An Improved Recombinase Polymerase Amplification Coupled with Lateral Flow Assay for Rapid Field Detection of 'Candidatus Liberibacter asiaticus'.

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.

The Impact of Diaphorina citri-Vectored 'Candidatus Liberibacter asiaticus' on Citrus Metabolism.

'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.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analysis of Citrus limon Response to Graft Inoculation by Candidatus Liberibacter asiaticus.

Presymptomatic detection of citrus trees infected with Candidatus Liberibacter asiaticus (CLas), the bacterial pathogen associated with Huanglongbing (HLB; citrus greening disease), is critical to controlling the spread of the disease. To test whether infected citrus trees produce systemic signals that may be used for indirect disease detection, lemon (Citrus limon) plants were graft-inoculated with either CLas-infected or control (CLas-) budwood, and leaf samples were longitudinally collected over 46 weeks and analyzed for plant changes associated with CLas infection. RNA, protein, and metabolite samples extracted from leaves were analyzed using RNA-Seq, mass spectrometry, and 1H NMR spectroscopy, respectively. Significant differences in specific transcripts, proteins, and metabolites were observed between CLas-infected and control plants as early as 2 weeks post graft (wpg). The most dramatic differences between the transcriptome and proteome of CLas-infected and control plants were observed at 10 wpg, including coordinated increases in transcripts and proteins of citrus orthologs of known plant defense genes. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic or early disease detection as part of efforts to control the spread of HLB into uninfected citrus groves.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Analyses of Citrus sinensis (L.) Osbeck Graft-Inoculated with "Candidatus Liberibacter asiaticus".

"Candidatus Liberibacter asiaticus" (CLas) is the bacterium associated with the citrus disease Huanglongbing (HLB). Current CLas detection methods are unreliable during presymptomatic infection, and understanding CLas pathogenicity to help develop new detection techniques is challenging because CLas has yet to be isolated in pure culture. To understand how CLas affects citrus metabolism and whether infected plants produce systemic signals that can be used to develop improved detection techniques, leaves from Washington Navel orange (Citrus sinensis (L.) Osbeck) plants were graft-inoculated with CLas and longitudinally studied using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry), and metabolomics (proton nuclear magnetic resonance). Photosynthesis gene expression and protein levels were lower in infected plants compared to controls during late infection, and lower levels of photosynthesis proteins were identified as early as 8 weeks post-grafting. These changes coordinated with higher sugar concentrations, which have been shown to accumulate during HLB. Cell wall modification and degradation gene expression and proteins were higher in infected plants during late infection. Changes in gene expression and proteins related to plant defense were observed in infected plants as early as 8 weeks post-grafting. These results reveal coordinated changes in greenhouse navel leaves during CLas infection at the transcript, protein, and metabolite levels, which can inform of biomarkers of early infection.

Evaluation of Insecticides, Entomopathogenic Nematodes, and Physical Soil Barriers for Control of Diaprepes abbreviatus (Coleoptera: Curculionidae) in Citrus.

The Diaprepes root weevil, Diaprepes abbreviatus L. (Coleoptera: Curculionidae), was discovered as the cause of early decline and death of citrus trees and ornamental plants along coastal Orange and Los Angeles Counties in 2005 and San Diego County in 2006. We established trials to evaluate the effectiveness of two tactics to manage life stages of Diaprepes root weevil that are found in the soil. The first tactic was soil applications of the pesticides bifenthrin or imidacloprid, with and without entomopathogenic nematodes, to kill larvae. The second tactic was attempting to block adult emergence from the soil using a thick wood-chip mulch layer, a small-meshed landscape fabric, and a dry soil barrier (created by subirrigation). We found that soil treatments with the two insecticides (bifenthrin and imidacloprid) at maximum label rates with and without Steinernema riobrave Cabanillas et al. at 5 million nematodes per tree significantly reduced numbers of soil-borne Diaprepes root weevil larvae in one trial compared with the control. Another entomopathogenic nematode, Heterorhabditis indica Poinar et al. applied in wax moth Galleria mellonella L. cadavers at 86 wax moth cadavers per tree was not effective. Adult emergence was reduced by almost 100% when the landscape fabric was kept intact (2009), but emergence occurred in 2010 when the fabric was torn by grove operations (60% reduction). Adult emergence was reduced to ≍70% compared with the control in the subirrigation treatment in 2009 when the summer was hot and dry, but emergence occurred in 2010 during periodic rains toward the end of summer. The mulch treatment kept the soil moist in between irrigations resulting in greater adult emergence rates compared with controls during 2009 and 2010. Health ratings of the trees were taken following several years of physical barrier treatments, and trees treated with the landscape fabric were significantly healthier (mean rating 0.9, scale 0 = healthy to 5 = dead) than those in the other treatments (mean rating of 3.6, 4.0, and 2.5 for control, mulch and subirrigation, respectively). Of the physical barrier methods studied, landscape fabric is most effective in reducing Diaprepes root weevil emergence, increases plant health, and is long lasting, but it is the most costly to install and can be damaged during harvest.