Avirulent Erwinia carotovora subsp.carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3′ end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5′ end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qβ-replicase; andAzorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coliDH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.
Plasmid-like DNA (plDNA) was found in 10 out of 61 field isolates of Fusarium oxysporum. These 10 isolates were distributed among 6 formae speciales. Electron microscopic analysis revealed that all these plDNAs were linear molecules. The sizes of plDNAs found were varied from 1.9 to 8.0kb. The sequence homology among four 1.9kb plDNAs, pFOA, pFOL, pFOC and pFOR found in four different F. oxysporum formae speciales was examined by Southern blot analysis, using nick-translated plDNAs as probes. Considerable sequence homology was observed among pFOA, pFOL and pFOC DNA, but pFOR had no homology with three other 1.9kb plasmid DNAs. pFOM and pFOB DNAs of the sizes of 8.0 and 2.2kb, respectively, had no sequence homology with four plDNAs of 1.9kb.
Two pathogenic strains of E. carotouora subsp. carotouora 2T-2 and TT-4 with high bacteriocin activity but low sensitivity to the bacteriocins of other strains were treated with ethyl methane sulphonate (EMS). Two avirulent mutants A-f-39 and B-e-19 developed from 2T-2 and TT-4, respectively, by this treatment had the same bacteriocin activity as their respective parents and inhibited the in uitro growth of pathogenic strains of this species. The disease control of these two mutant strains were compared in the field in 1995 and 1997 t o the control by CGE234M403 (M403) (a commercialized biocontrol agent), a mixture of A-f-39 and M403, and an agrochemical (basic dithianon-copper chloride). The protection obtained with A-f-39 was comparable to M403 and was better than that with the chemical. The mixture of A-f-39 and M403 consistently gave the best results in all the field trials.
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