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-

Bacterial fruit blotch of cucurbits is a destructive disease caused by Acidovorax avenae subsp. citrulli, which is a typical seedborne pathogen. In seed health testing for this disease, we have detected many strains of Acidovorax with some differences from A. avenae subsp. citrulli. Their 16S rRNA sequences were divided into six types. The most common sequence was completely consistent with that of A. avenae subsp. avenae originally isolated from rice. The other sequences were over 99% similar but not identical to those of A. avenae subsp. avenae and A. avenae subsp. citrulli. Some commercialized antibodies against A. avenae subsp. citrulli reacted with several of these strains. Some of these strains incited yellow spots or brownish water-soaked lesions mainly on young true leaves of cucumber and squash after spray inoculation. Histological observations showed that these strains entered the leaf tissues of cucurbit plants through stomata and multiplied in the intercellular spaces of parenchymatous tissues as well as in the vascular tissues. The amount of bacterial multiplication and spread in the tissues differed among the strains, presumably reflecting their ability to induce symptoms. These isolated strains are therefore different from A. avenae subsp. citrulli, and their potential threat to the cultivation of cucurbits is lower than that of A. avenae subsp. citrulli.

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Blast caused by the fungus Magnaporthe oryzae (Hebert) Barr. and bacterial leaf blight (BLB) caused byXanthomonas oryzae pv. oryzae (Xoo) are two major diseases of rice (Oryza sativa). The use of varietal resistance is the most appropriate strategy for controlling the diseases, and molecular assisted selection can potentially accelerate breeding programs. The objective of this study was to pyramid genes conferring resistance to blast and bacterial leaf blight diseases to rice cultivar RD6, using molecular assisted selection. Near-isogenic lines (NIL) derived from two blast resistant crosses (RD6 × P0489 and RD6 × Jao Hom Nin) were pyramided with IR62266 (xa5), to transfer bacterial leaf blight resistance to RD6 introgression lines. Five flanking sets of simple sequence repeat (SSR) markers (RM319/RM212, RM48/RM207, RM224/RM144, RM313/RM277 and RM122/RM159: four for blast and one for BLB resistance) were used for screening of introgression lines carrying five quantitative trait loci (QTLs) from the BC 1 F 2 generation through to BC 2 F 2:3 generation, and 12 pyramiding lines were identified. Gene validation for blast and bacterial leaf blight diseases was accomplished using artificial inoculation under greenhouse conditions. BC 2 F 2:3 2-8-2-24 and BC 2 F 2:3 2-8-2-25 showed greater levels of blast broad spectrum resistance (BSR) whereas BC 2 F 2:3 2-8-2-36 expressed the highest of bacterial leaf blight resistance with a high blast BSR.

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Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISAbased method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×10 5 to 1×10 6 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×10 4 to 1×10 7 CFU/mL and 5×10 4 to 5×10 5 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections.

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