The inhibition of Fusarium oxysporum f.sp. cubense race 4 by Burkholderia cepacia.
Inhibition of Fusarium oxysporum f. sp. cubense race 4 by Burkholderia cepacia was evident when grown on various media (TSA, PDA, PSA, YM, KMB, PPM, NYGA, LA) with different carbon sources and under various pH and temperature conditions. In addition, B. cepacia was able to inhibit several fungal pathogens in vitro. Antagonism of B. cepacia against F. oxysporum f. sp. cubense occured at high levels of Fe³+, which may suggest that antagonism by B. cepacia did not involve siderophore production. Thin layer chromatogram (TLC) examination showed that B. cepacia produced several substances, one of which had similar R[f] value to that described for pyrrolnitrin. Cell-free supernatant of a 4-day culture of 6. cepacia was applied to an Amberlite XAD-2 column and inhibitory activity co-eluted with the 95% methanol (pH 9.5) fraction. The concentrated activated fractions showed inhibitory activity against F. oxysporum f. sp. cubense. A GC-MS chromatogram indicated numerous components in the antifungal extracts. The only compound identified in the Wiley 138 library, was 1,2- Benzenedicarboxylic acid, bis (2-Ethylhexyl) ester. Observations by light microscopy indicated that B. cepacia inhibited spore germination in F. oxysporum f. sp. cubense race 4 and retarded the mycelial growth. The interaction between the endophytic bacterium, B. cepacia and F. oxysporum f. sp. cubense race 4 was investigated with aid of scanning and transmission electron microscopy. This demonstrated that the bacterium was able to colonize the surface of hypha and macrospore of F. oxysporum f. sp. cubense. Mycelial deformation, terminal and/or intercalary swelling were evident. At later stages, hyphae of F. oxysporum f.sp. cubense, colonized by B. cepacia, were found to have collapsed. Further studies in vivo confirmed that B. cepacia colonized the hypha of F. oxysporum f. sp. cubense which had invaded banana roots. TEM observation showed that in the banana plant B. cepacia was closely associated with the healthy banana roots and a matrix was frequently found to be present between the bacterium and the plant surface. In addition, B. cepacia exists mainly in the intercellular space of the banana roots. UV irradiation treatment of B. cepacia resulted in a mutant that had lost inhibitory activity against F. oxysporum f. sp. cubense on TSA agar. Transposon mutagenesis of B. cepacia was performed by Tn5 insertion. Six mutants which had lost or had reduced inhibitory activity against F. oxysporum f. sp. cubense were generated. These mutants showed no inhibitory zones on TSA medium in the presence of the fungus. It was observed that one mutants. cepacia :: Tn5-188 appeared to lose the ability to colonize the fungal hypha, whilst a different mutant B. cepacia ::Tn5 - 217 was still able to colonize the fungal hyphae. TLC analyses showed that there was a decrease in antibiotic production in mutants B. cepacia :: Tn5 - 217 and B. cepacia - UV - 34, compared with the wild type. GC- MS analyses showed that there was no evidence of the peaks at 14.62 minutes, 20.0 minutes and 20.46 minutes in both chromatograms of mutants B. cepacia :: Tn5 -217 and 8. cepacia -UV - 34, compared with the wild type B. cepacia. No PCR products were detected using primers that were developed from sequences within the biosynthetic loci for Phi of P.fluorescens Q2-87(GenBank accession no. U41818) and PCA of P. fluorescens 2-79 (GeneBank no. L48616). Colony hybridization suggested that genomic DNA from B. cepacia could contain both Phi- and PCA probes. It was found that hybridization of genomic DNA digested with Cla-I of B. cepaca with Phl2a probe only occurred at low stringency. A hybridization signal was detected from a Cla-l fragment of approximately 2800bp.