling and Root Diseases in the United States.

Fusarium graminearum is an economically important pathogen that causes Fusarium head blight of wheat, barley, and oat, and Gibberella ear and stalk rot of maize. More recently, F. graminearum was reported as a soybean seedling and root pathogen in North America (1,5), causing seed decay, damping-off, and brown to reddish-brown root rot symptoms. Type B trichothecene mycotoxins are commonly produced by F. graminearum, which can be categorized into three trichothecene genotypes; those that produce 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), or nivalenol (NIV). The 15-ADON genotype is dominant in populations from small grains and maize in North America (4), but the 3-ADON genotype has recently been found (4). F. graminearum was known as a pathogen of wheat and maize in North America for over a century before it was reported as a soybean pathogen. Therefore, we hypothesized that recent reports on soybean could be associated with the appearance of the 3-ADON genotype. The objective of this research was to determine the trichothecene genotype of F. graminearum isolates from soybean in the United States. Thirty-eight isolates from soybean were evaluated. Twenty-seven isolates came from a 3-year survey for Fusarium root rot from 2007 to 2009 in Iowa. Other isolates (Ahmad Fakhoury, Southern Illinois University, Carbondale) were collected from soybean seedlings during a multi-state survey in 2012, and included three isolates from Illinois, three from Indiana, and five from Nebraska. Species identification and lineage of F. graminearum were confirmed by sequencing the translation elongation factor gene (EF1-α) using EF-1H and EF-2T primers. A maximum likelihood analysis of the EF1-α, including voucher strains from nine lineages of F. graminearum (2), placed all 38 isolates into lineage 7, F. graminearum sensu stricto (representative GenBank accessions KJ415349 to KJ415352). To determine the trichothecene genotype of each isolate we used three multiplex PCR assays. The first two assays targeted a portion of trichothecene biosynthesis genes Tri3 and Tri12 (4), while the third assay targeted portions of the Tri3, Tri5, and Tri7 genes (3). The PCR for the first two assays was conducted as described by Ward et al. (4) using four sets of primers: 3CON, 3NA, 3D15A, and 3D3A; and 12CON, 12NF, 12-15F, and 12-3F for the Tri3 and Tri12 genes, respectively. The PCR for the third assay was conducted as described by Quarta et al. (3) using the following primers: Tri3F971, Tri3F1325, Tri3R1679, Tri7F340, Tri7R965, 3551H, and 4056H. The amplification products were analyzed by gel electrophoresis. All 38 isolates produced amplicons consistent with the 15-ADON genotype; ~610 and 670 bp for the Tri3 and Tri12 genes, respectively (4), and two amplicons of ~708 and 525 bp for the Tri3/Tri5 genes (3). Our results indicated that the dominant trichothecene genotype among isolates of F. graminearum from soybean is 15-ADON, and the introduction of 3-ADON isolates does not explain the recent host shift of F. graminearum to soybean in North America. To our knowledge, this is the first assessment of trichothecene genotypes in F. graminearum populations from soybean from the United States. References: (1) K. E. Broders et al. Plant Dis. 91:1155, 2007. (2) K. O'Donnell et al. Fungal Gen. Biol. 41:600, 2004. (3) A. Quarta et al. FEMS Microbiol. Lett. 259:7, 2006. (4) T. D. Ward et al. Fungal Gen. Biol. 45:473, 2008. (5) A. G. Zue et al. Can. J. Plant Pathol. 29:35, 2007.

First Report of Pythium schmitthenneri Causing Maize Seedling Blight in Iowa.

In spring 2012, maize farmers in southeast and south central Iowa reported stand losses due to pre- and post-emergence damping-off, and many of the fields had to be replanted. Symptoms of the disease included rotted seed, or brown, rotted, water-soaked mesocotyls and root tips. Maize seedlings with severe root and mesocotyl symptoms were yellow and wilted, stunted, or dead. The disease occurred approximately 2 weeks after cool, wet conditions. Symptomatic mesocotyls and roots were washed for 30 min, rinsed with sterile distilled water, and blotted dry on sterile paper towels. Isolation of the pathogen was performed by aseptically cutting 2- to 3-mm sections of tissue from the edge of a lesion, placing the segments under corn meal agar (CMA) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP), and incubating at 22°C in the dark. Colonies that developed were putatively identified as Pythium species based on morphological characteristics and cultural features when compared to published descriptions (2,3). Characteristics of isolate IAC12F21-3 included spherical and smooth-walled oogonia 18 to 26 µm in diameter, monoclinous or usually diclinous antheridia 10 to 22 µm long and 5 to 10 µm wide with one or occasionally two per oogonium, and plerotic oospores 15 to 25 µm in diameter. Sporangia were globose to ellipsoidal, 22 to 41 µm in diameter, and zoospores were 7 to 10 µm long. Primers ITS1 and ITS4 were used to amplify the ITS region within clade E1 of 88 isolates. The resultant amplicons were sequenced and a BLAST search in GenBank confirmed isolate IAC12F21-3 as Pythium schmitthenneri based on 100% similarity with GenBank accession numbers JF836869 and JF836870. Pathogenicity testing was conducted using seed and seedling assays (1,4). Koch's postulates was performed by sampling pieces of symptomatic mesocotyl and root tissue from the inoculated pots, placing segments under CMA + PARP, and incubating at 22°C. Symptoms were similar to those observed in the field and P. schmitthenneri was re-isolated successfully. Non-inoculated control plants showed no symptoms. This is the first report of P. schmitthenneri causing seedling blight on maize in Iowa. Previously, P. schmitthenneri was reported as a pathogen on maize in Ohio (2). References: (1) K. Broders et al. Plant Dis. 91:727, 2007. (2) M. Ellis et al. Mycologia, 104:477, 2012. (3) J. Middleton. Memoirs of the Torrey Botanical Club 20:171, 1943. (4) A. Rojas et al. Phytopathology, 102(Suppl):S5.8, 2012.

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.

Infection of Soybean Seed by Fusarium graminearum and Effect of Seed Treatments on Disease Under Controlled Conditions.

Fusarium graminearum causes seed decay and damping-off of soybean. This study evaluated the effect of inoculum density of F. graminearum, temperature, and fungicide seed treatments on disease development. To determine the optimum conditions for disease development, individual soybean seed was inoculated with 100 µl of a suspension of 2.5 × 102, 2.5 × 103, 2.5 × 104, or 2.5 × 105 macroconidia/ml in a rolled-towel assay at temperatures of 18, 22, and 25°C. Inoculum concentrations of 2.5 × 104 macroconidia/ml or higher were necessary for optimum disease development at all temperatures. The efficacy of captan, fludioxonil, mefenoxam + fludioxonil, azoxystrobin, trifloxystrobin, and pyraclostrobin as seed treatments was then evaluated with the same assay at 2.5 × 104 and 2.5 × 105 macroconidia/ml. Seed treated with captan at 61.9 g a.i. or fludioxonil at 2.5 or 5.0 g a.i. per 100 kg developed smaller lesions than other seed treatments and the nontreated control. Based on these results, there are limited choices in fungicide seed treatments for managing this seedling disease, and it is possible that shifts in seed treatment products may have played a role in the recent emergence of this soybean pathogen.

First Report of Phytophthora sansomeana Causing Wilting and Stunting on Corn in Ohio.

During the spring of 2004, corn seedlings with symptoms of wilting and stunting were observed in corn fields with emergence problems in Madison and Brown counties, Ohio. Phytophthora isolates were recovered from sections of root tissue of diseased seedlings placed on dilute V8 media amended with pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol. Colonies were rosaceous on potato dextrose agar, with a growth rate of 5 mm per day. Homothallic isolates with paragynous antheridia were observed on lima bean agar (LBA); oogonia were 35 to 50 µm in diameter. Sporangia were ovoid to obpyriform, nonpapillate, with an average size of 49 × 30 µm. Pathogenicity was tested on corn seeds using a petri dish assay with 3-day-old cultures on LBA and a sand-cornmeal cup test amended with inoculum from 7-day-old cultures on LBA (1). After 1 week in the petri dish assay, the seeds failed to germinate completely and were covered with white, fungal-like, aerial mycelia and the pathogen was recovered from brown discolored radicle roots. In the cup assay, 2-week-old seedlings developed the same symptoms observed in the field; the pathogen was also isolated from brown discolored roots. In both assays, no symptoms developed in the noninoculated controls. Both pathogenicity tests were repeated two times. Genomic DNA was extracted from mycelia of two isolates and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS6/ITS4 primers (2). Both isolates had identical ITS sequences (GenBank Accession No. GQ853880). A BLAST search of the NCBI database showed 100% homology with the sequence of the haplotype isolate of Phytophthora sansomeana (Accession No. EU925375). P. sansomeana is a new species characterized principally by a large oogonial diameter (37 to 45 µm), rapid growth rate (7 to 10 mm/day), and an ITS sequence falling in Cooke's clade 8 (4). Pathogenicity tests, morphological characteristics, and the ITS sequence analysis indicate that P. samsomena is the causal agent of the symptoms observed on corn seedlings. P. sansomeana has been reported as a pathogen of soybean in Indiana, Douglas-fir in Oregon, and weeds in alfalfa fields in New York (4). To our knowledge, this is the first report of P. sansomeana infecting corn in Ohio, albeit other isolates have previously been recovered from soybean in the state. There are four previous reports of Phytophthora spp. affecting corn in the United States and Mexico (3). Crop rotation will have little effect in management of this pathogen since corn and soybean are produced in the same fields continuously throughout the state. References: (1) K. E. Broders et al. Plant. Dis. 91:727, 2007. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. 1989. (4) E. M. Hansen et al. Mycologia 101:129, 2009.

Isolate-Cultivar Interactions, In Vitro Growth, and Fungicide Sensitivity of Fusarium oxysporum Isolates Causing Seedling Disease on Soybean.

Fusarium oxysporum is frequently associated with soybean root rot in the United States. Information about pathogenicity and other phenotypic characteristics of F. oxysporum populations is limited. The objective of the research described herein was to assess phenotypic characteristics of F. oxysporum isolates from soybean, including the interaction between isolates and soybean cultivars, fungal growth characteristics in culture, and sensitivity to fungicides commonly used as seed treatment products. The pathogenicity of 14 isolates was evaluated in rolled-towel and Petri-dish assays using 11 soybean cultivars. In the rolled-towel assay, seed were inoculated with a conidial suspension and disease severity was observed. In the Petri-dish assay, F. oxysporum isolates were grown on 2% water agar and seed were placed on the F. oxysporum colony to observe the symptoms that developed. Cultivars differed in susceptibility to F. oxysporum, and significant (P = 0.0140) isolate-cultivar interactions were observed. F. oxysporum isolates differed in radial growth on potato dextrose agar at 25°C. Pyraclostrobin and trifloxystrobin reduced conidial germination with average 50% effective concentration (EC50) of 0.15 and 0.20 µg active ingredient (a.i.)/ml, respectively. Ipconazole reduced fungal growth with average EC50 of 0.23 µg a.i./ml, whereas fludioxonil was ineffective. Our results illustrate soybean F. oxysporum isolate variability and the potential for their management through cultivar selection or seed treatment.

A new class of antihypertensive therapy: angiotensin II receptor antagonists.

Despite the availability of safe and efficacious antihypertensive agents, hypertension continues to be a major source of morbidity and mortality in the United States. Losartan, the first of a new class of agents, the angiotensin II receptor antagonists, can be administered as monotherapy in the treatment of hypertension or to complement existing therapy. The angiotensin II receptor antagonists block the effects of angiotensin II through preferential binding to angiotensin II receptor subtype AT1 on the cell membrane. Compared with angiotensin-converting enzyme inhibitors, they may provide more complete blockade of the renin-angiotensin system and be associated with a lower frequency of cough as a side effect.

An examination of the O and K specificity involved in the antibody-induced loss of the K88 plasmid from porcine enteropathogenic strains of Escherichia coli.

The heat-labile K88 antigen, a virulence determinant coded for by a transmissible plasmid, was eliminated from enteropathogenic strains of Escherichia coli by passage through media containing antibodies to the heat stable antigens of an Abbotstown (O149:K91,K88ac) strain. The plasmid-curing activity of O149 antisera was not O-antigen specific as O149, O45, O8 and O138 strains of E. coli could be 'cured' of their K88 plasmids by this technique. The curing activity was differentiated from the O-antibody by gel filtration, the O149 antibodies were eluted in the IgM peak while the curing activity was found in the IgG peak. In view of the lack of O-specificity and the absence of K88 antibodies it appears that antibodies to a common heat-stable antigenic determinant were involved in this phenomenon.

The AHCPR clinical practice guideline for heart failure revisited.

OBJECTIVE: To review the Agency for Health Care Policy and Research (AHCPR) clinical practice guideline for heart failure and comment on the guideline regarding pharmacotherapy from the perspective of the latest clinical trial data and the authors' clinical experience. DATA SOURCES: A MEDLINE search (1966 to June 1997) of English-language literature pertaining to the pharmacotherapy of heart failure was performed. Special emphasis was placed on literature published in the last 5 years. Additional literature was obtained from reference lists of key articles identified through the search. DATA SYNTHESIS: Pertinent clinical trials were reviewed and considered along with information from the authors' database of over 800 patients with heart failure. Evidence concerning the use of angiotensin-converting enzyme inhibitors at appropriate dosages in all New York Heart Association classes of heart failure and the inclusion of digoxin as part of triple therapy in all symptomatic patients with left ventricular systolic dysfunction are reviewed. Strategies to circumvent clinical problems that may limit the proper application of standard therapeutic agents are considered, and the possible future role of beta-blockers as the therapeutic agents in patients with heart failure is discussed. CONCLUSIONS: The AHCPR guideline provides the clinician with an excellent framework for treating the patient with heart failure. Building on the fundamentals of the guideline, the clinician can carefully apply current therapy at appropriate dosages and in the best combinations to individualize and thereby optimize pharmacologic therapy for this patient population.