Leaf epicuticular wax and hormone-mediated resistance to Alternaria brassicicola in broccoli

Alternaria leaf spot disease is a foliar disease of brassica crops, including broccoli, caused by Alternaria brassicicola. This disease is a serious problem causing unmarketable yields and millions of dollars in losses in broccoli and other crucifers across the globe. During the pathogenicity and whole plant inoculation assays, we observed significantly higher disease severity in the older leaves at the bottom, as compared to the younger leaves at the top. The symptoms seemed to appear first in the lower and older leaves and gradually progressed upwards to the younger leaves, ultimately reaching the broccoli head. Epicuticular wax analysis showed a significantly higher amount of wax deposition on the younger leaves at the top as compared to the older leaves at the bottom. Further regression analysis showed a negative correlation between wax per unit area and disease severity. Differential gene expression analysis showed upregulation of key wax biosynthesis genes, namely 3-ketoacyl-CoA synthase (Bol018447), alkane hydroxylase CYP96A15 (Bol016302) and O-acyltransferase WSD1 (Bol024738) in the younger leaves. The abscisic acid, salicylic acid and jasmonic acid were differentially accumulated in the older leaves as compared to the younger inoculated leaves in response to A. brassicicola inoculation. Transcriptome analysis uncovered large transcriptome reprogramming in the older leaves as compared to the younger leaves. Senescence-associated genes such as senescence regulator S40 (BolC7t42093H), stress up-regulated Nod 19 (BolC2t12223H), and late embryogenesis abundant protein (BolC8t47646H) were upregulated in the older leaves. These findings suggest that the older bottom leaves of broccoli are highly susceptible to A. brassicicola, potentially associated with lower wax deposition and increased modulation in senescence hormones.