|Keywords (Extracted from title, table of contents and abstract of thesis)
microbial antagonists, antifungal activity, phytopathogenic fungi, pesticides, pseudomonas, transposon mutagenesis, macrophomina phaseolina, rhizoctonia solani, sclerotium rolfsii, fusarium solani, fusarium oxysporum, t koningii, p. lilacinus
Twenty four bacterial strains were picked randomly from CMG stock for screening of antagonism against five phytopathogenic fungi viz Macrophomina phaseolina, Rhizoctonia solani, Sclerotium rolfsii, Fusarium solani and Fusarium oxysporum that are major pathogens of cash crops of Pakistan. Among the twenty four bacterial strains, twelve were isolated from different agriculture fields of Pakistan whereas twelve were metal resistant strains isolated from metal contaminated sites of industrial area of Karachi. Eight of the twelve soil strains showed variable inhibition against the fungi while four did not inhibit any of the tested fungi. Soil strain CMG 159 (P. aeruginosa) showed inhibition against all five phytopathogenic fungi and produced maximum inhibition against Sclerotium rolfsii, Fusarium oxysporum and Fusarium solani. Among all twelve metal resistant strains, five showed variable inhibition against the tested fungi while seven did not inhibit any of the tested fungi.
To determine the nature of antifungal activity, further experiments were conducted using the soil strains, CMG151 (Po aeruginosa), CMG152 (E. coli), CMG153 (P. aeruginosa), CMG154 (P. aeruginosa), CMG158 (P. aeruginosa), CMG159 (P. aeruginosa), CMG160 (P. aeruginosa) and CMG161 (P. aeruginosa) that showed antagonism towards fungi. Cell free supernatants of all strains did not show any antifungal activity. Continued antifungal activity in presence of protease enzyme showed that the antifungal activity was not protein in nature. Siderophores involvement was not possible as antifungal activity persisted at elevated concentrations of Fe3+ ions in the growth medium. Similarly, involvement of HCN as the cause of antifungal action was also ruled out, as picric acid test was negative.
Screening of extra chromosomal DNA showed that strains CMG 152, CMG 153, CMG 154 and CMG161 each harbored one single large plasmid DNA designated as pCMGI52, pCMGI53, pCMG154 and pCMG161 respectively. Size of pCMG152 was estimated to be 31.6 kb while other plasmids were 24.1 kb each. CMG 159 however, was also found to carry one small plasmid of approximately 8.8 kb, designated as pCMGI59.
CMG159 was selected for further genetic studies because it showed inhibition against all the tested fungi. Involvement of plasmid DNA in the antifungal activity of CMG 159 strain was ruled out, as elimination of pCMG 159 by curing did not abolish or reduce the antifungal action of the strain. In order to determine the gene(s) for the fungal antagonism, transposon mutagenesis of strain CMG 159 was carried out by introducing transposon Tn5 (km) into CMG159 to generate mutants defective for fungal inhibition (Af-). A mutant, CMG159m Af- was obtained which showed reduced inhibition and in some cases no inhibition against tested fungi. Southern blot analysis of EcoR1-digested chromosomal DNA of mutant CMG159m Af- confirmed transposon integration into the host genome as the probe for internal EcoR1 fragment of Tn5 hybridized to the complementary integrated DNA, thereby confirming, which inactivation of antifungal gene was caused by transposon mutagenesis.
To determine the chemical nature of the antifungal activity, extraction with organic solvents was done. Antifungal activity of CMG 159 could not be detected in hexane and ethyl acetate extracts. Vacuum layer chromatography was also performed with different polarity solvents but antifungal compound could not be extracted. Crude extraction from potato dextrose agar plates showed antifungal activity, which was correlated to a peak on high-pressure liquid chromatography analysis of the extract of wild type strain. The mutant CMG159m Af- showed reduction in the "antifungal" peak compared to the wild type strain, under same growth and extraction conditions. Therefore, loss of antifungal activity in mutant CMG 159m Af- suggested a link with the reduced peak seen on high pressure liquid chromatography analysis.
Finally, antifungal activities of the three soil strains, which produced good activity against tested fungi, were tested in vivo for the control of root rot infection of sunflower and mung bean. The strains were used along with a known biocontrol agent Trichoderma koningii by seed dressing and soil drench method. Soil strains showed significant (p
To compare the antifungal activity ofCMG159 and its derivative mutant CMG159m Af- experiments were done in vivo on root rot infection of sunflower caused by M phaseolina, R. solani, and Fusarium spp., by seed dressing and soil drench methods. CMG 159 significantly (p
In addition, antifungal activities of three metal resistant strains (CMG63, CMG52 and CMG58), were also checked in vivo against fungal infection in sunflower, by seed dressing and soil drench method. Metal resistant strains of Pseudomonas were observed not only to control the infection significantly but also increased the plant height and fresh weight of the shoot of sunflower. In vivo antifungal activity of metal resistant strains was also checked against cotton plants along with two known biocontrol agents T koningii and P. lilacinus. Combination of P. aeruginosa (CMG63, CMG52 and CMG58), T koningii and P. lilacinus produced significant (p