CRISPR/Cas9 Knockout Studies Implicate Phenazine-1-carboxylic Acid, but Not 2-Hydroxy Phenazine, in the Biocontrol Activity of Pseudomonas chlororaphis Subsp. phenazini Strain S1Bt23 Against Pythium arrhenomanes (Drechsler)

Liquid chromatography-mass spectrometry analysis of <i>Pseudomonas chlororaphis</i> subsp. <i>phenazini</i> S1Bt23 extracts detected phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) as the main phenazine derivatives. We investigated their relative contributions to the antagonistic activity of strain S1Bt23 against <i>Pythium arrhenomanes</i>, a root rot pathogen of corn. CRISPR-Cas9 knockouts were carried out on the <i>phz</i>F gene, required for PCA synthesis, and the <i>phz</i>O gene, which is involved in converting PCA to 2-OH-PHZ. Deletion of the <i>phz</i>F gene abrogated the production of PCA and 2-OH-PHZ, and the Δ<i>phz</i>F mutant lost the antagonism against <i>Pythium arrhenomanes</i>. In contrast, deletion of the <i>phz</i>O gene created a 2-OH-PHZ-negative mutant with intact antagonistic ability. Concordantly, S1Bt23 wild type and the Δ<i>phz</i>O mutant, but not the Δ<i>phz</i>F mutant, significantly bioprotected corn seeds of a susceptible inbred variety, CO441, from <i>P. arrhenomanes</i>. At equimolar amounts of 75 nM, synthetic PCA inhibited <i>Pythium</i> growth, whereas 2-OH-PHZ did not. This highlights the critical contribution of PCA to the biocontrol activity of strain S1Bt23 against <i>P. arrhenomanes</i>. Unexpectedly, deletion of <i>phz</i>O did not result in additional PCA accumulation. This suggests that the conversion of PCA to 2-OH-PHZ by S1Bt23 is a potential protective mechanism against the overproduction of lethal cellular doses. This study paves the way for bioengineering strain S1Bt23 into a more effective biopesticide.