Soybean Sudden Death Syndrome Research Articles

First Report of Sexual Reproduction by the Soybean Sudden Death Syndrome Pathogen Fusarium tucumaniae in Nature.

Of the four fusaria that have been shown to cause soybean sudden death syndrome (SDS), field surveys indicate that Fusarium tucumaniae is the most important and genetically diverse SDS pathogen in Argentina. Although none of the SDS fusaria have been shown to produce perithecia in nature, a heterothallic sexual cycle has been demonstrated for F. tucumaniae via laboratory crosses. Herein we report on the discovery of perithecia of F. tucumaniae on soybean in Argentina. Ascospores derived from these perithecia gave rise to colonies that produced sporodochial conidia diagnostic of F. tucumaniae. Sporodochial conidia were longer and narrower than those produced by the other SDS fusaria; these conidia also possessed a diagnostic acuate apical cell and a distinctly foot-shaped basal cell. Sixteen strains derived from single ascospores subjected to a validated multilocus genotyping assay (MLGT) for SDS species determination, together with 16 conidial isolates from two sites where teleomorphs were collected, independently confirmed the morphological identification as F. tucumaniae. This study represents the first authentic report of sexual reproduction by a soybean SDS pathogen in nature.

Carbon utilization profiles of Fusarium virguliforme isolates

Fusarium virguliforme is the cause of sudden death syndrome in soybean. Physiological variability among isolates of the fungus is unknown. One way to measure physiologic variability is to analyze growth on different carbon sources. The carbon source utilization profiles of 18 F. virguliforme isolates were examined using the Biolog FF 96-well microplate, which contains 95 different carbon sources. The utilization of dextrin,D-mannitol, maltotriose,D-lactic acid methyl ester, N-acetyl-D-galactosamine, salicin, D-trehalose, and L-alanine differed significantly among isolates (P = 0.05). Carbon sources were grouped into 3 clusters based on their ability to promote growth of F. virguliforme, after calculating Euclidean distances among them. About 12% of the carbon sources promoted a high amount of mycelial growth, 39% promoted a medium amount of growth, and 49% promoted a low amount of mycelial growth; the latter was not significantly different from the water blank control. A hierarchical tree diagram was produced for the 18 isolates based on their carbon source utilization profiles using Ward's hierarchical analysis method. Two main clusters of isolates were formed. One cluster represented greater average mycelial growth on all of the carbon sources than the other cluster. In this study, variability in carbon source utilization among F. virguliforme isolates was evident, but the results were not associated with geographic origin of the isolates, year collected, or published data on aggressiveness. Additional research is needed to determine if these carbon utilization profiles are associated with other biological characteristics, like spore germination, propagule formation, and saprophytic competitiveness.

First Report and Confirmed Distribution of Soybean Sudden Death Syndrome Caused by Fusarium virguliforme in Southern Michigan.

Leaf lesions and root rot symptoms typical of soybean sudden death syndrome (SDS) caused by Fusarium virguliforme O'Donnell & T. Aoki were observed in commercial soybeans (Glycine max (L.) Merr.) in southern Michigan. Leaf symptoms ranged from chlorotic spots to severe interveinal chlorosis and necrosis, no foliar pathogens were noted. In 2008, isolates were collected from Berrien and St. Joseph counties. In 2009, isolates were collected from Cass, St. Joseph, Van Buren, Allegan, and Monroe counties. Pieces of roots with root rot symptoms were washed prior to surface disinfestation with 70% ethanol for 30 s and 0.5% NaOCl for 1 min and incubated on water agar (WA) in petri plates amended with 50 μg/ml of chloramphenicol for the production of sporodochia. Alternatively, spores were collected directly from nondisinfested roots expressing blue sporodochia. Single-spore cultures were derived by streaking macroconidia with a bacterial loop onto 3% WA + chloramphenicol and incubated overnight. With a dissecting microscope, single germinated macroconidia were collected with a sterile 0.2-mm-diameter insect pin and transferred to potato dextrose agar (PDA). Cultures on PDA grew slowly and developed blue-to-purple masses of sporodochia typical of F. virguliforme descriptions and similar to a representative isolate, Mont-1, grown alongside (1,2). Size of macroconidia from the six representative isolates, one from each county (including isolates derived from surface-disinfested and nondisinfested roots), and Mont-1 were determined to be within the range for F. virguliforme (42 to 56 × 5 to 6 μm), with an average of four septa per macroconidia. Identity of the representative isolates was confirmed by partial DNA sequencing of both strands of the internal transcribed spacer (ITS) region of the ribosomal RNA gene, translation elongation factor 1-α, and β-tubulin loci. All six representative isolates were identical in each of the three loci and matched with 100% similarity F. virguliforme accessions in GenBank and Fusarium-ID database searches, except for the β-tubulin locus in which a single nucleotide insertion was noted (Accession Nos. HM453328-HM453330). Sequences were 98 to 99% similar to other SDS Fusarium spp. not yet recorded in the United States. Koch's postulates were performed in the greenhouse according to Malvick and Bussey (3). Infested sorghum seed (~20 g) was placed 2 cm below soybean seed of susceptible cv. Williams 82 in plastic pots. Noninfested sorghum seed was used as a negative control and sorghum infested with Mont-1 as a positive control. Chlorotic spots developed 2 weeks after establishing the trial, and 3 to 4 weeks postinoculation, severe SDS symptoms of foliar interveinal chlorosis and necrosis and severe root rot developed. Koch's postulates were completed by reisolating F. virguliforme from a subset of infected plants. In addition, an isolate of F. virguliforme collected in 2008 was used to inoculate a 2009 field trial in East Lansing, MI with no history of SDS. Typical SDS symptoms developed in the field trial and F. virguliforme was isolated from a symptomatic plant that was identified as described above. Despite being reported across the majority of soybean-producing states, to our knowledge, this is the first confirmation and distribution report for SDS in Michigan.

Soybean Sudden Death Syndrome Species Diversity Within North and South America Revealed by Multilocus Genotyping

Sudden death syndrome (SDS) of soybean has become a serious constraint to the production of this crop in North and South America. Phenotypic and multilocus molecular phylogenetic analyses, as well as pathogenicity experiments, have demonstrated that four morphologically and phylogenetically distinct fusaria can induce soybean SDS. Published molecular diagnostic assays for the detection and identification of these pathogens have reported these pathogens as F. solani, F. solani f. sp. glycines, or F. solani f. sp. phaseoli, primarily because the species limits of these four pathogens were only recently resolved. In light of the recent discovery that soybean SDS and Phaseolus and mung bean root rot (BRR) are caused by four and two distinct species, respectively, multilocus DNA sequence analyses were conducted to assess whether any of the published molecular diagnostic assays were species-specific. Comparative DNA sequence analyses of the soybean SDS and BRR pathogens revealed that highly conserved regions of three loci were used in the design of these assays, and therefore none were species-specific based on our current understanding of species limits within the SDS-BRR clade. Prompted by this finding, we developed a high-throughput multilocus genotyping (MLGT) assay which accurately differentiated the soybean SDS and two closely related Phaseolus and mung BRR pathogens based on nucleotide polymorphism within the nuclear ribosomal intergenic spacer region rDNA and two anonymous intergenic regions designated locus 51 and 96. The single-well diagnostic assay, employing flow cytometry and a novel fluorescent microsphere array, was validated by independent multilocus molecular phylogenetic analysis of a 65 isolate design panel. The MLGT assay was used to reproducibly type a total of 262 soybean SDS and 9 BRR pathogens. The validated MLGT array provides a unique molecular diagnostic for the accurate identification and molecular surveillance of these economically important plant pathogens.

Comparative analysis and characterization of the soybean sudden death syndrome pathogen Fusarium virguliforme in the northern United States

Sudden death syndrome (SDS), caused by Fusarium virguliforme, has occurred in several US states and Ontario since 1985 but is a new problem in the northern states, including Minnesota. The characteristics and distribution of F. virguliforme and SDS in Minnesota and other northern soybean production areas were unknown. In 2006 and 2007, SDS was confirmed in 21 counties from the eastern to western borders of Minnesota and to 45°N based on symptoms and a polymerase chain reaction assay. It was also confirmed that F. virguliforme and Phialophora gregata, the cause of brown stem rot, can simultaneously infect a single plant. Isolates of F. virguliforme from soybeans in Minnesota were morphologically identical to those from other states and induced typical SDS symptoms on soybean seedlings. Isolates (n = 15) obtained from 15 fields in 10 Minnesota counties were compared genotypically and phenotypically to a group of 13 isolates of F. virguliforme from other states. Genetic similarity was very high among isolates from Minnesota, Iowa, Missouri, and Illinois based on DNA sequences from the intergenic spacer region, the translation elongation factor 1α gene, and the internal transcribed spacer region. Growth rates and production of macroconidia at several temperatures differed among isolates. Virulence varied among isolates from different fields and states based on disease reactions on susceptible and moderately resistant soybean cultivars in greenhouse tests. Results support the hypothesis that SDS in Minnesota may stem from the establishment of a clonal US population of F. virguliforme that is spreading north and west, thus increasing production risk for soybean grown in northern states. Key words: Fusarium virguliforme, Fusarium solani, root rot, population biology, DNA sequences, soybean. Le syndrome de la mort subite (SMS), causé par Fusarium virguliforme, a été documenté dans plusieurs États américains et en Ontario depuis 1985, mais dans les États du nord, incluant le Minnesota, c’est un problème récent. Les caractéristiques et les aires de distribution de F. virguliforme et du SMS au Minnesota et dans les autres régions productrices de soja du nord étaient inconnues. En 2006 et 2007, la présence du SMS a été confirmée, par reconnaissance des symptômes et PCR, dans 21 comtés du Minnesota s’étendant de l’est à l’ouest de l’État, et ce, jusqu’au 45° de latitude Nord. On a également confirmé que F. virguliforme et Phialophora gregata, responsable de la pourriture brune des tiges, peuvent infecter simultanément un même plant. Des isolats de F. virguliforme provenant de plants de soja du Minnesota étaient morphologiquement identiques à ceux provenant d’autres États et induisaient l’apparition des symptômes typiques du SMS chez les plantules de soja. Des isolats (n = 15) obtenus de 15 champs situés dans 10 comtés du Minnesota ont été comparés, sur les plans génotypiques et phénotypiques, à 13 isolats de F. virguliforme provenant d’autres États. En ce qui a trait aux séquences d’ADN extraites de la région de l’espaceur intergénique, du gène du facteur d’élongation 1α de la traduction et de la région de l’espaceur transcrit interne, elles présentaient une similarité génétique frappante malgré que les isolats provenaient du Minnesota, de l’Iowa, du Missouri et de l’Illinois. Par ailleurs, le taux de croissance ainsi que la production de macroconidies variaient chez les isolats exposés à des conditions différentes de température. En outre, selon des tests visant à évaluer les réactions à la maladie, tests effectués en serre sur des cultivars de soja réceptifs et moyennement réfractaires, le taux de virulence variait aussi chez les isolats provenant de différents champs et de divers États. Les résultats supportent l’hypothèse voulant qu’au Minnesota le SMS provienne de l’établissement d’une population clonale états-unienne de F. virguliforme qui s’étend vers le nord et l’ouest, ce qui accroît les risques relatifs à la production de soja dans les États du nord. Mots-clés : Fusarium virguliforme, Fusarium solani, pourridié, biologie des populations, séquences d’ADN, soja.

Morphological and Molecular Characterization of Fusarium solani Isolates

Abstract Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF‐1α) distinguished the ‘F. solani complex’ in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment‐length polymorphism (AFLP) and mini‐microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum, were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well‐supported clade including F. solani f. sp. pisi, nine into a clade including only isolates of F. solani f. sp. radicicola and four into a clade including F. solani f. sp. cucurbitae, but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.

Separate loci underlie resistance to root infection and leaf scorch during soybean sudden death syndrome

Soybean [Glycine max (L.) Merr.] cultivars show differences in their resistance to both the leaf scorch and root rot of sudden death syndrome (SDS). The syndrome is caused by root colonization by Fusarium virguliforme (ex. F. solani f. sp. glycines). Root susceptibility combined with reduced leaf scorch resistance has been associated with resistance to Heterodera glycines HG Type 1.3.6.7 (race 14) of the soybean cyst nematode (SCN). In contrast, the rhg1 locus underlying resistance to Hg Type 0 was found clustered with three loci for resistance to SDS leaf scorch and one for root infection. The aims of this study were to compare the inheritance of resistance to leaf scorch and root infection in a population that segregated for resistance to SCN and to identify the underlying quantitative trait loci (QTL). "Hartwig", a cultivar partially resistant to SDS leaf scorch, F. virguliforme root infection and SCN HG Type 1.3.6.7 was crossed with the partially susceptible cultivar "Flyer". Ninety-two F5-derived recombinant inbred lines and 144 markers were used for map development. Four QTL found in earlier studies were confirmed. One contributed resistance to leaf scorch on linkage group (LG) C2 (Satt277; P = 0.004, R2 = 15%). Two on LG G underlay root infection at R8 (Satt038; P = 0.0001 R2 = 28.1%; Satt115; P = 0.003, R2 = 12.9%). The marker Satt038 was linked to rhg1 underlying resistance to SCN Hg Type 0. The fourth QTL was on LG D2 underlying resistance to root infection at R6 (Satt574; P = 0.001, R2 = 10%). That QTL was in an interval previously associated with resistance to both SDS leaf scorch and SCN Hg Type 1.3.6.7. The QTL showed repulsion linkage with resistance to SCN that may explain the relative susceptibility to SDS of some SCN resistant cultivars. One additional QTL was discovered on LG G underlying resistance to SDS leaf scorch measured by disease index (Satt130; P = 0.003, R2 = 13%). The loci and markers will provide tagged alleles with which to improve the breeding of cultivars combining resistances to SDS leaf scorch, root infection and SCN HG Type 1.3.6.7.

Foliar Symptom Expression in Association with Early Infection and Xylem Colonization by Fusarium virguliforme (formerly F. solani f. sp. glycines), the Causal Agent of Soybean Sudden Death Syndrome

Soybean sudden death syndrome (SDS), caused by Fusarium virguliforme (FV), is a root disease that results in severe foliar symptoms during the reproductive stage. In a regular growing season, an epidemic of SDS is highly correlated with the planting date and the disease tends to be more severe in earlier planted soybeans. Occurrence of infection early in the season is likely to result in colonization in the xylem and phloem tissues, a process essential for foliar symptom expression because xylem tissues are upward pathways in soybean plants. To demonstrate the biology of this infection, we used an effective and quantifiable seedling inoculation technique in which germinated seeds in a Petri dish were spray-inoculated with conidial suspension before being transplanted. Plants that had foliar symptoms showed both external and internal discolored taproots and basal stems, while plants with no foliar symptoms had only superficial discoloration. Microtome sectioning of taproots of plants that had foliar symptoms revealed the presence of fungal structures in both xylem and phloem tissues, while plants that had no foliar symptoms revealed fungal structures only in phloem tissue. A scanning electron microscope study showed a higher penetration frequency of FV near the root-cap zone where few or no root hairs of the radicle were found. These results indicate that fungal penetration into the xylem tissue plays a role in foliar symptom expression. Accepted for publication 10 December 2007. Published 22 February 2008.

First Report of Sudden Death Syndrome of Soybean in Wisconsin.

In August of 2006, soybean (Glycine max (L.) Merr.) plants collected from Columbia, Dane, Green Lake, Walworth, Jefferson, and Waushara counties in southern Wisconsin exhibited symptoms typical of sudden death syndrome (SDS) caused by Fusarium virguliforme O'Donnell & Aoki [synonym F. solani (Mart.) Sacc. f. sp. glycines] (1). Foliar symptoms ranged from chlorotic spots to severe interveinal chlorosis and necrosis. Taproots of symptomatic plants were necrotic and stunted and stems exhibited a light tan discoloration, but never the dark brown discoloration typical for brown stem rot, a disease with similar foliar symptoms. Isolations from root and crown tissue of symptomatic plants were made using one-quarter-strength potato dextrose agar (PDA) amended with 100 ppm of streptomycin. Slow-growing, white-to-cream fungal colonies with blue and turquoise sporodochia were observed. Spores produced in sporodochia grown on PDA ranged in size from 32.5 to 70 μm long (average 53.1 μm) and 3 to 6 μm wide (average 4.4 μm) and with 3-5 septa (mode of 3). Isolates were characteristic of F. virguliforme based on colony morphology, spore morphology and size, and the absence of microconidia (3). The identity of F. virguliforme was confirmed by PCR amplification and DNA sequencing of the ITS, BT1, Act, and EF1B regions. All isolate sequences exhibited single nucleotide polymorphisms that matched the sequences of these regions of F. virguliforme. Koch's postulates were conducted to confirm that the causal agent of the observed symptoms was F. virguliforme. Inoculum of single-spore isolates was produced on sterilized sorghum seed. After 14 days of incubation at 20 to 22°C and a 12-h photoperiod, the sorghum seed was assayed to determine colonization incidence by transferring seeds to PDA. In all trials, sorghum seed was 100% infested. Infested sorghum seeds (35) were placed in potting soil at 2 cm beneath each seed of the susceptible soybean cv. Williams 82 (4). Noninfested sorghum seed was used for a noninoculated control. Three trials were performed, each using 15 replicates of several fungal isolates and 15 replicates of the noninoculated control. Plants were grown in water baths located in a greenhouse (trial 1) and in a growth chamber (trial 2) and both maintained at an average temperature of 25°C with a 14-h photoperiod (2). The third trial was conducted in the growth chamber without a water bath with the same temperature and light regimen. In all environments, inoculated plants developed chlorotic spots 14 days after planting. After 21 days, symptoms progressed to a range of chlorotic mottling to interveinal chlorosis and necrosis. Foliar and root symptoms that resembled those on the original plant samples infected with F. virguliforme appeared on 88% of inoculated plants. Isolates that resembled the original F. virguliforme were recovered from 75% of inoculated plants and from 88% of plants showing symptoms. No symptoms were observed and no isolates were recovered from noninoculated plants. There was a statistically significant difference between inoculated and control plants (P < 0.001) based on the presence of symptoms and isolation success using the Goodman χ2 analysis. The confirmation of the presence of SDS in five counties suggests that the disease is widespread in Wisconsin and could become a serious threat to soybean production in the future.

Sexual reproduction in the soybean sudden death syndrome pathogen Fusarium tucumaniae

We investigated the sexual reproductive mode of the two most important etiological agents of soybean sudden death syndrome, Fusarium tucumaniae and Fusarium virguliforme. F. tucumaniae sexual crosses often were highly fertile, making it possible to assign mating type and assess female fertility in 24 South American isolates. These crosses produced red perithecia and oblong-elliptical ascospores, as is typical for sexual members of the F. solani species complex. Genotyping of progeny from three F. tucumaniae crosses confirmed that sexual recombination had occurred. In contrast, pairings among 17 U.S. F. virguliforme isolates never produced perithecia. Inter-species crosses between F. tucumaniae and F. virguliforme, in which infertile perithecia were induced only in one of the two F. tucumaniae mating types, suggest that all U.S. F. virguliforme isolates are of a single mating type. We conclude that the F. tucumaniae life cycle in S. America includes a sexual reproductive mode, and thus this species has greater potential for rapid evolution than the F. virguliforme population in the U.S., which may be exclusively asexual.

Irrigation and Inoculation Treatments that Increase the Severity of Soybean Sudden Death Syndrome in the Field

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 &lt; 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.

Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae

The objective of this work was to identify genomic regions that underlie resistance to Fusarium tucumaniae sp. nov., the causing agent of sudden death syndrome (SDS) in soybean in South America, using a population with a genetic background different from that previously reported for Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), also responsible for SDS in soybean. Although major genes and quantitative trait loci (QTL) for SDS resistance have been identified, little is known about the same disease caused by Fusarium tucumaniae sp. nov., in South America. To identify genetic factors related to resistance to F. tucumaniae and DNA markers associated with them, a QTL analysis was performed using recombinant inbred lines. The map locations of the four loci, here identified, differed from those SDS resistance QTL previously described. It was screened a residual heterozygous line (RHL), which was heterozygous around the most effective QTL, RSDS1, and homozygous for the other genomic regions. The genetic effect of RSDS1 was confirmed using near-isogenic lines (NIL) derived from the RHL. The line which was homozygous for the Misuzudaizu genotype showed resistance levels comparable with that of the line homozygous for the Moshidou Gong 503 genotype.

Modification of phenolic metabolism in soybean hairy roots through down regulation of chalcone synthase or isoflavone synthase

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.

Response of soybean sudden death syndrome to tillage and cultivar

Sudden death syndrome (SDS), caused by Fusarium solani f. sp. glycines, is a season-long root disease of soybean (Glycine max) with foliar symptoms beginning in late vegetative and early reproductive stages of plant growth. The disease has been reported in most soybean-growing regions of the United States of America and the world. Previous research found that SDS was less severe in areas that were subsoiled when compared with those with no-till. The objectives of the current study were to determine (i) if cost-effective tillage practices, i.e., chisel plowing and disk tillage, can be efficient in reducing SDS and (ii) if cultivars with a range of SDS resistance or susceptibility respond differently in SDS expression across various tillage practices. In the fall of 2000 and 2001, plots were established in a field with a history of severe SDS and with a soil type consisting of an Armiesburg silty clay loam. The experiment was organized in a split-plot design with four replications. Main plots were tillage (chisel tillage, disk tillage, and no tillage), and subplots were cultivars randomized in main plots. Twelve cultivars were selected based on resistance or susceptibility to SDS. In 2000, chisel tillage reduced the area under the foliar disease progress curve (AUFDPC) when compared with no-till or disk tillage. In 2001, both chisel and disk tillage reduced AUFDPC similarly when compared with no-till. In 2000, the rate of root infection by the fungus, that is, the area under the root colonization progress curve (AURCPC), was higher in both disk- and chisel-tillage treatments than in no-tillage treatments. In 2001, higher rates of root infection occurred with chisel tillage compared with disk tillage and no-till. Among the cultivars, foliar disease ratings, AUFDPC, and soybean yield differed, but there were no differences in the AURCPC.

Sudden death syndrome of soybean in South America is caused by four species of Fusarium : Fusarium brasiliense sp. nov., F. cuneirostrum sp. nov., F. tucumaniae, and F. virguliforme

Soybean sudden death syndrome (SDS) pathogens and dry bean root-rot pathogens were studied taxonomically, phylogenetically, and pathologically. Detailed phenotypic comparisons of macro- and microscopic features and phylogenetic analyses of multilocus DNA sequence data, including those on the nuclear ribosomal intergenic spacer region and the single copy nuclear gene translation elongation factor 1-a, indicated that they comprised five distinct species of Fusarium. Two new species causing soybean SDS in Brazil, F. brasiliense and F. cuneirostrum, are formally described. Fusarium cuneirostrum is responsible for soybean SDS in Brazil and dry bean or mung bean root-rot in the United States, Canada, and Japan. Strains of each species, including F. cuneirostrum isolates from dry bean and mung bean and F. phaseoli isolates from dry bean, were inoculated on soybean cultivar Pioneer 9492RR to determine their pathogenicity. Although intraspecific variation in pathogenicity was observed, all the species were able to induce typical SDS symptoms on soybean plants in the artificial inoculation tests. Comparisons of the key diagnostic morphological features reveal that all five species can be diagnosed using conidial morphology.

Genomic analysis of a region encompassingQRfs1andQRfs2: genes that underlie soybean resistance to sudden death syndrome

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.

Morphological and molecular characterization of the sudden‐death syndrome pathogen of soybean in Brazil

Brazilian Fusarium isolates causing soybean sudden death syndrome (SDS) were characterized by comparing them with other Fusarium isolates associated with soybean root rot, as well as F. solani f.sp. glycines isolates associated with the disease in the USA, using molecular (mitochondrial and nuclear rDNA), morphological, cultural and pathogenic characteristics. On the basis of pathogenicity data, and restriction fragment length polymorphism and sequence analysis of the rDNA internal transcribed spacer (ITS) regions, isolates formed a group distinct from nonSDS F. solani isolates, as well as other Fusarium species. ITS sequence analysis also revealed that Brazilian isolates were distinct from the majority of SDS pathogens from the USA (Fusarium virguliforme) and conformed to Fusarium tucumaniae.

Detection and Quantification of Fusarium solani f. sp. glycines in Soybean Roots with Real-Time Quantitative Polymerase Chain Reaction

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 cyclophilin 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 × 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.

Mapping soybean sudden death syndrome as related to yield and soil/site properties

Soybean sudden death syndrome (SDS) appears as a mid to late season foliar disease. Presently, little on-farm research has been conducted to study soil and site factors as related to the SDS disease. The objectives of this study were to study the influence of SDS disease on soybean yield and to compare soil and site factors with the SDS disease incidence and severity so that the factors that influence the disease could be identified. This was a 2-year study conducted on a farm near Desoto, IL, USA. Various site and soil parameters were collected, mapped and analyzed statistically to correlate with the SDS foliar index, DX (= disease incidence × severity/9). Soybean yield declined 7% for every 10% increase in DX. Statistically, no significant relationship was found between DX and organic matter content in soils. However, DX was positively related to soil pH, bulk density and moisture content at field capacity, but inversely related to available potassium, and macro-porosity. Both rooting depth and topography of the field had mixed relationships with the SDS index.

Recent Outbreak of Soybean Sudden Death Syndrome Caused by Fusarium virguliforme and F. tucumaniae in Argentina.

Sudden death syndrome (SDS) of soybean was detected initially in Argentina during 1991-1992 in the Pampas Region and 1992-1993 in the Northwest Region. The first report of the fulfillment of Koch's postulates of SDS caused by Fusarium solani f. sp. glycines in Argentina was published in 2003 (3). Subsequently, analyses have shown that F. solani f. sp. glycines represents several morphologically and phylogenetically distinct species, including F. tucumaniae in Argentina and F. virguliforme in the United States (1). Isolations were made from plants that exhibited typical SDS symptoms (interveinal foliar chlorosis and necrosis leading to defoliation of the leaflets but not the petioles) from fields in Santa Fe and Buenos Aires provinces in 2001, 2002, and 2003. To determine which species are responsible for SDS in Argentina, cultures of eight slow growing isolates that developed bluish pigmentation and produced abundant macroconidia in sporodochia on potato dextrose agar were subjected to morphological and molecular phylogenetic analyses and pathogenicity tests. Morphological analyses demonstrated that three of the isolates were F. virguliforme and five were F. tucumaniae. Isolates of F. tucumaniae produced long and narrow sporodochial conidia while F. virguliforme produced diagnostic comma-shaped conidia. Molecular phylogenetic analyses of DNA sequences from multiple loci confirmed morphology-based identifications and showed that the soybean SDS pathogen in the United States, F. virguliforme, was also present in Argentina. To our knowledge, this is the first report of F. virguliforme in Argentina and of this pathogen outside the United States. Five isolates of F. tucumaniae and three isolates of F. virguliforme were used for pathogenicity tests. F. virguliforme isolate 171 provided by J. Rupe (University of Arkansas, Fayetteville) was used as a positive control. Soybean cultivars Ripley, RA 702, Pioneer 9492RR, Spencer, and A-6445RG were inoculated with each of the isolates tested in a greenhouse assay using soil infestation and toothpick methods (2). All eight isolates produced typical foliar SDS symptoms 15 to 25 days after inoculation. Severity of foliar symptoms averaged 3.3 for F. virguliforme, 2.6 for F. tucumaniae, and 3.3 for the positive control using a disease severity scale in which 1 = no symptoms and 5 = severely infected or dead plants. Under these conditions, F. virguliforme appeared to be more virulent than F tucumaniae. Noninoculated plants remained symptomless. Koch's postulates were confirmed with soybean cultivars RA 702 and A6445RG. Isolates recovered from symptomatic plants inoculated by the soil infestation and toothpick methods were identical to those used to inoculate the plant. Strains were recovered at frequencies of 100 and 60% from plants inoculated by the toothpick and soil infestation methods, respectively. To our knowledge, this is the first report of the fulfillment of Koch's postulates for F. tucumaniae and F. virguliforme in Argentina.