Biological control of a plant pathogen and pest by expression of a cloned Serratia marcescens chiA gene and Bacillus thuringiensis cryIA(c) gene in endophytic bacteria
Protection of plants from pathogens and pests by introduced microorganisms provides an alternative to environmentally hazardous chemical pesticides and fungicides. Plant associated, free living, non-pathogenic bacteria found in the rhizosphere and phyllo plane have been the emphasis of research on biological control· agents. These regions however pose problems of competition between introduced microorganisms and native microflora and environmental extremes. Endophytic bacteria, present in the interior regions of healthy plants, offer a solution to these problems. Since little work has been done to date with endophytes, the work reported in this thesis comprises a novel approach to achieving biological control of a plant pest and pathogens .An endophytic strain of Pseudomonas fluorescens was isolated from micro propagated apple plantlets and shown to be present in the roots of beans at a level of 1.2 x 10⁵ CFU/g fresh weight 10 days after introduction. Generation of spontaneous rifampicin resistant mutants of this strain resulted in P. fluorescens Rifl. Isolates of P. fluorescens and Aeromonas caviae were recovered from the interior regions of surface sterilized bean seeds. In addition, two sugarcane endophytes, Acetobacter diazotrophicus and Herbaspirillum seropedicae, which has also been isolated from rice, maize and sorghum, as well as an endophytic Citrobacter sp. of pine seeds and bean plants were used in this work. The gene coding for the major chitinase of S. marcescens, chiA, was cloned under the control of the tac promoter by PCR amplification. The gene contained the endogenous Shine Dalgarno sequence 7 bases upstream of the ATG start codon in the plasmid pTC33. The plasmid ptacchiA had previously been constructed with a distance of 20 bases between the ribosome binding site of this vector and the A TG start codon of the gene. Gene expression of chiA carried on pTC33 was shown to be approximately 16-foldhigher than that of ptacchiA. This clearly illustrated that the distance between the Shine Dalgarno sequence and ATG start codon was critical and that the shorter distance of 7 bases significantly increased the translation efficiency of chiA. The first and second generation tacchiA cassettes from ptacchiA and pTC33 respectively were cloned into the broad host range plasmids pKT240, pDER405 and pML122 and into the integration vector pJfF350.These plasmids were introduced into the endophytes P.fluorescens Rift, H. seropedicae and Citrobacter sp.AJl by conjugative transfer and electroporation and were shown to express the gene at varying levels. pKTCl and pMTC33 carrying the 1 and 2nd generation tacchiA cassettes on pKT240 and pML122respectively were not stably maintained in P. fluorescens Rift or H. seropedicae.
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Department of Molecular and Cell Biology