Soybean Sudden Death Syndrome Pathogen Research Articles

Genome-wide transcriptional response of the causal soybean sudden death syndrome pathogen Fusarium virguliforme to a succinate dehydrogenase inhibitor fluopyram.

Succinate dehydrogenase inhibitors (SDHIs) have been widely used to manage plant diseases caused by phytopathogenic fungi. Although attention to and use of SDHI fungicides has recently increased, molecular responses of fungal pathogens to SDHIs have often not been investigated. A SDHI fungicide, fluopyram, has been used as a soybean seed treatment and has displayed effective control of Fusarium virguliforme, one of the causal agents of soybean sudden death syndrome. To examine genome-wide gene expression of F. virguliforme to fluopyram, RNA-seq analysis was conducted on two field strains of F. virguliforme with differing SDHI fungicide sensitivity in the absence and presence of fluopyram. The analysis indicated that several xenobiotic detoxification-related genes, such as those of deoxygenase, transferases and transporters, were highly induced by fluopyram. Among the genes, four ATP-binding cassette (ABC) transporters were characterized by the yeast expression system. The results revealed that expression of three ABCG transporters was associated with reduced sensitivity to multiple fungicides including fluopyram. In addition, heterologous expression of a major facilitator superfamily (MFS) transporter that was highly expressed in the fluopyram-insensitive F. virguliforme strain in the yeast system conferred decreased sensitivity to fluopyram. This study demonstrated that xenobiotic detoxification-related genes were highly upregulated in response to fluopyram, and expression of ABC or MFS transporter genes was associated with reduced sensitivity to the SDHI fungicide. This is the first transcriptomic analysis of the fungal species response to fluopyram and the finding will help elucidate the molecular mechanisms of SDHI resistance. © 2021 Society of Chemical Industry.

Soybean SDS in South Africa is Caused by Fusarium brasiliense and a Novel Undescribed Fusarium sp.

Soybean sudden death syndrome (SDS) was detected in South Africa for the first time during pathogen surveys conducted in 2013 to 2014. The primary objective of this study was to characterize the 16 slow-growing Fusarium strains that were isolated from the roots of symptomatic plants. Molecular phylogenetic analyses of a portion of translation elongation factor 1-α (TEF1) and the nuclear ribosomal intergenic spacer region (IGS rDNA) indicated that the etiological agents were Fusarium brasiliense and a novel, undescribed Fusarium sp. This is the first report of F. brasiliense outside of Brazil and Argentina and the novel Fusarium sp. causing soybean SDS. Koch's postulates were completed for both fusaria on seven soybean cultivars that are commercially available in South Africa. Results of the pathogenicity experiment revealed that the strains of F. brasiliense and Fusarium sp. differed in aggressiveness to soybean, as reflected in differences in foliar symptoms, root rot, and reduction in shoot length. Cell-free culture filtrates of the two soybean SDS pathogens from South Africa and two positive control strains of F. virguliforme from the United States induced typical SDS symptoms on susceptible soybean cultivars in a whole-seedling assay, indicating that they contained phytotoxins.

Fusarium paranaense sp. nov., a member of the Fusarium solani species complex causes root rot on soybean in Brazil

Isolates of Fusarium obtained from soybean plants showing symptoms of root rot collected in subtropical southern and tropical central Brazil were characterized based on phylogenetic analyses, sexual crossing, morphology, and pathogenicity tests. A novel species within the Fusarium solani species complex (FSSC) causing soybean root rot is formally described herein as Fusarium paranaense. This species can be distinguished from the other soybean root rot pathogens in the FSSC, which are commonly associated with soybean sudden death syndrome (SDS) based on analyses of the combined DNA sequences of translation elongation factor 1-α and the second largest subunit of RNA polymerase II and on interspecies mating compatibility. Bayesian and maximum parsimony phylogenetic analyses showed that isolates of F. paranaense formed a distinct group in clade 3 of the FSSC in contrast to the pathogens currently known to cause SDS, which are in clade 2. Female fertile tester strains were developed that can be used for the identification of this new species in the FSSC based on sexual crosses. All isolates were heterothallic and belonged to a distinct mating population. Fusarium tucumaniae, a known SDS pathogen, was found in the subtropical southern region of the country.

Phenotypic, molecular phylogenetic, and pathogenetic characterization of Fusarium crassistipitatum sp. nov., a novel soybean sudden death syndrome pathogen from Argentina and Brazil

A novel soybean sudden death syndrome (SDS) pathogen from Argentina and Brazil is formally described herein as Fusarium crassistipitatum based on detailed phenotypic analyses of macro- and microscopic characters and phylogenetic analyses of multilocus DNA sequence data. Fusarium crassistipitatum can be distinguished from the other soybean SDS and bean (Phaseolus/Vigna) root rot pathogens (BRR) phenotypically by the production of yellowish colonies on PDA; and tall, stout, and mostly unbranched conidiophores with a thick-walled base, which form multiseptate conidia apically. Phylogenetic species recognition based on genealogical concordance of a sixgene dataset strongly supported the reciprocal monophyly of F. crassistipitatum with respect to the other SDS and BRR pathogens. Isolates of F. crassistipitatum were able to induce typical SDS foliar and root rot symptoms on soybean that were indistinguishable from those caused by three other SDS pathogens (i.e., F. virguliforme, F. brasiliense, and F. tucumaniae) on susceptible cultivars A-6445RG and N-4613RG in a pathogenicity experiment.

Fusarium azukicola sp. nov., an exotic azuki bean root-rot pathogen in Hokkaido, Japan

We report on the phenotypic, molecular phylogenetic and pathogenic characterization of a novel azuki bean (Vigna angularis) root-rot (BRR) pathogen from Hokkaido, Japan, which formally is described herein as Fusarium azukicola. This species can be distinguished phenotypically from the other Phaseolus/Vigna BRR and soybean sudden-death syndrome (SDS) pathogens by the production of wider and longer four-septate conidia cultured on SNA. Molecular phylogenetic analyses of four anonymous intergenic loci, a portion of the translation elongation factor (EF-1α) gene and the nuclear ribosomal intergenic spacer region (IGS rDNA) strongly support the genealogical exclusivity of F. azukicola with respect to the other soybean SDS and BRR pathogens within Clade 2 of the F. solani species complex (FSSC). Evolutionary relationships of F. azukicola to other members of the SDS–BRR clade, however, are unresolved by phylogenetic analyses of the individual and combined datasets, with the exception of the IGS rDNA partition, which strongly supports it as a sister of the soybean SDS pathogen F. brasiliense. A multilocus genotyping assay is updated to include primer probes that successfully distinguish F. azukicola from the other soybean SDS and BRR pathogens. Results of a pathogenicity experiment reveal that the F. azukicola isolates are able to induce root-rot symptoms on azuki bean, mung bean (Vigna radiata), kidney bean (Phaseolus vulgaris) and soybean (Glycine max), as well as typical SDS foliar symptoms on soybean. Our hypothesis is that F. azukicola evolved in South America and was introduced to Hokkaido, Japan, on azuki bean but its possible route of introduction remains unknown.

Phenotypic, molecular phylogenetic, and pathogenetic characterization of <i>Fusarium crassistipitatum</i> sp. nov., a novel soybean sudden death syndrome pathogen from Argentina and Brazil

A novel soybean sudden death syndrome (SDS) pathogen from Argentina and Brazil is formally described herein as Fusarium crassistipitatum based on detailed phenotypic analyses of macro- and microscopic characters and phylogenetic analyses of multilocus DNA sequence data. Fusarium crassistipitatum can be distinguished from the other soybean SDS and bean (Phaseolus/Vigna) root rot pathogens (BRR) phenotypically by the production of yellowish colonies on PDA; and tall, stout, and mostly unbranched conidiophores with a thick-walled base, which form multiseptate conidia apically. Phylogenetic species recognition based on genealogical concordance of a sixgene dataset strongly supported the reciprocal monophyly of F. crassistipitatum with respect to the other SDS and BRR pathogens. Isolates of F. crassistipitatum were able to induce typical SDS foliar and root rot symptoms on soybean that were indistinguishable from those caused by three other SDS pathogens (i.e., F. virguliforme, F. brasiliense, and F. tucumaniae) on susceptible cultivars A-6445RG and N-4613RG in a pathogenicity experiment.

Comparison of inoculation methods for characterizing relative aggressiveness of two soybean sudden-death syndrome pathogens, Fusarium virguliforme and F. tucumaniae

Fusarium tucumaniae and F. virguliforme are the primary etiological agents of sudden-death syndrome (SDS) of soybean in Argentina and the United States, respectively. Five isolates of F. tucumaniae and four isolates of F. virguliforme were tested for relative aggressiveness to soybean, using a toothpick inoculation method and two versions of a soil infestation inoculation method. Partially resistant soybean cultivar RA629 and susceptible cultivar A6445RG were inoculated separately with each of the nine isolates. Two experiments for each inoculation method were performed. Analysis of variance identified a significant three-way interaction of soybean cultivar*experiment*SDS pathogen (P = 0.01) using the different methods. When the two soil infestation methods were used, F. virguliforme was more aggressive than F. tucumaniae; however, when using the toothpick method, isolates of F. virguliforme and F. tucumaniae were equally aggressive. Although all three methods discriminated levels of partial resistance of the genotypes to SDS, results of the present study indicated that soil inoculations with sorghum infested grain represent the best method for evaluating soybean cultivar resistance to SDS. The existence of interactions among the host, pathogen and environmental conditions highlights the need for additional studies to improve the reproducibility of tests for screening soybean germplasm for resistance to SDS.

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.

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.

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.

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.

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.

Phylogenetic relationships of the soybean sudden death syndrome pathogen Fusarium solani f. sp. phaseoli inferred from rDNA sequence data and PCR primers for its identification.

Phylogenetic relationships of several species within the Fusarium solani-complex were investigated using characters from the nuclear ribosomal DNA. Genetic variation within 24 isolates, including 5 soybean sudden death syndrome (SDS) strains, was assessed using rDNA sequence data and restriction fragment length polymorphic markers. By these techniques, the causal agent of soybean SDS was identified as F. solani f. sp. phaseoli. In separate cladistic analyses, Plectosphaerella cucumerina and Nectria cinnabarina or F. ventricosum were used for rooting purposes. Monophyly of the F. solani-complex was strongly supported by bootstrap and decay analyses. Parsimony analysis indicates that this complex is composed of a number of phylogenetically distinct species, including Neocosmospora vasinfecta, F. solani f. sp. phaseoli, and biological species designated as MPI, MPV, and MPVI of N. haematococca. The results demonstrate complete congruence between biological and phylogenetic species within the N. haematococca-complex. In addition, DNA sequence data were used to design a PCR primer pair which could specifically amplify DNA from isolates of the SDS pathogen from infected plants.