Petcharat Thummabenjapone

Pyramiding of blast and bacterial leaf blight resistance genes into rice cultivar RD6 using marker assisted selection

Blast caused by the fungus Magnaporthe oryzae (Hebert) Barr. and bacterial leaf blight (BLB) caused by Xanthomonas 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 BC1F2 generation through to BC2F2:3 generation, and 12 pyramiding lines were identified. Gene validation for blast and bacterial leaf blight diseases was accomplished using artificial inoculation under greenhouse conditions. BC2F2:3 2-8-2-24 and BC2F2:3 2-8-2-25 showed greater levels of blast broad spectrum resistance (BSR) whereas BC2F2:3 2-8-2-36 expressed the highest of bacterial leaf blight resistance with a high blast BSR. Keywords: Gene pyramiding, introgression lines, molecular marker, Near-isogenic lines, SSR. African Journal of Biotechnology Vol. 12(28), pp. 4432-4438

Novel and Highly Specific Monoclonal Antibody to Acidovorax citrulli and Development of ELISA-Based Detection in Cucurbit Leaves and Seed

A novel monoclonal antibody (MAb) specific to the seedborne 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, Comamonas 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 test. In Western blot analysis, MAb 11E5 reacted with an A. citrulli protein of a molecular mass >170 kDa. MAb 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-sELISA for detection of A. citrulli in cucurbit leaf and seed extracts. The detection limit of the MC-sELISA was 5 × 104 CFU/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 of A. citrulli in cucurbits and can be applied for routine field inspection.

Genetic diversity and pathogenicity of cucurbit-associated Acidovorax

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.

Evaluation of antigen preparation methods for polyclonal antibody production against Streptomyces spp.

Aim: To determine the optimum antigen preparation method for producing specific polyclonal antibody specific for Streptomyces species. Study Design: Experimental study. Place and Duration of Study: Faculty of Science, Mahasarakham University, Mahasarakham Province, Thailand, between May 2011 and December 2011. Methodology: Two Streptomyces isolates were used for antisera production. The sonication method was chosen for antigen preparation. Antigen suspensions were emulsified with incomplete Freund’s adjuvant and 1 ml was injected into rabbit thigh muscle for the first, second and third immunization. The fourth and fifth immunizations were injected intravenously. Antibody titer, detection limit and specificity were measured using indirect-ELISA. Streptomyces antigen mixed with soil was investigated. Results: The 5 min sonication method gave a higher protein than other test methods, so this protocol was chosen for all subsequent work. The sonicated Streptomyces antigen was