Effect of Producing Different Phenazines on Bacterial Fitness and Biological Control in Pseudomonas chlororaphis 30-84

Abstract

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

Keywords: Pseudomonas chlororaphis 30-84; biofilm; biological control; eDNA; phenazine.

Fig. 1

Production of phenazines by isogenic derivatives of P. chlororaphis 30-84. (A) Phenazine production in PPMD broth at pH 7.0 and (B) on PPMD agar plate. Strains include: P. chlororaphis 30-84 wild-type (WT), 30-84ZN (phenazine non-producer, phzB::lacZ), 30-84PCA (PCA only producer, phzO::Tn5), 30-84O* (2OHPCA overproducer), 30-84H (PCA and PCN producer), 30-84M (PCA and 5MPCA producer), 30-84S (PCA and 1OHPZ producer), and 30-84MS (PCA and PYO producer). 30-84WT, 30-84ZN, 30-84PCA, and 30-84O* contain the empty vector pGT2-P_psp.

Fig. 2

LC-MS analyses of phenazines extracted from isogenic derivatives of P. chlororaphis 30-84. (A) 30-84PCA, (B) 30-84WT, (C) 30-84H, (D) 30-84M, (E) 30-84S, and (F) 30-84MS. One microliter samples were used for the LC-MS separation and the identities of peaks were confirmed by monoisotopic mass analysis accompanied by comparisons to available positive control strains and standards (i.e., PCA only producer P. synxantha 2-79, PCN producer P. chlororaphis ATCC17411, and purified PYO). The assay was repeated three times.

Fig. 3

Suppression of take-all disease symptoms on wheat roots by isogenic derivatives of P. chlororaphis 30-84. 30-84WT, 30-84PCA and altered phenazine produces were seed inoculated and planted into a soil mix infested with Ggt. After 20 days of growth, roots were thoroughly washed and evaluated for disease severity on scale of 0 (no disease) – 5 (nearly dead). The values are the mean of two separate experiments (8 plants per each experiment) with standard error bars. Values with the same letter do not differ significantly as determined by a Fishers protected Least Significantly Difference (LSD) test (P > 0.05).

Fig. 4

Growth curve, biofilm formation and eDNA production by isogenic derivatives of P. chlororaphis 30-84. (A) Biofilm production by cultures grown in 96-well plates containing AB-C for 48 and 72 h. Attached cells with crystal violet staining were quantified at optical density 540 nm. (B) Bacterial cultures were grown in 96-well microtiter polystyrene plates containing AB-C up to 80 h without agitation. Cell density was measured periodically by optical density of 620 nm. (C) Quantification of non-attached floating biofilm matrix by cultures grown in 24-well plates containing AB-C for 72 h at 28°C without agitation. Non-attached floating biofilm matrix was determined by weight. (D) Quantification of extracellular DNA produced by cultures grown in 24-well polystyrene plates containing AB-C for 72 h at 28°C without agitation; extracellular DNA concentration was quantified using a Qubit fluorometer (invitrogen Life Technologies). Quantification of (E) non-attached floating biofilm matrix and (F) extracellular DNA produced by the phenazine non-producing strain 30-84ZN when grown for 72 h in cultures containing supernatants from each of the phenazine-producing strains or 30-84ZN (negative control). The designation on the X axis indicates the source of the supernatants. All data represent the average of 6 biological replicates and error bars indicate the standard error. Values with the same letter do not differ significantly as determined by a Fishers protected Least Significantly Difference (LSD) test (P > 0.05).