Jacqueline K. Burns

Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: comparison with girdled fruit

Distribution of viable Candidatus Liberibacter asiaticus (CaLas) in sweet orange fruit and leaves (‘Hamlin’ and ‘Valencia’) and transcriptomic changes associated with huanglongbing (HLB) infection in fruit tissues are reported. Viable CaLas was present in most fruit tissues tested in HLB trees, with the highest titre detected in vascular tissue near the calyx abscission zone. Transcriptomic changes associated with HLB infection were analysed in flavedo (FF), vascular tissue (VT), and juice vesicles (JV) from symptomatic (SY), asymptomatic (AS), and healthy (H) fruit. In SY ‘Hamlin’, HLB altered the expression of more genes in FF and VT than in JV, whereas in SY ‘Valencia’, the number of genes whose expression was changed by HLB was similar in these tissues. The expression of more genes was altered in SY ‘Valencia’ JV than in SY ‘Hamlin’ JV. More genes were also affected in AS ‘Valencia’ FF and VT than in AS ‘Valencia’ JV. Most genes whose expression was changed by HLB were classified as transporters or involved in carbohydrate metabolism. Physiological characteristics of HLB-infected and girdled fruit were compared to differentiate between HLB-specific and carbohydrate metabolism-related symptoms. SY and girdled fruit were smaller than H and ungirdled fruit, respectively, with poor juice quality. However, girdling did not cause misshapen fruit or differential peel coloration. Quantitative PCR analysis indicated that many selected genes changed their expression significantly in SY flavedo but not in girdled flavedo. Mechanisms regulating development of HLB symptoms may lie in the host disease response rather than being a direct consequence of carbohydrate starvation.

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.