Antioxidant enzymes activity associated with resistance to Phytophthora melonis-pumpkin blight

Numerous Cucurbitaceae species are seriously threatened by the hemibiotrophic oomycetes with a severe negative impact on plant yields. In particular, Phytophthora melonis causes severe symptoms that significantly reduce cucumber productions. However, there is currently little known about the Cucurbita pepo-P. melonis interaction. This study analyzed enzymatic activity of defense-related antioxidant enzymes peroxidase (POX), superoxide dismutase (SOD), polyphenol oxidase (PPOs), catalase (CAT); hydrolytic enzymes- beta-1,3-glucanase; phenylalanine ammonia-lyase (PAL) and phenolic content in two resistant pumpkin "Tanbal" and "Ghelyani" and two susceptible, "Marmai" and "Khoreshti", in response to Phytophthora blight disease (PBD), caused by Phytophthora melonis over the time courses of 24, 48 and 72 h post inoculation (hpi). All enzymes were highly upregulated in the resistant C. pepo accessions "Tanbal" and "Ghelyani" inoculated with P. melonis. The mean level of enzyme activity varied from 1.3 to 6-fold higher in the resistant genotypes, of which SOD was increased by 6fold, followed by PPOs and PAL 5.3-fold in comparison to untreated controls. Conversely, the lower or negative expression of these enzymes was observed in susceptible pumpkins. The highest beta-1,3-glucanases activity, marker of salicylic acid pathway, was recorded in resistant "Tanbal" with 3-fold increase, followed by 2.4-fold increase in Ghelyani 24 hpi following inoculation which shows the involvement of salicylic acid (SA)-mediated defense. Furthermore, significantly higher activities of the ROS-scavenging-defense-enzymes CAT (1.6-fold), and POX (6fold), CAT (6-fold (, and POX (6-fold) were observed in the roots of resistant-cultivars at different-wpis, compared with non-inoculated-controls. We also analyzed the quantitative-gene-expression-changes of threecandidate-gene-markers; PR-1, POD and WRKY21 by qRT-PCR along the related time-courses revealed the considerable transcriptomic-response in the resistant-genotypes, 1.1-2.7-fold in the roots and leaves, respectively. Surprisingly, PR-1-gene-marker, basically resistant to wilting agent- Verticillium dahlia was overexpressed in resistant "Tanbal" and "Ghelyani" against P. melonis. Similar results were obtained on POD-gene-marker resistant to powdery mildew, Podosphaera xanthii that similarly modulates immunity against P. melonis. There was also a highly significant correlation between the related enzymes and gene-markers, predominantly between PPO-enzyme and PR-1, POD and WRKY21; r = 0.851**r = 0.878** and r = 0.866**, followed by PAL and SOD respectively. Thus, molecular pathways associated to P. melonis resistance may be considered as future targets for manipulation in the development of resistant cultivars.