Abstract
‘Candidatus Liberibacter asiaticus’ (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the α-proteobacteria. In this study serologically based methods for the detection of CaLas were developed. An anti-outer membrane protein A (OmpA) polyclonal antibody previously produced (in our laboratory) was highly effective for the detection of CaLas from citrus tissues in a simple tissue printing format. The antibody was also used to capture bacteria from periwinkle extracts. About 80% of all field samples analyzed tested positive with both immune tissue printing and qPCR; whereas 95% were positive with at least one of these two methods. When asymptomatic citrus tissues were tested, the tissue printing method gave a higher rate of detection (83%) than the qPCR method (64%). This is consistent with a lower concentration of CaLas DNA, but a higher proportion of viable cells, in the asymptomatic tissues. The immune tissue printing method also highlights the detail of the spatial distribution of ‘Ca. Liberibacter asiaticus’ in diseased citrus tissues. Both the immune capture PCR and immune tissue printing methods offer the advantages of low cost, high throughput, ease of scaling for multiple samples and simplicity over current PCR-based methods for the detection of ‘Ca. Liberibacter asiaticus’.
Highlights
Citrus huanglongbing (HLB), known as citrus greening, is one of the most devastating diseases of citrus and threatens the citrus industry worldwide[1,2,3], leading to large reductions in fruit production and quality as well as decline of infected trees
We report the optimization of a simple immune tissue print and demonstrate an immune capture-Polymerase Chain Reaction (PCR) (IC-PCR) assay based on a polyclonal antibody (Pab) raised in rabbit against the major outer membrane protein (OmpA) of Candidatus Liberibacter asiaticus’ (CaLas)
When the anti-outer membrane protein A (OmpA) Pab was diluted to 1:5,000 and 1:6,000 (Fig. S1e and f), the difference between diseased and healthy control petiole sections was very pronounced: very strong purple colored spots were seen in the CaLas infected phloem cells, but only a weak pink background was present in the healthy controls
Summary
Citrus huanglongbing (HLB), known as citrus greening, is one of the most devastating diseases of citrus and threatens the citrus industry worldwide[1,2,3], leading to large reductions in fruit production and quality as well as decline of infected trees. Owing to inadequate disease and vector control methods, and the extreme difficulty in protecting citrus from infection by CaLas, commercial citrus industries have suffered great economic losses from HLB and continue to decline[2]. Serological assays are widely used to diagnose plant diseases, but have not been widely used for HLB because the pathogen has not been available in culture to produce antibodies against CaLas cells. Due to the limitations of current assays, and the large numbers of trees that must be sampled in citrus production areas where the disease is either present or feared, it is important to develop fast, efficient and inexpensive methods to accurately detect CaLas. Previously, we constructed and produced a highly specific anti-OmpA polyclonal antibody against CaLas[30,31]. These optimized immune tissue print and IC-PCR methods complement existing PCR-based methods and will meet the urgent need for large scale detection of CaLas for the continued sustainability of the United States citrus industry
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