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.

Himananto, O., Thummabenjapone, P, Luxananil, P, Kumpoosiri, M., Hongprayoon, R., Kositratana, W., and Gajanandana, O. 2011. Novel and highly specific monoclonal antibody to Acidovorax citrulli and development of ELISA-based detection in cucurbit leaves and seed Plant Dis 95:1172-1178.A novel monoclonal antibody (MAb) specific to the seedbome bacterium Acidovorax citrulli was produced. MAb 11E5 reacted specifically with 19 strains of A. citrulli but not with three closely related bacteria in the family Comamonadaceae (i.e., A. facilis, Comctmonas acidovorans, and C. testosteroni) and another seven phytopathogenic bacteria. Moreover, this MAb detected a strain of A. citrulli that was not detected by a commercial enzyme-linked immunosorbent assay (ELISA)-based kit and a commercial immunochromatographic strip te.st. In Western blot analysis, MAb 11E5 reacted with an A. citrtilli protein of a molecular mass >I7O kDa. M Ah 11E5 was employed to develop two sandwich ELISA systems: MAb captured-sandwich ELISA (MC-sELISA) and polyclonal antibody captured-sandwich ELISA (PC-sELISA). MC-sELISA was 10 times more sensitive than PC-sELlSA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5x10" CEU/ml. Detection of A. citrulli in naturally infected cucurbit leaves, fruit, and seed was also feasible using MC-sELISA. The newly established MCsELISA provides another alternative for specific detection oí A. citrulli in cucurbits and can be applied for routine field inspection.Acidovorax citrulli (originally Pseudomonas pseudoalcaligenes subsp. citrulli and subsequently changed to A. avenae subsp. citrulli) (15,16,23) is a gram-negative bacterium that causes bacterial fruit blotch (BEB) of cucurbits, resulting in severe losses in cucurbit production worldwide. This bacterium causes serious concerns for the vegetable seed industry because it is naturally borne and transmitted by seed (2,7,13). BEB can be devastating for seed producers because it can result in 100% yield reduction (9). In Thailand, cucurbit seed, such as watermelon, cantaloupe, cucumber, gourd, squash, and pumpkin seed, account for 30% (approximately U.S.$29 million) of the total seed exported (approximately U.S.$98 million) in 2009 (The Office of Agricultural Regulation, Department of Agriculture Thailand, Retrieved from, http:// www.oae.go.th/ewtadmin/ewt/oae_web/ewt_news.php?nid=8115& filename=index). Phytosanitary certification is required for cucurbit seed export. Eield inspection prior to harvest or seed testing is required depending on regulation of each country. Standard methods for seed testing to detect A. citrulli are the seedling grow-out assays (SGO) and bacterial isolation on semiselective media, which are laborious and time consuming and require large areas of greenhouse space. To alleviate this problem, most of the previous studies have focused on developing new methods for seed testing such as immuno-

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Recent developments in smartphone-based strip readers have further improved the performances of lateral flow test kits. Most smartphone cameras encode an unaltered and nonlinear power-law transfer function that maps the light intensity to a pixel value; this poses some limitations for camera-based strip readers. For faint-color test lines which are almost as white such as with nitrocellulose pads, the slope of the transfer function is low. Therefore, it is difficult to differentiate between the faint test lines and the white background. We show that by manually setting the camera exposure time—instead of using the automatic settings—to the high-slope region of the transfer function, the reader’s sensitivity can be improved. We found that the sensitivity and the limit of detection of the Acidovorax avenae subsp. citrulli (Aac) test kit were enhanced up to 3-fold and 5-fold, respectively, when using the readers at the optimal camera settings, compared to the automatic mode settings. This simple technique can be readily applied to any existing camera-based colorimetric strip reader to significantly improve its performance.

Rapid and economical screening of plant pathogens is a high-priority need in the seed industry. Crop quality control and disease surveillance demand early and accurate detection in addition to robustness, scalability, and cost efficiency typically required for selective breeding and certification programs. Compared to conventional bench-top detection techniques routinely employed, a microfluidic-based approach offers unique benefits to address these needs simultaneously. To our knowledge, this work reports the first attempt to perform microfluidic sandwich ELISA for Acidovorax citrulli (Ac), watermelon silver mottle virus (WSMoV), and melon yellow spot virus (MYSV) screening. The immunoassay occurs on the surface of a reaction chamber represented by a microfluidic channel. The capillary force within the microchannel draws a reagent into the reaction chamber as well as facilitates assay incubation. Because the underlying pad automatically absorbs excess fluid, the only operation required is sequential loading of buffers/reagents. Buffer selection, antibody concentrations, and sample loading scheme were optimized for each pathogen. Assay optimization reveals that the 20-folds lower sample volume demanded by the microchannel structure outweighs the 2- to 4-folds higher antibody concentrations required, resulting in overall 5–10 folds of reagent savings. In addition to cutting the assay time by more than 50%, the new platform offers 65% cost savings from less reagent consumption and labor cost. Our study also shows 12.5-, 2-, and 4-fold improvement in assay sensitivity for Ac, WSMoV, and MYSV, respectively. Practical feasibility is demonstrated using 19 real plant samples. Given a standard 96-well plate format, the developed assay is compatible with commercial fluorescent plate readers and readily amendable to robotic liquid handling systems for completely hand-free assay automation.

Little is known about immunogenicity after ChAdOx1 nCov‐19 vaccination after transplantation. We assessed the vaccine response by antibody testing, surrogate neutralization test (sVNT) against wild‐type (WT) and delta variant (DT), and T cell assay in 83 kidney transplant recipients (KTRs) and 52 healthy volunteers (HVs). For KTRs, a positive anti‐RBD antibody was seen in 2.8% after one dose and 15.7% after two doses of the vaccine. After two doses, the positivity rate by sVNT was equal (4.9% each, for WT and DT) and was 13.4% by T cell response. Post two doses, KTRs had significantly lower geometric mean titer than HVs (1.93 [95% CI: 1.39–2.69] vs. 248.3 [95% CI: 203.7–302.6] BAU/ml, respectively, p < .001). Daily mycophenolate dose of ≥1000 mg significantly associated with negative seroconversion [risk ratio (RR) of 0.33, 95% CI: 0.15–0.72, p = .005]. Compared with cyclosporine, daily tacrolimus dose of ≤3 mg and >3 mg of tacrolimus significantly associated with negative seroconversion [RR = 0.38 (95% CI, 0.17–0.85, p = .018) and RR = 0.16 (95% CI, 0.37–0.73, p = .018)], respectively. The vaccine was safe and well‐tolerated but the immune response after the two doses of ChAdOx1 nCov‐19 vaccine in KTRs was very low.

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