| By analysis of the rDNA intergenic spacer (IGS) regions,a polymerase chain reaction(PCR)-based method was developed to detect rDNA of Fusarium tucumaniae andFusarium virguliforme, the cause of soybean sudden death syndrome. Three pairs ofspecific primers, FT1/FT9 or FT1/FT6 detected for Fusarium tucumaniae produced PCRproducts of about 655bp or 250bp; and FV1/FV1A detected for Fusarium virguliformeproduced PCR products of 228bp respectively. Primer specificity was tested with DNAfrom 3 F. tucumaniae,1 Fusarium cuneirostrum, 1 Fusarium brasiliense, 5Evirguliforme, 1 F. phaseoli, 6 isolates of the oomycete Phytophthora sojae, 21 isolates ofFusarium sp. from soybean or soil . PCR products of Fusarium tucumaniae-specificPrimers FT1/FT6 were digested with Alu I or Sac I into 232bp and 18bp or 158bp and92bp. PCR products of Fusarium virguliforme -specific Primers FV1/FV1A were digestedwith Alu I. into 144bp and 84bp. The sensitivity of primers FT1/FT6 was 1pg whenusing F. tucumaniae total genomic DNA or down to 10 super(6) macroconidia g super(-1)soil. Nested PCR increased the sensitivity of the PCR assay 10000-fold to 10 fg usingprimers FT1/FT6 after using primers FT1/FT9 when using F. tucumaniae totalgenomic DNA or down to 10 super(1) macroconidia g super(-1) soil. The sensitivity ofprimer FV1/FV1A was lpg when using F. virguliforme total genomic DNA or down to 10super(3) macroconidia g super(-1) soil. |
