Research Article Untargeted metabolite analysis of healthy and Huanglongbing-infected orange leaves by CE-DAD Huanglongbing (HLB) is considered the most destructive bacterial citrus disease worldwide. Early detection of HLB is crucial for minimizing its spread. CE was used for the discovery of potential biomarkers for HLB. Optimization of extraction and separation allowed resolving 24 compounds of which 6 were present in significantly higher (p o 0.05) concentrations in HLB-infected samples collected monthly for 6 months during the 2007–2008 season. Three of these compounds were identified by mobility and UV spectra as hesperidin, naringenin, and quercetin with mean increase in concentration of 154, 555, and 467%, respectively, above that in healthy leaves. Results support the potential of CE-DAD for untargeted plant metabolomic analysis. CZE, NACE, and MEEKC were compared for metabolic differentiation of healthy and HLB-infected citrus leaves. CZE in a semi-aqueous BGE solution consisting of 8.5 mM of sodium borate (pH 9.3), 15% ACN, and 9% 1-butanol yielded the best peak separation with detection at 190 nm. Keywords: Biomarkers / CE / Huanglongbing / Metabolomics / Plant diseases DOI 10.1002/elps.200800594 1 Introduction Citrus Huanglongbing (HLB) is the most significant threat of the citrus industry worldwide [1]. It originated in Asia and first detected in the Americas in 2004 (Brazil), reaching the United States in 2005 [2]. The causal agent is Candidatus Liberibacter, a gram-negative bacteria not yet cultured to Koch’s postulates. Present methods for its quantification based on real-time PCR are currently being optimized [3]. The disease is transmitted through a psyllid (Diaphorina citri) vector that feeds on citrus and other tropical and sub-tropical plants making vector eradication impos- sible. Symptoms of the disease include marked yellow regions on leaves and poor quality, small, inedible, and misshapen fruit [4]. HLB has had serious damaging impact in the citrus industry in many Asian countries, wiped out a great number of trees in Brazil, and as of 2008, has seriously affected groves in 26 Florida counties making Candidatus Liberibacter the most dangerous citrus pathogen in the world [5]. Since the appearance of HLB, many efforts have been directed toward controlling this disease including frequent pesticide spray to reduce psyllid population, the use of pheromone traps, and psyllid repellents. Early detection of HLB-infected trees is crucial to managing HLB’s spread by reducing inoculums through tree elimination Currently, PCR is the only approved method for diag- nosis of HLB; however, this is an expensive, laborious, and time-consuming alternative that does not allow in-field analysis. Other limitations such as the low concentration and uneven distribution of the bacteria in the tree [6] make PCR detection very difficult, especially at early stages. Research on new methods such as the isothermal and chimeric primer-initiated amplification of nucleic acids combined with cycling probe technology [7], application of nested PCR [8], comparisons of primers for PCR and nested PCR [9], along with the improvement of DNA isolation for conventional PCR [10] has been reported. However, PCR amplification of the bacteria is very weak during spring and summer seasons [11], increasing the probability of obtaining false negatives during these periods. Therefore, methods that do not rely on the presence of the bacteria in the sample are sought as more reliable throughout the year. To the best of our knowledge, only two non-PCR methods have been comprehensively researched. One relies on the presence of excessive amounts of starch [12], whereas the other on overproduction of gentisic acid [13] in infected trees. The disadvantages of these methods are that they are not HLB- specific, because excessive amounts of starch are noticed with other stress situations such as girdling [14] and gentisic acid overproduction is caused by other infections in several Juan Manuel Cevallos- Cevallos 1 Russell Rouseff 1 Jose ´ I. Reyes-De-Corcuera 1 Citrus Research and Education Center, IFAS, University of Florida, FL, USA Received September 12, 2008 Revised October 31, 2008 Accepted November 2, 2008 Abbreviations: CW, cold water; HLB, Huanglongbing; HW, hot water; IS, internal standard; MWC, methanol/water/ chloroform Correspondence: Assistant Professor Jose ´ I. Reyes-De-Corcuera, 700 Experiment Station Road, Lake Alfred, FL 33850, USA E-mail: jireyes@ufl.edu Fax: 11-863-956-4631 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com Electrophoresis 2009, 30, 1–8 1