Abstract
Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection.
Highlights
Seed export is a major agricultural industry worldwide with a total of 57 countries exporting vegetable seed, accounting for 106 thousand metric tons and contributing to $2,851 million in 2010
This study describes for the first time in detail how the multiplex microsphere-based assay was optimized and used to detect the widespread plant pathogens: Acidovorax avenae subsp. citruli (Aac), chili vein-banding mottle virus (CVbMV), watermelon silver mottle virus (WSMoV) and melon yellow spot virus (MYSV)
To select antibody pairs for multiplex detection of Acidovorax avenae subsp. citruli (Aac), chili vein-banding mottle virus (CVbMV), watermelon silver mottle virus (WSMoV) and melon yellow spot virus (MYSV), all possible combinations of the available antibodies specific to these pathogens were coupled to different microsphere sets as capture antibodies and labeled with fluorescent R-phycoerythrin (RPE) as a detecting antibody (2.0 mg mL21 of each antibody) (Table 2)
Summary
Seed export is a major agricultural industry worldwide with a total of 57 countries exporting vegetable seed, accounting for 106 thousand metric tons and contributing to $2,851 million in 2010 (www.worldseed.org, last accessed in November 2012). Conventional detection methods rely upon a symptom and morphology identification of plant disease followed by further characterization such as isolation, culturing, pathogenicity testing [1], enzyme linked immunosorbent assay (ELISA) or real-time polymerase chain reaction (PCR) [2,3,4]. Multiplex PCR assays were developed to detect multiple plant viruses such as two clades of tomato leaf curl virus (TYLCV) in tomato [5], and cucumber vein yellowing virus (CVYV) and cucurbit yellow stunting disorder virus (CYSDV) in the whitefly vector Bemisia tabaci [6] These molecular techniques are sensitive but require high-skilled workers for tedious DNA extraction and purification steps which become an additional cost for sample testing. Until now, there has never been a report on the optimization of a method to develop a multiplex plant pathogens detection using the microsphere immunoassay
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