Quantitative RT-PCR Expression Analysis of Lipodepsipeptides Synthetase and Defence-related Genes in Orange Fruit in Response to Antagonist–pathogen Interaction

Strains of Pseudomonas syringae are effective in controlling postharvest diseases of citrus fruits, and antagonistic activity has been correlated with in vitro production of lipodepsipeptides. Additionally, biocontrol agents can induce a range of defence mechanisms of resistance in citrus tissue that result in a broad spectrum of metabolic modifications, such as systemic acquired resistance, induced systemic resistance and production of reactive oxygen species. The aim of this study was to evaluate the expression of syringomycin (syrB1) and syringopeptin (sypA) synthetase genes from P. syringae pv. syringae biocontrol strains in vitro on different culture media and in vivo on citrus fruits (Citrus sinensis cv. Tarocco) during the interaction with Penicillium digitatum by quantitative RT-PCR. Similarly, gene transcript levels of chitinase (CHI1), allene oxide synthase (AOS), glutathione peroxidase (GPX1) and phenylalanine ammonia-lyase (PAL1) were measured. SyrB1 and sypA genes were more actively expressed when antagonistic Pseudomonas strains were grown on orange peel broth as compared to NB and PDB. Penicillium digitatum resulted to be strongly stimulatory only to syrB1 expression, thus suggesting that syrB1 gene could be involved in biocontrol activity. QRT-PCR showed that both P. s. pv. syringae and P. digitatum strains increase CHI1 transcription in inoculated flavedo tissues relative to the untreated control. Interestingly, CHI1 transcription was markedly induced by co-inoculation of P. s. pv syringae and P. digitatum strains. Pseudomonas syringae pv. syringae, alone or co-inoculated with P. digitatum, was weakly effective in enhancing GPX1, AOS and PAL1 gene expression, whereas P. digitatum alone strongly enhanced GPX1, AOS and PAL1 expression. Moreover, we assume that CHI1 gene is most likely part of the molecular mechanisms involved in pathogen defence responses in citrus fruit. This is the first example of monitoring the expression of syrB1-sypA and CHI1, GPX1, AOS and PAL1 genes in interactions between P. s. pv. syringae biocontrol agents and P. digitatum in vitro and in vivo.