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.

Fusarium graminearum and F. avenaceum often co-contaminate Canadian durum wheat grain, resulting in the co-deposition of species-specific mycotoxins, including trichothecenes produced by F. graminearum and enniatins produced by F. avenaceum. These mycotoxins pose significant risks to human and animal health. Although these fungi commonly co-occur in infected wheat, relatively little is known about how they interact during host infection. Interactions between the two species were examined using co-inoculation experiments on durum wheat spikes. In pathology trials, co-inoculations often reduced both disease severity and trichothecene accumulation compared with inoculations of F. graminearum alone, despite F. graminearum greatly out-competing F. avenaceum in total fungal biomass. Transcriptomic profiling identified strong induction of the F. graminearum ABC transporter gene FgABCB8 during co-inoculation with an enniatin-producing F. avenaceum strain. When F. graminearum was grown in vitro, FgABCB8 was induced upon exposure to F. avenaceum culture filtrate, or the related cyclohexadepsipeptides enniatin B1 or beauvericin. Heterologous expression of FgABCB8 in yeast provided partial protection against enniatin and beauvericin toxicity. Gene disruption of FgABCB8 increased F. graminearum sensitivity to enniatins. These findings demonstrate that FgABCB8 expression enhances the ability of F. graminearum to tolerate enniatin-producing fungi.