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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