2013 Annual Report
1a.Objectives (from AD-416):
(1) Establish baseline metabolic profiles of commercial citrus products through the use of modern analytical methods.
(2) Determine the influence of cultivar variety, environment and plant health on the metabolite composition by comparing the profiles obtained for these samples to those obtained for other citrus products.
(3) Identify diagnostically significant metabolites.
1b.Approach (from AD-416):
Pepsico will provide the majority of the samples for this study. Additional samples may be obtained from commercial citrus growers or from citrus variety collections. Samples will be processed into juice and stored as such until analyzed by GC-MS, LC-MS, HPLC and NMR. Additional analyses will be performed as needed. Initial data analysis and the subsequent statistical analysis will be conducted at the Agricultural Research Service, Western Regional Research Center, Albany, CA.
This project has been a multi-prong approach to identify naturally occurring metabolites found in Citrus fruits that could be used diagnostically to distinguish healthy trees from those suffering from Citrus Greening disease (HBL). GC-MS, H1-NMR and HPLC analyses have been conducted for the entire sample set provide by the cooperator and for samples provided from the ARS laboratory located at Fort Pierce, Florida. Due to financial and time restraints only a subset of the samples were analyzed by LC-MS. By using four different analytical techniques we were able to evaluate volatiles (GC-MS) non-volatiles (LC-MS and HPLC) and primary metabolites, thereby covering a wide spectrum of metabolites. We used metabolite profiles from GC-MS and NMR analyses to identify distinct metabolite differences between fruits harvested from healthy trees (healthy) and fruits showing symptoms of HLB that were harvested from infected trees (symptomatic fruit). Flavonoids and limonoids were the notable metabolites detected by LC-MS analysis and statistical analyses of the profile results from healthy and symptomatic fruits failed identify diagnostically significant metabolites. GC-MS and NMR analyses of asymptomatic fruits taken from infected trees were also conducted. Unlike the profiles resulting from healthy fruits and symptomatic fruits which were clearly distinguishable, profiles from asymptomatic fruits as a group were less uniform and the lack of uniformity prevented them from being classified into their own group. In fact, the profiles of some asymptomatic samples were very similar to profiles found for healthy samples, whereas the metabolic profiles of other asymptomatic samples were closer to the profiles found for symptomatic samples. These results suggest that asymptomatic samples represent a continuum between healthy and symptomatic profiles and that a tree’s response to HLB is based upon a complex mixture of environmental factors. In order to better distinguish between asymptomatic fruits, additional work will need to be done with sample sets in which these factors, including the extent of infection and time since infection, are better understood.
The HPLC analysis for limonin, a major contributor to citrus bitterness, was conducted on all samples. For each sample, analyses of freshly thawed juice and juice incubated for 24 hrs at 4 C were conducted to determine the initial levels of limonin and the amount a limonin that could form during incubation. On average, samples that came from asymptomatic and symptomatic fruits had higher limonin concentrations for both freshly thawed and incubated samples, however for almost all of the samples tested the limonin concentrations that were found were below the level that would result in perceptible bitterness. These results suggest the limonin alone is not responsible for the off-taste found in citrus fruits harvest from trees infected with HLB.
This project was initiated during the previous parent Project (5325-41430-009-00D & 5325-41430-010-00D) and was in support of that parent Project’s objectives to establish the role of naturally occurring biologically active citrus phytochemicals and to enhance citrus co-product utilization.