[Study on the polymerase chain reaction methods for the detection of fumonisin-producing strains of Fusarium moniliforme].

Three pairs of PCR primers P1/P2, P3/P4 and Fum5F21/Fum5R1 specific for Fumonisin-producing were designed, based on the polyketide synthase gene fum5 of Fusarium moniliforme involved in fumonisin biosynthesis. PCR methods for detecting Fumonisin-producing F. moniliforme strains were developed. The specificity of PCR with 3 pairs of primers was detected with 5 standard F. moniliforme strains from ATCC. 880 bp, 702 bp and 1040 bp DNA fragments were amplified with P1/P2, P3/P4 and Fum5F21/Fum5R1, respectively, by using ATCC 52539 (Fumonisin-producing strain) as DNA template. Negative results were obtained by using ATCC 38946, ATCC 26263, ATCC 12763, ATCC 38016 (Fumonisin-non-producing strains), and other negative control strains such as F. gramnearum, F. poae, F. equiseti, F. tricinctum, A. flavus and E. coli O157. The sensitivity of PCR with 3 pairs of primers was detected by applying different dilutions of ATCC 52539 DNA template. The detection limits for PCR with P1/P2 was 100 pg per PCR assay, and with P3/P4 and Fum5F21/Fum5R1 were all 10 pg, equivalently 10(4) and 10(3) spores per PCR assay, respectively. Total 32 strains of F. moniliforme and F. moniliforme var isolated from different regions in China were identified by using PCR with P1/P2, P3/P4 and Fum5F21/Fum5R1. 26 out of them were identified as Fumonisin-producing strains, and other 6 isolates as Fumonisin-non-producing strains. Restriction fragment length polymorphism (RFLP) analysis for PCR (P3/P4) products of some Fumonisin-producing strains were performed by using restriction endonuclease EcoR V and Bam HI. Two DNA fragments 181 bp, 521 bp and three fragments 116 bp, 258 bp, 328 bp were produced, respectively, which accorded with reported reference value. Reliability of the results was confirmed. The results indicated that only one strain, ZJ-fm052, had Fumonisin biosynthesis gene, but negative in detecting fumonisin B1 in culture. Further studies are required. It proved that PCR methods are rapid and reliable to detect Fumonisin-producing strains.