A black-pigmented pseudomonad isolate with antibacterial activity against phyllospheric pathogens

Abstract

Bacteria of the Pseudomonas genus have been widely studied due to their antagonistic potential against a diverse group of fungal and bacterial phytopathogens, and their competence to colonize different plant tissues. We have isolated a rhizospheric pseudomonad that produced a black pigment, which is not a widespread trait within this genus. We confirmed that the isolate belonged to the P. putida complex through a MLSA analysis. We observed that the pigment synthesis was enhanced under high C:N ratios (25:1) and it was dependent of the carbon source, being maximized when we added glucose to M9. Besides, the supplementation of M9 with tryptophan inhibited the pigment production under C:N ratios of 4:1, and the addition of kojic acid reduced notably the pigment under favorable conditions. Ps. black presented several traits associated with plant-growth promoting potential with classical in vitro assays. Through a Tn5 mutagenesis approach, we found 2 representative clones, PB1 and PB5, that were consistently unable to produce the pigment under several growth conditions and were not altered in their in vitro probiotic traits. When comparing with PB1 and PB5 performances, we observed that the pigment gives Ps. black a higher tolerance to oxidative stress and UV radiation exposure. When confronting Ps. black with different bacterial phytopathogens, we demonstrated that Ps. black could inhibit in vitro the growth of Xanthomonas vesicatoria Bv5-4a, Pseudomonas syringae pv. tomato DC3000, P. syringae pv. syringae B728a, P. savastanoi pv. glycinea B076 and Clavibacter michiganensis subsp. michiganensis Cm9. Except for Psg B076, this antagonism was lost for PB1 and PB5 and when performing the test for Ps. black with tryptophan supplementation. Thus, we suggest that the pigment should be involved in the bacterial antagonisms, and that Ps black contains more than one antibacterial mechanism.