Genetic characterization of surfactant-producing bacteria with lysing activity against Pythium zoospores in hydroponic lettuce

Abstract

Pythium root rot is one of the most severe diseases in hydroponic lettuce worldwide. Several species of Pythium may cause the disease, which is characterized mainly by root browning and poor plant growth. Application of chemical fungicides and disinfectants is the most common method used to manage these pathogens. Hydroponic systems offer an interesting opportunity to apply biological control agents or biologically derived products to control waterborne pathogens due to the controlled environment and the absence of soil. Despite the advantages of biological agents, products specifically developed to control Pythium in hydroponics are not common. Our goals in this study were to select, characterize and apply surfactant-producing bacteria to control Pythium aphanidermatum on hydroponically grown lettuce. From six bacterial strains initially known to produce surfactants, strain 88A secreted compounds with the highest activity. Genome sequencing of strain 88A and analyses of genomic indices revealed that it is a Pseudomonas areruginosa. A comparative genomic analysis of 309 genomes of this species showed that all genomes harbored genes rhlA and rhlB in one operon, which encode enzymes responsible for the synthesis of mono-rhamnolipids. An additional gene, rhlC located in another locus encodes for the conversion of mono- into di-rhamnolipids. Only one of these genomes had two copies of rhlAB and rhlC genes and seven genomes did not harbour the rhlC gene. The precipitated dry crude surfactants produced by strain 88A had properties that were similar to that of a mixture containing rhamnolipids, such as surface activity, foaming and capacity to lyse Pythium zoospores at concentrations equal and higher than 1 mg/ml. The identity of the rhamnolipid-encoding genes among 301 genomes of P. aeruginosa in relation to strain 88A varied from 98.9 to 99.9% for rhlA, from 98.7 to 100% for rhlB and from 97 to 100% for rhlC. The 303 sequenced strains deposited in databases and used in this study were isolated from animals (84.5%), plants (3.6%), soil (2.6%) and from environmental samples (9.2%). Strain 88A or the precipitated dry crude rhamnolipids decreased Pythium severity in hydroponic lettuce by approximately 60% and increased fresh weight of lettuce plants by 68%, when compared with the plants inoculated with Pythium. Although this bacterial species is frequently associated with immunocompromised patients, the purified rhamnolipids may be applied in the control of Pythium in hydroponic lettuce.