An Investigation of the Impact of the Stringent Response on the Growth Inhibition of Sclerotinia sclerotiorum by Biocontrol Pseudomonads Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23
Manuel, Jerrylynn Laguras
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The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.