Phenazines and cyclic lipopeptides produced by Pseudomonas sp. CMR12a are involved in the biological control of Pythium myriotylum on cocoyam (Xanthosoma sagittifolium)

Root rot disease caused by Pythium myriotylum is the most devastating disease of the tropical tuber crop cocoyam (Xanthosoma sagittifolium) with production losses of up to 90%. This study was conducted in order to determine the role of phenazines (PCN), and two cyclic lipopeptides namely sessilins and orfamides, produced by Pseudomonas sp. CMR12a, in the suppression of the Cocoyam Root Rot Disease (CARD). Previously generated biosynthetic mutants of CMR12a that were deficient in either one, two or all three metabolites were used in in vitro, microscopic and plant experiments. Using CMR12a and its biosynthetic mutants, plant experiments revealed that mutants, which produced sessilins, orfamides or phenazines alone or at least any two of the metabolites, could successfully suppress the cocoyam root rot disease caused by P. myriotylum albeit in varying capacities. In vitro assays using filter sterilized bacterial supernatants showed that besides the WT strain, all mutants except the null mutant, which makes none of the metabolites, could successfully inhibit P. myriotylum. Following contact between the mycelium of P. myriotylum and purified/semi-purified metabolites, microscopic analysis showed that a concentration of 1 nM and higher of crude sessilins resulted in cell damage including vacuolization and lysis whereas amounts of 25 nM PCN and higher could produce a similar phenotype including mycelial bloating. Treatments with orfamide at 10 nM and higher resulted in mycelial lysis. Interestingly, during plant experiments, the null mutant was able to give some level of disease suppression suggesting that CMR12a produces other compound(s) which could be antagonistic towards our target pathogen. In summary, phenazines, sessilins and orfamides, produced by Pseudomonas sp. CMR12a, can play independent and additive roles in the suppression of Pythium-mediated CARD