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

First and second authors: Microbial Genomics Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agriculture Research Service, 1815 North University Street, Peoria IL 61604; third author: Laboratorio Agrícola Río Paraná, San Pedro, Buenos Aires, Argentina; fourth to sixth authors: Centro de Referencia de Micología (CEREMIC), Fac. de Cs. Bioquímicas y Farmacéuticas, UNR, Suipacha 531, 2000, Rosario, Santa Fe; seventh author: Estación Experimental Agropecuaria Marcos Juárez, Instituto Nacional de Tecnología Agropecuaria, (EEA INTA Marcos Juárez), Casilla de Correo 21, 2580 Marcos Juárez, Córdoba; eighth author: Nidera S.A, Catamarca 2597, 4000 San Miguel de Tucumán; ninth and tenth authors: Estación Experimental Agroindustrial “Obispo Colombres”, Casilla de Correo 9, 4101, Las Talitas, San Miguel de Tucumán; eleventh author: Estación Experimental Agropecuaria Paraná, Instituto Nacional de Tecnología Agropecuaria, (EEA INTA Paraná), Ruta 11 km 12,5, 3101, Oro Verde, Entre Ríos; twelfth author: Cátedra de Fitopatología, Fac. de Cs. Agrarias-UNR, Campo Experimental Villarino CC 14, 2125 Zavalla, Santa Fe, Argentina; thirteenth author: National Institute of Agrobiological Sciences, Genetic Diversity Department, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan; fourteenth author: Department of Plant Pathology, Iowa State University, Ames, IA 50011; and fifteenth author: Department of Biological Sciences, University of Idaho, Moscow, ID 83844.

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.

Additional keywords:Argentina, Brazil, EF-1α, Glycine max, Phaseolus vulgaris, United States, Vinga radiata.