Role of Reactive Oxygen Species (ROS) in Tritrophic Interactions in Postharvest Biocontrol Systems

Abstract

To elucidate the role of ROS in the tri-trophic interactions in postharvest biocontrol systems a detailed molecular and biochemical investigation was undertaken. The application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. The expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. The yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate relatively high levels of super oxide anion (O2−) following its interaction with wounded fruit surface. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H2O2 accumulation in host tissue. The present data, together with our earlier discovery of the importance of H₂O₂ production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases. Effect of ROS on yeast cells was also studied. Pretreatment of the yeast, Candida oleophila, with 5 mM H₂O₂ for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H₂O₂), high temperature (40 °C), and low pH (pH 4). Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semi quantitative reverse transcription-PCR. Seven antioxidant genes were up regulated. Pretreatment of the yeast antagonist Candida oleophila with glycine betaine (GB) increases oxidative stress tolerance in the microenvironment of apple wounds. ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. A mild heat shock (HS) pretreatment (30 min at 40 1C) improved the tolerance of M. fructicola to subsequent high temperature (45 1C, 20–30 min) and oxidative stress (0.4 mol-¹) hydrogen peroxide, 20–60 min). HS-treated yeast cells showed less accumulation of reactive oxygen species (ROS) than non-treated cells in response to both stresses. Additionally, HS-treated yeast exhibited significantly greater (P≥0.0001) biocontrol activity against Penicillium expansum and a significantly faster (Po0.0001) growth rate in wounds of apple fruits stored at 25 1C compared with the performance of untreated yeast cells. Transcription of a trehalose-6-phosphate synthase gene (TPS1) was up regulated in response to HS and trehalose content also increased.