Godfrey, Kris

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

‘Candidatus Liberibacter asiaticus’ is associated with the devastating citrus disease Huanglongbing (HLB). It is transmitted by grafting infected material to healthy plants and by the feeding of the Asian citrus psyllid (Diaphorina citri). Previously, we demonstrated that a metabolomics approach using proton-nuclear magnetic resonance spectroscopy discriminates healthy from diseased plants via grafting. This work assessed the capability of this technology in discriminating healthy and diseased plants when the bacterium is vectored by psyllids. One-year-old greenhouse-grown ‘Lisbon’ lemon trees were exposed to either carrier psyllids (exposed, n = 10), or psyllids that were free of ‘Candidatus Liberibacter asiaticus’ (control, n = 6). Leaf metabolites were tracked for 1 year and disease diagnosis was made using quantitative PCR. Overall, 31 water-soluble metabolites were quantified in leaves, including four sugars and 12 amino acids. Analysis via nonmetric multidimensional scaling and principal component analysis revealed significant differences between the leaf metabolome of control versus infected trees beginning at 8 weeks postexposure, including alterations in glucose and quinic acid concentrations. These findings provide a longitudinal overview of the metabolic effects of HLB during the early phases of disease, and confirm previous experimental work demonstrating that infection elicits changes in the leaf metabolome that enables discrimination between healthy and infected plants. Here we demonstrate that the mode of inoculation (i.e., graft versus psyllid) results in a similar pathology.

Comparison of the metabolic changes prior to symptom development upon infection with Candidatus Liberibacter asiaticus (CLas), the bacterium associated with citrus greening disease, between citrus hosts with different tolerances is lacking. The objective of this study was to compare the early response of Lisbon lemon (Citrus limon) and Washington navel orange (Citrus sinensis [L.] Osbeck), two citrus species commercially important to California, to CLas through graft inoculation. Here, we compare the transcriptome, proteome, and metabolome response, using RNA sequencing, liquid chromatography tandem mass spectrometry, and 1H nuclear magnetic resonance, respectively, from our two recently published studies examining the response of the lemon and navel oranges separately, and introduce new micronutrient data from inductively coupled plasma mass spectrometry analysis, focusing on lemons at 10 and 14 weeks postgrafting (wpg), and navels at 8 and 18 wpg, prior to symptom development. Several micronutrients accumulated in presymptomatic infected lemons compared with controls, whereas little change was observed in the navels. Photosynthesis proteins were substantially altered by CLas infection in navels, with fewer changes observed in lemons. The metabolome differed between control and infected navels throughout infection, although differences between control and infected lemons were identified only after symptom expression. Taken together, these findings highlight differences in response to CLas between two varieties with differing tolerances. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .