# 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)

## TL;DR

This study shows that phenazine-1-carboxylic acid, not 2-hydroxyphenazine, is responsible for the biocontrol activity of a Pseudomonas strain against a corn root rot pathogen.

## Contribution

The study identifies PCA as the key biocontrol compound and reveals the role of phzO in protecting the bacteria from its own toxins.

## Key findings

- PCA is essential for antagonism against Pythium arrhenomanes, while 2-OH-PHZ is not.
- Deleting phzF eliminates both PCA and 2-OH-PHZ production and antagonistic activity.
- Deleting phzO prevents 2-OH-PHZ production but does not affect biocontrol ability.

## Abstract

Liquid chromatography–mass spectrometry analysis of Pseudomonas chlororaphis subsp. phenazini 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 Pythium arrhenomanes, a root rot pathogen of corn. CRISPR-Cas9 knockouts were carried out on the phzF gene, required for PCA synthesis, and the phzO gene, which is involved in converting PCA to 2-OH-PHZ. Deletion of the phzF gene abrogated the production of PCA and 2-OH-PHZ, and the ΔphzF mutant lost the antagonism against Pythium arrhenomanes. In contrast, deletion of the phzO gene created a 2-OH-PHZ-negative mutant with intact antagonistic ability. Concordantly, S1Bt23 wild type and the ΔphzO mutant, but not the ΔphzF mutant, significantly bioprotected corn seeds of a susceptible inbred variety, CO441, from P. arrhenomanes. At equimolar amounts of 75 nM, synthetic PCA inhibited Pythium growth, whereas 2-OH-PHZ did not. This highlights the critical contribution of PCA to the biocontrol activity of strain S1Bt23 against P. arrhenomanes. Unexpectedly, deletion of phzO 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.