Genetically Engineered Microbial Agents for Biocontrol of Plant Fungal Diseases
Authors: Thomashow Linda , Chernin Leonid , Chet Ilan , Weller David M. , Mavrodi Dmitri 
Published: March 2022

The objectives of the project were: a) to construct the site-specific integrative expression cassettes carrying: (i) the chiA gene for a 58-kDa endochitinase, (ii) the pyrrolnitrin biosynthesis operon, and (iii) the acdS gene encoding ACC deaminase; b) to employ these constructs to engineer stable recombinant strains with an expanded repertoire of beneficial activities; c) to evaluate the rhizosphere competence and antifungal activity of the WT and modified strains against pathogenic fungi under laboratory and greenhouse conditions; and d) to monitor the persistence and impact of the introduced strains on culturable and nonculturable rhizosphere microbial populations in the greenhouse and the field. The research generally support our concepts that combining strategically selected genes conferring diverse modes of action against plant pathogens into one organism can improve the efficacy of biological control agents. We hypothesized that biocontrol agents (BCAs) engineered to expand their repertoire of beneficial activities will more effectively control soilborne plant pathogens. In this work, we demonstrated that biocontrol activity of Pseudomonas fluorescens Q8r1-96 and Q2-87, both producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) effective against the plant pathogenic fungus Rhizoctonia solani, can be improved significantly by introducing and expressing either the 1.6-kb gene chiA, encoding the 58-kDa endochitinase ChiA from the rhizosphere strain SerratiaplymuthicaIC1270, or the 5.8-kb prnABCDoperon encoding the broad-range antibiotic pyrrolnitrin (Prn) from another rhizosphere strain, P. fluorescens Pf-5. The PₜₐcchiAandPₜₐcprnABCDcassettes were cloned into the integrative pBK-miniTn7-ΩGm plasmid, and inserted into the genomic DNA of the recipient bacteria. Recombinant derivatives of strains Q8r1-96 and Q2-87 expressing the PₜₐcchiA or PₜₐcprnABCD cassettes produced endochitinase ChiA, or Prn, respectively, in addition to 2,4-DAPG, and the recombinants gave significantly better biocontrol of R. solani on beans under greenhouse conditions. The disease reduction index increased in comparison to the parental strains Q8r1-96 and Q2-87 to 17.5 and 39.0% from 3.2 and 12.4%, respectively, in the case of derivatives carrying the PₜₐcchiAcassette and to 63.1 and 70% vs. 2.8 and 12,4%, respectively, in the case of derivatives carrying the PₜₐcprnABCDcassette. The genetically modified strains exhibited persistence and non-target effects comparable to those of the parental strains in greenhouse soil. Three integrative cassettes carrying the acdS gene encoding ACC deaminase cloned under the control of different promoters were constructed and tested for enhancement of plant growth promotion by biocontrol strains of P. fluorescens and S. plymuthica. The integrative cassettes constructed in this work are already being used as a simple and efficient tool to improve biocontrol activity of various PGPR bacteria against fungi containing chitin in the cell walls or highly sensitive to Prn. Some parts of the work (e. g., construction of integrative cassettes) was collaborative while other parts e.g., (enzyme and antibiotic activity analyses) were fully synergistic. The US partners isolated and provided to the Israeli collaborators the original biocontrol strains P. fluorescens strains Q8r1-96 and Q2-87 and their mutants deficient in 2,4-DAPG production, which were used to evaluate the relative importance of introduction of Prn, chitinase or ACC deaminase genes for improvement of the biocontrol activity of the parental strains. The recombinant strains obtained at HUJI were supplied to the US collaborators for further analysis.