The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

Virulence and double-stranded RNA (dsRNA) profiles of 44 isolates of Fusarium virguliforme were compared. When grouped according to dsRNA profiles, isolates with large dsRNAs were significantly (P≤0.05) less virulent than isolates without dsRNAs. High-throughput sequence analysis of total RNA prepared from cultures with large dsRNAs identified two novel RNA viruses with genome sequences of approximately 9.3 kbp, which were named Fusarium virguliforme dsRNA mycovirus 1 and Fusarium virguliforme dsRNA mycovirus 2. The new viruses were most closely related to a group of unclassified viruses that included viruses of F. graminearum and Phlebiopsis gigantic and are related to members of the family Totiviridae.

Sudden death syndrome (SDS) of soybean is caused by a soilborne pathogen, Fusarium virguliforme. Phytotoxins produced by F. virguliforme are translocated from infected roots to leaves, in which they cause SDS foliar symptoms. In this study, additional putative phytotoxins of F. virguliforme were identified, including three secondary metabolites and 11 effectors. While citrinin, fusaric acid, and radicicol induced foliar chlorosis and wilting, Soybean mosaic virus (SMV)-mediated overexpression of F. virguliforme necrosis-inducing secreted protein 1 (FvNIS1) induced SDS foliar symptoms that mimicked the development of foliar symptoms in the field. The expression level of fvnis1 remained steady over time, although foliar symptoms were delayed compared with the expression levels. SMV::FvNIS1 also displayed genotype-specific toxicity to which 75 of 80 soybean cultivars were susceptible. Genome-wide association mapping further identified three single nucleotide polymorphisms at two loci, where three leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes were found. Culture filtrates of fvnis1 knockout mutants displayed a mild reduction in phytotoxicity, indicating that FvNIS1 is one of the phytotoxins responsible for SDS foliar symptoms and may contribute to the quantitative susceptibility of soybean by interacting with the LRR-RLK genes.

The occurrence of sudden death syndrome (SDS), caused by the fungus Fusarium solani (Mart.) Sacc. f. sp. glycines (FSG) (syn. Fusarium virguliforme Akoi, O’Donnell, Homma and Lattanzi), is unpredictable in soybean [Glycine max (L.) Merr.] field trials making it difficult to evaluate soybean for resistance to the pathogen. Our objective was to evaluate the effect of field inoculation, soil compaction, and irrigation on the occurrence and severity of SDS symptoms. Six inoculation treatments were tested which included applications of FSG-infested grain planted in the furrow with the soybean seed, broadcasted and incorporated into the soil before planting, or placed below the soybean seed just before planting. Soil was compacted by driving a tractor across the field once in early spring. Irrigation treatments were applied at combinations of growth stages V3, V7, R3, R4, and/or R5. Significant increases in foliar SDS severity were observed from inoculation and irrigation treatments (P , 0.05), but not from compaction treatments. The inoculation treatments that placed inoculum close to the seed resulted in the greatest foliar severity. Irrigation treatments during mid to late reproductive growth stages resulted in significant increases in SDS foliar symptom development. These results increase our understanding of what environmental conditions increase SDS field symptoms and will be useful to researchers establishing SDS field nurseries.

Soybean cyst nematode, Heterodera glycines, and the fungus that causes sudden death syndrome of soybean (SDS), Fusarium solani f. sp. glycines, frequently co-infest soybean (Glycine max) fields. The interactions between H. glycines and F. solani f. sp. glycines were investigated in factorial greenhouse experiments with different inoculum levels of both organisms on a soybean cultivar susceptible to both pathogens. Responses measured included root and shoot fresh and dry weights, H. glycines reproduction, area under the SDS progress curve (AUDPC), and fungal colonization. Results of experimets conducted in two greenhouse environments were analyzed separately by 2-way, and 3-way analysis of variance (ANOVA) models that were developed that included the effect of greenhouse environment. Both H. glycines and F. solani f. sp. glycines affected the growth of soybeans in an additive pattern. Reproduction of H. glycines was suppressed (P=0.05) by high inoculum levels of F. solani f. sp. glycines, whereas the low the level had no significant impact. Real-time PCR assays were used to quantify fungal infection of soybean roots by F. solani f. sp. glycines and indicated that the infection of soybean roots by H. glycines did not impact colonization of F. solani f. sp. glycines.

Fusarium solani infects roots of a number of different plant species and some strains produce phytotoxins. F. solani f. sp. glycines, the causal organism of sudden death syndrome (SDS) of soybean (Glycine max), colonizes soybean roots and produces toxin(s) that are translocated to leaves and cause intervienal chlorosis and necrosis. Several experiments evaluated the phytotoxicity of cell-free culture filtrates of F. solani f. sp. glycines by immersing cuttings of soybean seedlings into filtrates to determine what in vitro growth conditions alter the phytotoxicity, and to determine the specificity of toxicity of F. solani f. sp. glycines and other F. solani on cuttings of soybean and other legume species. Foliar disease severity ratings of soybean cuttings in cell-free culture filtrates diluted 25- and 50-fold were higher than when diluted 100-fold or more. Cell-free culture filtrates originating from cultures grown at 15, 20, and 25˚C caused greater (P = 0.05) foliar disease severity ratings on cutting than when the fungus was grown at 30˚C. Cell-free culture filtrates of F. solani isolates from cucumber (Cucumis sativus) and pumpkin (Cucurbita pepo) did not cause symptoms on soybean cuttings while filtrates of F. solani isolates obtained from other hosts caused some leaf chlorosis and/or necrotic spots. F. solani f. sp. glycines inoculated on eight legume species caused symptoms such as leaf chlorosis, defoliation, wilt or death on most of the species, but azuki bean (Vigna angularis), common pea (Pisum sativum) and vetch (Vicia sativa) were symptomless. Cell-free culture filtrates of F. solani f. sp. glycines caused foliar symptoms on all cuttings of legume species except for mung bean (Vigna radiata), although none of the symptoms matched the SDS symptoms observed on soybean foliage.

A phytotoxic polypeptide identified in culture filtrates of Fusarium solani, the causal agent of soybean sudden death syndrome, was heat unstable, negatively charged, absorbed by 10% charcoal, and destroyed by proteinase K. The toxicity of the culture filtrates and fractions obtained during purification was bioassayed by measuring browning of soybean calli. Purification of the phytotoxin was achieved by Sephadex G-50 gel filtration chromatography followed by ion exchange chromatography on a DE-52 column. The purified protein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 17,000. The sequence of the N-terminal 15 amino acids was determined and indicated that a peptide was present. Samples containing this single protein caused browning of soybean calli, necrosis on detached soybean cotyledons and leaves, and yellowing, curling, and drying of attached soybean cotyledons and leaves.

An isolate of Fusarium solani that caused sudden death syndrome (SDS) of soybean was grown in a semidefined liquid medium. The phytotoxicity oi cell-free fungal culture filtrates was determined on soybean calli grown on tissue culture medium amended with fungal culture filtrate. The mean calli brown rating increased as culture filtrate increased in the medium. There were significant (P < 0.05) positive correlations between SDS severity from field microplot data and calli browning due to culture filtrates. Seedlings of five soybean cultivars inoculated with the fungus under greenhouse conditions had significantly (P < 0.05) different foliar severity ratings and relative plant fresh weights. Sensitivity of calli of the same five cultivars to culture filtrate also had significantly (P < 0.05) different brown ratings and relative fresh weights. Then were significant (P < 0.05) correlations between variables measured on inoculated soybean seedlings and calli sensitivity, Pathogenicity and phytotoxicity of F. solani isolates from different hosts were tested on soybean plants and calli, respectively. Isolates from hosts other than soybean did not cause SDS symptoms, and their culture filtrates had significantly lower toxicity to soybean calli than did that of soybean SDS-causing isolates.

A polymerase chain reaction (PCR)-based method was developed to detect DNA of Fusarium solani L sp. glycines, the cause of soybean sudden death syndrome. Two pairs of primers, Fsg1/Fsg2 designed from the mitochondrial small subunit ribosomal RNA gene, and FsgEF1/FsgEF2 designed from the translation elongation factor 1-± gene, produced PCR products of 438 and 237 bp, respectively. Primer specificity was tested with DNA from 82 F. solani f. sp. Glycines, 55 F. solani non-SDS isolates, 43 isolates of 17 soybean fungal pathogens and the oomycete Phytophthora soja, and soybean. The sensitivity of primer Fsg1/Fsg2 was 10 pg while that of FsgEF1/FsgEF2 was 1 ng when using F. solani f. sp. glycines total genomic DNA or down to 103 macroconidia g-1 soil Nested PCR increased the sensitivity of the PCR assay 1000-fold to 10 fg using primers Fsg1/Fsg2, and 1 pg using primers FsgEF1/FsgEF2. f. solani f. sp. glycines DNA was detected in field-grown soybean roots and soil by PCR using either single pairs of primers or the combination of two pairs of primers. The occurrence of F. solani f. sp.glycines was determined using nested PCR for 47 soil samples collected from soybean fields in 20 counties of Illinois in 1999. F. solani f. sp. glycines was detected in soil samples from all five Illinois Agricultural Statistic Districts including 100, 89, 50, 92 and 50% of the samples from East, Central, North-east and West District, respectively.

Fusarium virguliforme (Akoi, O’Donnell, Homma & Lattanzi), formerly named F. solani (Mart.) Sacc. f.sp. glycines, is the cause of soybean sudden death syndrome (SDS). Over the last 6 years, an international collection of F. virguliforme isolates has been established and maintained at the National Soybean Pathogen Collection Center, University of Illinois at Urbana-Champaign. Using part of the collection, aggressiveness of F. virguliforme isolates to a susceptible soybean cultivar, Great Lakes 3202, was evaluated under controlled conditions in the greenhouse. After an initial evaluation of 123 isolates on soybean, 30 isolates from different geographic origins with different levels of foliar severity were selected to further evaluate both foliar and root severities. Variability of aggressiveness based on measurements of SDS foliar severity, shoot, root, and root lesion lengths, shoot and root dry weights, and total dry weights was found among isolates (P ≤ 0.01). Isolate FSG1(Mont-1), a reference isolate that has been widely used by the soybean community for basic and applied research, caused the greatest reduction in shoot weight and shoot length compared to the non-inoculated control plants, but six isolates caused higher foliar severity and 15 isolates caused longer root lesion length than the isolate FSG1. Isolate FSG5 caused the greatest reduction in root weight among isolates. Knowledge about the variability of the pathogen is important for selection of isolates for testing for broadbased SDS resistant soybean lines.

Six isolates of Fusarium solani f. sp. glycines, that cause soybean sudden death syndrome were examined for chlamydospore formation, production, and nuclear status, Chlamydospores formed from macroconidia either terminally, laterally by outward protrusion, or intercalarily. They also formed from germinated macroconidia and hyphae. Occasionally, a single macroconidium produced more than one chlamydospore. The percentage of chlamydospores produced differed significantly (P < 0.0001) by incubation temperature and varied among fungal isolates. More Chlamydospores formed at 30°C followed by 25°C, 20°C and 4°C. Nuclear stained Chlamydospores usually were uninucleate but occasionally multinucleate. Nuclei migrated from macroconidia into Chlamydospores through germ tubes.

Fusarium solani is a soilborne plant pathogen that infects many different hosts. Within the species, there is some specialization, and a number of forma specialis have been described based on host affiliation. One of these, F. solani f. sp. glycines, infects soybean and causes sudden death syndrome. To differentiate between F. solani f. sp. glycines and other F. solani isolates, a partial sequence of the mitochondrial small subunit (mtSSU) rRNA gene was amplified by polymerase chain reaction and sequenced from 14 F. solani f. sp. glycines and 24 F. solani isolates from various plant hosts. All F. solani f. sp. glycines isolates had identical sequences. A single, unique insertion of cytosine occurred in all F. solani isolates but not in any of the F. solani f. sp. glycines isolates. Two major lineages, distinguished by sequence divergence and the presence or absence of multiple insertions, occurred in F. solani isolates. Cladistic analysis produced a single most-parsimonious tree with three major clades. The first clade contained all F. solani f. sp. glycines isolates. A second clade grouped together all of the F. solani isolates that had only a single nucleotide insertion difference from the first clade. Genetic distance between these two clades was 0.016. A third clade was formed by five F. solani isolates that had multiple insertions. Isolates in the third clade had a genetic distance of 0.040 from the first and second clades. Based on the sequence data, it is likely that F. solani f. sp. glycines has a shorter evolutionary history than other F. solani isolates that have either single or multiple nucleotide insertions. The differences in nucleotide insertions in part of the mtSSU rRNA gene between F. solani f. sp. glycines and other F. solani isolates provide a direct and reliable way to distinguish isolates off solani.

Plant cell wall degrading enzymes (PCWDEs) are a subset of carbohydrate-active enzymes (CAZy) produced by plant pathogens to degrade plant cell walls. To counteract PCWDEs, plants release PCWDEs inhibitor proteins (PIPs) to reduce their impact. Several transgenic plants expressing exogenous PIPs that interact with fungal glycoside hydrolase (GH)11-type xylanases or GH28-type polygalacturonase (PG) have been shown to enhance disease resistance. However, many plant pathogenic Fusarium species were reported to escape PIPs inhibition. Fusarium virguliforme is a soilborne pathogen that causes soybean sudden death syndrome (SDS). Although the genome of F. virguliformewas sequenced, there were limited studies focused on the PCWDEs of F. virguliforme. Our goal was to understand the genomic CAZy structure of F. viguliforme, and determine if exogenous PIPs could be theoretically used in soybean to enhance resistance against F. virguliforme. Results F. virguliforme produces diverse CAZy to degrade cellulose and pectin, similar to other necrotorphic and hemibiotrophic plant pathogenic fungi. However, some common CAZy of plant pathogenic fungi that catalyze hemicellulose, such as GH29, GH30, GH44, GH54, GH62, and GH67, were deficient in F. virguliforme. While the absence of these CAZy families might be complemented by other hemicellulases, F. virguliforme contained unique families including GH131, polysaccharide lyase (PL) 9, PL20, and PL22 that were not reported in other plant pathogenic fungi or oomycetes. Sequence analysis revealed two GH11 xylanases of F. virguliforme, FvXyn11A and FvXyn11B, have conserved residues that allow xylanase inhibitor protein I (XIP-I) binding. Structural modeling suggested that FvXyn11A and FvXyn11B could be blocked by XIP-I that serves as good candidate for developing transgenic soybeans. In contrast, one GH28 PG, FvPG2, contains an amino acid substitution that is potentially incompatible with the bean polygalacturonase-inhibitor protein II (PvPGIP2). Conclusions Identification and annotation of CAZy provided advanced understanding of genomic composition of PCWDEs in F. virguliforme. Sequence and structural analyses of FvXyn11A and FvXyn11B suggested both xylanases were conserved in residues that allow XIP-I inhibition, and expression of both xylanases were detected during soybean roots infection. We postulate that a transgenic soybean expressing wheat XIP-I may be useful for developing root rot resistance to F. virguliforme.

Sudden death syndrome (SDS), caused by the fungus Fusarium virguliforme, is a widespread mid- to late-season soybean disease with distinctive foliar symptoms that in some extreme cases may cause nearly 100% yield loss. This article reports on the development of an image analysis method to quantify SDS severity using an RGB camera under natural light in a laboratory environment. An HSI (hue, saturation, and intensity) color space based image processing approach was developed. A lesion index (LI) was defined as the ratio of affected leaf area to healthy area for evaluating SDS severity levels on leaves of infected plants. A method of using the frequency distribution of hue values of whole leaf area to measure the severity of SDS was also investigated. Preliminary results obtained from laboratory-scale validation demonstrated that the developed method could provide a feasible means of recording SDS severity in situ that could potentially be used for high-throughput greenhouse evaluation of breeding lines or commercial soybean cultivars.

The objectives of this work were to evaluate two greenhouse screening methods for sudden death syndrome (SDS) and to determine which one is best correlated with field resistance of soybean genotypes. The evaluations were done with three sets of genotypes that were classified as partially resistant, intermediate, and susceptible to SDS based on previous field evaluations. These three sets were independently evaluated for greenhouse SDS reactions using cone and tray inoculation methods. Plants were infected using grains of white sorghum [Sorghum bicolor (L.) Moench] infested with Fusarium solani f. sp. glycines. Foliar symptom severity was rated 21 days after emergence. The cone and field SDS ratings were significantly correlated and ranged from 0.69 for set 1 to 0.51 for set 3. Correlations of SDS ratings of genotypes between field and greenhouse tray ratings were significant for set 1 and not significant for set 2. The cone method showed the highest correlation with field results and is recommended to screen soybean genotypes for SDS resistance.

Fusarium solani f. sp. glycines is the causal organism of soybean sudden death syndrome (SDS). This organism is difficult to detect and quantify because it is a slow-growing fungus with variable phenotypic characteristics. Reliable and fast procedures are important for detection of this soybean pathogen. Protocols were optimized for extraction of DNA from pure fungal cultures and fresh or dry roots. A new procedure to test polymerase chain reaction (PCR) inhibitors in DNA extracts was developed. Novel real-time quantitative PCR (QPCR) assays were developed for both absolute and relative quantification of F. solani f. sp. glycines. The fungus was quantified based on detection of the mitochondrial small-subunit rRNA gene, and the host plant based on detection of the cyctophilin gene of the host plant. DNA of F. solani f sp. glycines was detected in soybean plants both with and without SDS foliar symptoms to contents as low as 9.0 x 10-5 ng in the absolute QPCR assays. This is the first report of relative QPCR using the comparative threshold cycle (Ct) method to quantify the DNA of a plant pathogen relative to its host DNA. The relative QPCR assay is reliable if care is taken to avoid reaction inhibition and it may be used to further elucidate the fungus-host interaction in the development of SDS or screen for resistance to the fungus.

The cultivated soybean [Glycine max (L.) Merr.] has a relatively narrow genetic base and most commercial cultivars are susceptible to Sclerotinia sclerotiorum (Lib.) de Bary and Fusarium solani (Mart.) Sacc. f. sp. glycines, which, respectively cause Sclerotinia stem rot (SSR) and sudden death syndrome (SDS). The objective of this study was to screen all the available accessions of the perennial Glycine species for resistance to the pathogens that cause SSR and SDS. For SSR evaluations, five seedlings of each of 787 accessions were screened once in a series of eight non-replicated runs. Seedlings were inoculated with an agar plug cut from the edge of a 1-d-old fungal culture by placing the plug next to the stem. Of the 787 accessions, 183 had partial resistance with 144 of these accessions being G. tabacina (Labill.) Benth. A selected set of 53 accessions was further screened in two replicated trials with five plants per each of four replications. Glycine tabacina had several accessions that were consistently rated as partially resistant. For SDS evaluations, five plants of each of 767 accessions were screened once in a series of eight runs. Plants were inoculated by a layered technique in which infested sorghum seed were placed below the transplanted seedlings. In the initial evaluation of 767 accessions, 134 had partial resistance with 65 of these accessions being G. tomentella Hayata. In a replicated set of selected accessions, G. tomentella had several accessions that were consistently rated as partially resistant. These perennial Glycine species represent potential untapped sources for improving disease resistance in soybean.

Sudden death syndrome (SDS) is an important soybean disease that potentially can be controlled by host plant resistance. In this study, over 800 soybean plant introductions (PIs), lines, and cultivars were screened for resistance to Fusarium solani. Of 728 PIs from China, PI 567.374 had mean foliar SDS severities significantly (P = 0.05) lower than PI 520.733 (resistant check) in both growth-chamber and greenhouse tests. In addition, PIs 567.315, 567.441C, 567.650B, and 567.664 had mean SDS severity ratings significantly (P = 0.05) lower than PI 520.733 in a growth-chamber test. Of 16 soybean cyst nematode-resistant entries tested, 5 had values lower than the resistant check, PI 520.733, with cv. Hartwig significantly lower in the first trial. In trial two, no entries were lower than the resistant check, although cvs. Bell and Hartwig were not significantly different from PI 520.733. In another experiment, few soybean cultivars or experimental lines had SDS severity ratings lower than PI 520.733 in any one of three trials. Some of the newly acquired PIs from China that exhibited low foliar SDS severity ratings may provide the sources of resistance needed to develop new SDS-resistant soybean breeding lines and cultivars.

Four soybean plant introductions, PI 520.733, PI 567.374, PI 567.650B, and PI 567.659, and one soybean cultivar, Great Lakes 3202, were inoculated under greenhouse conditions with four isolates of Fusarium solani f. sp. glycines. Foliar disease severity rating was greatest on PI 567.659, followed by Great Lakes 3202, PI 520.733, PI 567.650B, and PI567.374. There was no significant interaction between isolates and soybean entries for foliar disease severity ratings. Experiments also were conducted to determine if disease development and root colonization differed among entries. Root infection of the five entries did not differ (P = 0.05). Foliar disease progress curves increased faster for PI 567.659 and Great Lakes 3202 than for PI 567.374. The area under the disease progress curve (AUDPC) value for PI 567.374 was the lowest and differed (P = 0.01) from AUDPC values for Great Lakes 3202 and PI 567.659. There were no differences (P = 0.01) in length of taproot lesions, losses in root dry weight, and vascular stem length discoloration among the entries, and there was no correlation (P = 0.05) between these measurements and foliar AUDPC values. Cut seedling stems immersed in culture filtrate developed interveinal chlorosis on leaves of each entry within 2 days. Disease severity on cut seedlings of PI 567.374 was lower (P = 0.01) than on the other entries. There was a positive correlation (r = 0.94, P = 0.05) between AUDPC values of the five entries inoculated with the fungus and the cut seedling test using culture filtrate.

Soybean embryogenic suspension cultures established from immature cotyledons of four cultivars were selected for resistance to a toxic culture filtrate of Fusarium solani, the causal agent of sudden death syndrome (SDS) of soybean. The embryogenic cultures were challenged with the fungal culture filtrates for 1 to 2 months. Many well-developed somatic embryos formed when the toxin-selected embryogenic cultures were incubated on regeneration medium; however, only a few plants were regenerated from three cultivars, whereas 98 plants were regenerated from cv. Jack embryos. The regenerants that survived were grown to maturity to obtain more seeds for screening plants for resistance to SDS. In the first experiment, the R1 (187, first-selfed generation) and R2 (225, second-selfed generation) plants of cv. Jack regenerants, resistant line PI520.733, and cvs. Jack, Great Lakes 3202, and Ripley were inoculated with a F. solani SDS isolate and rated on a 1 to 5 scale. The regenerants had significantly (P < 0.05) lower mean disease ratings than two of the three cultivars but did not differ from PI520.733. In the second experiment, R3 plants (990 from cv. Jack and 53 from cv. Spencer) were screened for SDS resistance. The regenerants did not differ significantly from the other cultivars/lines in mean disease severity ratings, although a greater percentage of regenerants (Jack and Spencer R3 combined) had disease severity ratings of 1 and 2 than did the soybean cultivars tested but not greater than PI520.733. The regenerants with lower disease severity ratings were grown to maturity to produce more seeds that will be used to screen plants for SDS resistance and for crossing to determine how heritable and useful. the resistance is in these regenerants.

The phytotoxicity of culture filtrates of Fusarium solani f. sp. glycines, the fungus causing sudden death syndrome (SDS) of soybean (Glycine max), was tested with a viability stain of soybean suspension-cultured cells and a stem cutting assay of soybean seedlings. Suspension-cultured cells from a SDS-susceptible soybean cultivar were exposed to cell-free culture filtrates of F. solani f. sp. glycines or other F. solani isolates for 2, 4, 6, and 8 days and then stained with 0.1% phenosafranin. The percentage of dead soybean suspension-cultured cells was greater (P < 0.001) with filtrates prepared from F. solani f. sp. glycines than from other F. solani isolates, and dead cells increased over time and with higher concentrations of culture filtrate. Cuttings of soybean seedlings with their stems immersed in culture filtrates of F. solani f. sp. glycines isolates developed SDS-like foliar symptoms, but not when immersed in filtrates of other isolates. There was a positive correlation (r = 0.94, P < 0.001) between soybean foliar symptom severity and percentage of stained soybean suspension-cultured cells. Both methods were used to determine the phytotoxicity of fungal culture filtrates.

Sudden death syndrome of soybean (Glycine max) is caused by the soilborne fungus, Fusarium solani f. sp. glycines, that infects soybean roots. Besides root necrosis, symptoms include interveinal leaf chlorosis, necrosis and premature defoliation. It is proposed that a fungal toxin is produced in soybean roots and translocated to foliage. In this study, we isolated compounds from soybean stem exudates from plants that were either inoculated or not inoculated with F. solani f. sp. glycines. A protein with an estimated molecular mass of 17 kDa and designated as FISP 17 for F. solani f. sp. glycines-induced stress protein was identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein occurred only in F. solani f. sp. glycines-infected soybean stem exudates. The N-terminal amino acid sequence of the purified protein had 100 % identity with a starvation-associated message 22 protein, and 80 and 78 % identity with purified bean pathogenesis-related proteins, PvPR1 and PvPR2, respectively. To determine if the protein was of plant or fungal origin, a synthetic peptide was designed based on the N-terminal sequence and used to raise a polyclonal antibody from rabbit. Western blot analysis showed that the antibody only reacted with a 17-kDa protein in F. solani f. so. glycines-infected plant exudates, but no reaction occurred with healthy plant exudates or with culture filtrates of F. solani f. Sp. glycines. This is the first report of the presence of a stress-induced protein in stem exudates of soybean seedlings root-infected with F. solani f. sp. glycines.

Fusarium solani f. sp. glycines, a soilborne fungus, infects soybean roots and causes sudden death syndrome. The response of 13 soybean genotypes to F. solani f. sp. glycines infection was tested with potted greenhouse grown plants and with cultured hairy roots. The taproots of all genotypes grown in the greenhouse had dark brown lesions following inoculation. Foliar disease severity for greenhouse grown plants measured 21 days after planting was greatest for Peking, followed by Spencer, Ripley, P3981, Williams 82, Essex, Forrest, Iroquois, PI 520733, Hartwig, PI 567650B, Jack, and PI 567374. There were significant negative correlations between foliar disease severity and shoot length (r = -0.422, P = 0.0018), shoot weight (r = -0.857, P < 0.0001), root weight (r = -0.732, P <0.0001), and total plant dry weights (r = -0.855, P < 0.0001). The taproot lesion length was not correlated with foliar disease severity indicating that soybean resistance may not be fully controlled at the root level. When cultured hairy roots were inoculated with F. solani f. sp. glycines mycelial plugs, the colony diameters after 10 days were significantly (P = 0.05) different among soybean genotypes ranging from 17 to 40 mm. Fungal colony diameters on hairy roots of Spencer and Peking were greater (P = 0.05) than on PI 567374 and PI 520733. In another experiment, following inoculation of Spencer and PI 567374 hairy roots with 10 µL of F. solani f. sp. glycines macroconidial suspensions, 10-day-colony diameters were 50 and 38 mm, respectively (P = 0.05). While there was generally a correlation between the growth of F. solani f. sp. glycines on the cultured hairy roots and the whole plant symptoms of the different genotypes, this was not always the case. The exceptions may be due to the fact that none of the genotypes showed clear root resistance even though they may show toxin resistance that would result in fewer foliar symptoms.

The soil-borne fungus, Fusarium solani f. sp. glycines (FSG), infects soybean roots and causes the disease sudden death syndrome. The biochemical response of soybean roots to FSG infection, that has not been studied before, was investigated by comparing FSG-inoculated and non-inoculated roots of two partially resistant (PI520.733 and PI567.374) and susceptible (Spencer) genotypes. Activity of phenylalanine ammonia-lyase, the first enzyme in the phenylpropanoid biosynthetic pathway, was greater in inoculated than non-inoculated plants of all three genotypes. The phytoalexin glyceollin increased to much higher levels in roots of the partially resistant cvs. PI520.733 and PI567.374 than in the susceptible Spencer. The changes in phenolic metabolism were much greater in lesion containing areas of roots than in the new portion growing under the FSG inoculum. No clear correlation was found between the glyceollin precursor daidzein and its conjugates and glyceollin levels in root tissues; however, isoflavone levels increased only in roots of inoculated plants of partially resistant lines, even though constitutive isoflavone levels were higher in the susceptible control. FSG growth on potato dextrose agar medium was inhibited by increasing concentrations of glyceollin. Induction of lignin synthesis was found in the inoculated roots of all three lines, with the highest rate of lignification observed in roots of the partially resistant genotypes, especially PI567.374. These studies show that FSG inoculation of soybean roots in soil induces the phenylpropanoid pathway to synthesize isoflavones, the phytoalexin glyceollin and lignin, indicating that these compounds may be involved in the partial resistance response.

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible ‘Spencer’ and partially resistant ‘PI567.374’) to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [14C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus.

Soybean hairy roots, transformed with the soybean chalcone synthase (CHS6) or isoflavone synthase (IFS2) genes, with dramatically decreased capacity to synthesize isoflavones were produced to determine what effects these changes would have on susceptibility to a fungal pathogen. The isoflavone and coumestrol concentrations were decreased by about 90% in most lines apparently due to gene silencing. The IFS2 transformed lines had very low IFS enzyme activity in microsomal fractions as measured by the conversion of naringenin to genistein. The CHS6 lines with decreased isoflavone concentrations had 5 to 20-fold lower CHS enzyme activities than the appropriate controls. Both IFS2 and CHS transformed lines accumulated higher concentrations of both soluble and cell wall bound phenolic acids compared to controls with higher levels found in the CHS6 lines indicating alterations in the lignin biosynthetic branch of the pathway. Induction of the soybean phytoalexin glyceollin, of which the precursor is the isoflavone daidzein, by the fungal pathogen Fusarium solani f. sp. glycines (FSG) that causes soybean sudden death syndrome (SDS) showed that the low isoflavone transformed lines did not accumulate glyceollin while the control lines did. The (iso)liquritigenin content increased upon FSG induction in the IFS2 transformed roots indicating that the pathway reactions before this point can control isoflavonoid synthesis. The lowest fungal growth rate on hairy roots was found on the FSG partially resistant control roots followed by the SDS sensitive control roots and the low isoflavone transformants. The results indicate the importance of phytoalexin synthesis in root resistance to the pathogen.

Sudden death syndrome, caused by Fusarium solani f. sp. glycines, has caused increased losses in soybean production in recent years. This study was done to identify potential sources of sudden death syndrome resistance. Over 6000 soybean plant introductions (PIs) were compared to a susceptible check, Great Lakes 3302, and two moderately resistant checks, PI 520.733 and PI 567.374. Only 60 PIs had foliar disease ratings equal to or lower than (P<0.05) PI 520.733 and PI 567.374 three weeks after inoculation. Also, 14 PIs had significantly lower disease severity ratings than PI 520.733 and six PIs had lower ratings than PI 567.374 four weeks after inoculation. Lesion lengths were measured four weeks after inoculation and ranged from 25.2 to 41.5 mm for all the PIs. None of the accessions had smaller lesions lengths than the susceptible check Great Lakes 3302. The correlation between lesion length and disease foliar severity rating was not significant. There also was no plant morphological characteristic associated with higher sudden death syndrome foliar symptoms. Eighteen moderately resistant PIs with differing agronomic traits were inoculated with five different isolates of Fusarium solani f. sp. glycines. There was very little variation for foliar symptoms among PIs within each isolate. However, there was variation for symptom intensity among isolates. Isolate Mont-1 caused the greatest disease severity ratings.

Sudden death syndrome, caused by Fusarium solani f. sp. glycines, has caused severe damage to soybean production in recent years. One way to control sudden death syndrome is with resistant cultivars. Over a 3-year period, 2335 publicly and privately developed soybean entries were inoculated and evaluated for their response to F. solani f. sp. glycines under greenhouse conditions. The entries were compared to the susceptible check, Great Lakes 3302 (GL3302), and the moderately resistant checks, plant introductions (PI) 520733 and 567374. Thirty-eight entries were identified with moderate levels of resistance. Based on foliar ratings, there were no differences (P<0.05) between the Roundup Ready and conventional cultivars. Ninety ancestral lines that represent 99% of the genes in modern U.S. cultivars and 55 lines found in the pedigrees of public cultivars reported to have some resistance were evaluated for their response to F. solani f. sp. glycines. Nine ancestral lines (Aoda , Kim, Jackson, Sioux, Mammoth Yellow, T117, PI 171450, PI 54615-1, and PI 71506) and 12 cultivars or experimental lines (Ina, D83-3349, LN98-4340, LN83-2356, Hartwig, Harosoy, Bedford, Merit, Cutler, Calland, Hill, and Evans) had disease ratings not significantly different (P<0.05) from PI 520733 or PI 567374. PI 54610, a putative ancestral line, was also found to be moderately resistant.

Several plant introductions (PIs) and cultivars have been classified as partially resistant (PR) to sudden death syndrome. However, little is known about the nature of resistance to this disease. Seedlings of two PR PIs and two susceptible cultivars were inoculated with Fusarium solani f. sp. glycines in aeroponic chambers. Plants were inoculated by taping two sorghum seeds infested with F. solani f. sp. glycines to the main root. Foliar symptoms of the susceptible cultivars were higher than those on the PR PIs and were associated with lower root and plant dry weight. Root lesion lengths of the four soybean lines differed (P < 0.05), but did not correlate with foliar disease or any other variable. To better understand the resistance mechanism by distinguishing between root and plant resistance, three partially resistant PIs (PI 520.733, PI 567.374, and PI 567.650B) and one susceptible soybean cultivar (GL3302) were compared using different grafting combinations in aeroponic chambers. Results of sudden death syndrome evaluation indicated that resistance is conditioned by both the scion and the rootstock. All three PIs evaluated had resistance associated with the scion; resistance in PI 567.650B also was associated with the rootstock. Although the PR PIs used appear to have little or no root resistance, an aeroponic system and grafting may help identify new sources of resistance to F. solani f. sp. glycines with root- or whole-plant resistance.

Cell-free toxic culture filtrates from Fusarium virguliforme, the causal fungus of soybean sudden death syndrome (SDS), cause foliar symptoms on soybean stem cuttings similar to those obtained from root inoculations in whole plants and those observed in production fields. The objectives of this study were to (i) optimize the production conditions for F. virguliforme cell-free toxic culture filtrates and the incubation conditions of the stem cutting assay used to test the toxicity of the cell-free toxic culture filtrates, and (ii) use the optimized assay and a whole plant root inoculation assay to compare four SDS-causing isolates on a panel of selected soybean genotypes. Area under the disease progress curve (AUDPC) values were highest (P = 0.05) when cuttings were immersed in culture filtrate of fungus grown in soybean dextrose broth, in filtrate produced from the fungus grown for 18 or 22 days, and when stem cuttings were incubated at 30°C. AUDPC values and shoot dry weights from the whole plant root inoculations and the AUDPC values from the stem cutting assay differed (P < 0.05) among nine soybean genotypes tested with F. virguliforme and F. tucumaniae isolates, and the AUDPC values from the two assays were positively correlated (r = 0.44 at P < 0.0001).

Fusarium virguliforme causes soybean sudden death syndrome (SDS) in the United States. The disease was first observed in Arkansas in 1971, and since has been reported in most soybean-producing states, with a general movement from the southern to the northern states. In addition to F. virguliforme, three other species, Fusarium brasiliense, Fusarium crassistipitatum, and Fusarium tucumaniae, have been reported to cause SDS in South America. Yield losses caused by F. virguliforme range from slight to 100%. Severely infected plants often have increased flower and pod abortion, reduced seed size, increased defoliation, and prematurely senescence. Foliar symptoms observed in the field are most noticeable from mid to late reproductive growth stages. To manage SDS, research on crop rotations, soil types, tillage practices, seed treatments, and the development and utilization of host resistance has been investigated. This review focuses on what is known about F. virguliforme, the management of SDS in the United States, and how genetic engineering along with other traditional management options may be needed as integrated approaches to manage SDS.

Sudden death syndrome (SDS) of soybeans, causal organism Fusarium solani, occurred for the first time in epidemic proportions in east-central Illinois in 1993. SDS occurred in 46% of the soybean fields, based on air and ground surveys. Within fields containing plants with SDS, the percent area affected ranged from 1 to 70% with an average of 7.3 and 13.9% for ground and aerial surveys, respectively. Soil samples taken in 25 fields in areas where all plants showed SDS symptoms and in adjacent areas where plants appeared healthy did not differ in cyst populations of soybean cyst nematode (Heterodera glycines). Disease severity and yield components were compared from replicated plots at an experimental farm and in a commercial field and in a nonreplicated commercial field. Diseased plants from infested areas had fewer pods and seeds and less 300-seed weights. Plant yields were 46, 41, and 20% less for plants in plots with a high incidence of SDS than were the yields for those with a low incidence of SDS in a nonreplicated commercial field, replicated plots at an experimental farm, and a commercial field, respectively. Seed germination was less and the frequency of Phomopsis spp. was greater on seeds harvested from plants in plots with a high occurrence of SDS compared to plots with a low occurrence of SDS.

Sudden death syndrome (SDS), caused by Fusarium virguliforme (Fv), is a major yield-limiting disease of soybean in North America. Infection of soybean seedling roots by Fv results in severe root damage; therefore, fungicide seed treatments could potentially reduce these early-season infections and reduce severity of foliar symptoms that typically occur later in the season. Multiple fungicide seed treatment combinations were evaluated for their effects on Fv infection, DNA concentrations in roots, soybean root development, and SDS development in the field, greenhouse and laboratory trials. Several seed treatments decreased root disease symptoms compared with the non-treated inoculated control in the laboratory assay, and the biological seed treatment, Bacillus pumilus, significantly decreased seedling development and increased SDS root disease compared with the non-treated inoculated control. In the greenhouse, Fv DNA concentrations in roots were reduced by a treatment combining mefenoxam + thiophanate-methyl + azoxystrobin + Bacillus pumilus + prothioconazole + fludioxonil compared with the non-treated control; however, the reduction in Fv DNA did not improve root growth or decrease SDS symptoms compared with the non-treated control. Field trials were conducted in Valmeyer, IL in 2008 and in Urbana, IL in 2008 and 2009. Seed treatments had no effect on the concentration of Fv DNA in soybean roots and had very little effect on root morphology. At the Valmeyer location, most seed treatments significantly decreased SDS symptoms compared with the control. In summary, no consistent, significant effects of the seed treatments evaluated in this study on SDS or Fv root infection were observed. Therefore, soybean growers should continue to utilize other practices for SDS management until new seed treatments with consistent efficacy in controlling SDS are available.