Variations of the chemical composition of Citrus sinensis Osbeck cv. Newhall fruit in relation to the symptom severity of Huanglongbing

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.

Huanglongbing (HLB) or citrus greening is the most severe citrus disease, currently devastating the citrus industry worldwide. The presumed causal bacterial agent Candidatus Liberibacter spp. affects tree health as well as fruit development, ripening and quality of citrus fruits and juice. Fruit from infected orange trees can be either symptomatic or asymptomatic. Symptomatic oranges are small, asymmetrical and greener than healthy fruit. Furthermore, symptomatic oranges show higher titratable acidity and lower soluble solids, solids/acids ratio, total sugars, and malic acid levels. Among flavor volatiles, ethyl butanoate, valencene, decanal and other ethyl esters are lower, but many monoterpenes are higher in symptomatic fruit compared to healthy and asymptomatic fruit. The disease also causes an increase in secondary metabolites in the orange peel and pulp, including hydroxycinnamic acids, limonin, nomilin, narirutin, and hesperidin. Resulting from these chemical changes, juice made from symptomatic fruit is described as distinctly bitter, sour, salty/umami, metallic, musty, and lacking in sweetness and fruity/orange flavor. Those effects are reported in both Valencia and Hamlin oranges, two cultivars that are commercially processed for juice in Florida. The changes in the juice are reflective of a decrease in quality of the fresh fruit, although not all fresh fruit varieties have been tested. Earlier research showed that HLB-induced off-flavor was not detectable in juice made with up to 25% symptomatic fruit in healthy juice, by chemical or sensory analysis. However, a blend with a higher proportion of symptomatic juice would present a detectable and recognizable off flavor. In some production regions, such as Florida in the United States, it is increasingly difficult to find fruit not showing HLB symptoms. This review analyzes and discusses the effects of HLB on orange juice quality in order to help the citrus industry manage the quality of orange juice, and guide future research needs.