HPLC-MS Analysis of Secondary Metabolites in Leaves from Orange Trees Infected with Huanglongbing: A 9-Month Time Series Study

Abstract

Huanglongbing (HLB) disease, presumably caused by CandidatusLiberibacter asiaticus (CLas), is threatening one million acres of commercial citrus groves that have an annual production value of approximately $3 billion across the United States. The objectives of this study were to identify the earliest significant difference in the metabolome of leaves from citrus affected with HLB, and to characterize the evolution of differences in metabolite profiles as related to bacteria titer and HLB symptom development in planta. Twenty each of 8-month-old ‘Valencia’ and ‘Hamlin’ sweet orange trees were graft inoculated with budwood from a PCR-positive HLB source tree. Leaves from five inoculated trees of each variety and three control trees were sampled biweekly and analyzed by HPLC-MS and PCR. Fourteen weeks after inoculation, CLas was detected in newly growing flushes in 55% and 42% of the inoculated ‘Valencia’ and ‘Hamlin’ trees, respectively. Inoculated trees remained visibly asymptomatic in the first 20 weeks but HLB symptoms were evident 30 weeks after grafting. No metabolomic differences were detected in leaves from HLB-infected trees 24 weeks after inoculation. However, 28 weeks after inoculation, just prior to the appearance of visible symptoms, metabolomic differences between control leaves and those from HLB-infected trees were clear. The abundance of 27 out of the 38 detected metabolites in leaves from infected ‘Valencia’ trees increased with time, two metabolites decreased with time, and nine did not change significantly. The response of ‘Hamlin’ metabolites to HLB was similar to ‘Valencia’; 24 out of the 38 detected metabolites increased with symptoms development, five metabolites decreased as symptoms increased, and the rest did not change significantly.