Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) disease in cereal crops worldwide. Infection with this fungal phytopathogen can regularly cause severe yield and quality losses and mycotoxin contamination in grains. In previous other studies, one research group reported that pyrrolnitrin had an ability to suppress of mycelial growth of F. graminearum. Other groups revealed that phenazine-1-carboxamide, a derivative of phenazine-1-carboxylic acid, could also inhibit the growth of F. graminearum and showed great potentials in the bioprotection of crops from FHB disease. In our recent work with Pseudomonas chlororaphis strain G05, however, we found that although the phz operon (phenazine biosynthetic gene cluster) was knocked out, the phenazine-deficient mutant G05Δphz still exhibited effective inhibition of the mycelial growth of some fungal phytopathogens in pathogen inhibition assay, especially including F. graminearum, Colletotrichum gloeosporioides, Botrytis cinerea. With our further investigations, including deletion and complementation of the prn operon (pyrrolnitrin biosynthetic gene cluster), purification and identification of fungal compounds, we first verified that not phenazines but pyrrolnitrin biosynthesized in P. chlororaphis G05 plays an essential role in growth suppression of F. graminearum and the bioprotection of cereal crops against FHB disease.

In previous studies with Pseudomonas chlororaphis G05, two operons (phzABCDEFG and prnABCD) were confirmed to respectively encode enzymes for biosynthesis of phenazine-1-carboxylic acid and pyrrolnitrin that mainly contributed to suppression of some fungal phytopathogens. Although some regulators were identified to govern their expression, it is not known how two operons coordinately interact. By constructing the phz- or/and prn- deletion mutants, we found that in comparison with the wild-type strain G05, phenazine-1-carboxylic acid production in the mutant G05Δprn obviously decreased in GA broth in the absence of prn, and pyrrolnitrin production in the mutant G05Δphz remarkably declined in the absence of phz. By generating the phzA and prnA transcriptional and translational fusions with a truncated lacZ on shuttle vector or on the chromosome, we found that expression of the phz or prn operon was correspondingly increased in the presence of the prn or phz operon at the post-transcriptional level, not at the transcriptional level. These results indicated that the presence of one operon would promote the expression of the other one operon between the phz and prn. This reciprocal enhancement would keep the strain G05 producing more different antifungal compounds coordinately and living better with growth suppression of other microorganisms.

In our recent work, we found that pyrrolnitrin, and not phenazines, pyrrolnitrin contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

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