No abstract

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-

Five hundred twenty-seven isolates of Pyricularia grisea were collected from trap rice cultivars of indigenous and exotic origin across three seasons at five sites in Thailand. Single conidium isolates were inoculated onto 15 rice lines near-isogenic (NILs) for resistance genes, one recurrent parent, and two local cultivars. One hundred seventy-five pathotypes were identified, of which 160 were represented by fewer than eight isolates. Predicted pathotype number was estimated at greater than 450 for the study region. Significant differences in pathotype diversity were detected across sites, seasons, and among isolates collected from exotic versus indigenous hosts. Isolates and pathotypes with greater numbers of virulence genes (as inferred from compatibility with NILs) were less common than those with fewer virulence genes. Analysis of virulence distributions among isolates grouped according to their MGR586 DNA-fingerprint similarities (i.e., “lineages”) also showed that, for the most commonly represented lineages, isolates with fewer virulence genes predominated. Lineages represented by one or a few isolates had greater numbers of virulence genes. Lower frequency of recovery of isolates with accumulated virulence genes is consistent with an associated fitness penalty. Resistance genes Pi 1, Pi z-5, and Pi ta2 were broadly effective across this population, compatibility with Pi 1 and Pi z-5 was very rare, and no isolate combined compatibility with both genes. Well-represented (more than 20 isolates) MGR586 lineages showed specific incompatibilities with some NILs, but these were restricted to Pi 1 and Pi z-5. No combination of resistance genes would confer resistance across all lineages.

Sexual fertility and mating type distribution of Magnaporthe grisea field isolates collected in Thailand were analyzed from sites previously found to harbor diverse populations of the pathogen. Three hundred forty-one single conidium isolates of M. grisea collected from five sites in north, northeast, and central Thailand were evaluated for in vitro sexual fertility and mating type by pairing with strains of known mating type. Most isolates (67%) were infertile when crossed with the hermaphrodite tester strains; but fertile isolates of each mating type that yielded viable ascospores were detected in all sites from the northeastern and northern regions. MAT1-2 predominated over MAT1-1 in bioassay mating type. Male fertility (female sterility) predominated in fertile MAT1-1 (50 to 75%) and MAT1-2 (50 to 85%) isolates from all locations in Thailand; however, hermaphroditic and/or female fertile isolates were also detected in all but one site. Fertility, as determined by perithecia density, was low (<10 perithecia cm-2) for most isolates, although a few produced in excess of 20 perithecia cm-2.

The nucleotide sequences of genome segments S7 and S10 of a Thai-isolate of rice ragged stunt virus (RRSV) were determined. The 1938 bp S7 sequence contains a single large open reading frame (ORF) spanning nucleotides 20 to 1843 that is predicted to encode a protein of M(r) 68,025. The 1,162 bp S10 sequence has a major ORF spanning nucleotides 142 to 1,032 that is predicted to encode a protein of M(r) 32,364. This S10 ORF is preceded by a small ORF (nt 20-55) which is probably a minicistron. Coupled in vitro transcription-translation from the two major ORFs gave protein products of the expected sizes. However, no protein was visualised from S10 when the small ORF sequence was included. Proteins were expressed in Escherichia coli from the full length ORF of S7 (P7) and from a segment of the S10 ORF (P10) fused to the ORF of glutathione S-transferase (GST). Neither fusion protein was recognised by polyclonal antibodies raised against RRSV particles. Furthermore, polyclonal antibodies raised against GST-P7 fusion protein did not recognise any virion structural polypeptides. These data strongly suggest that the proteins P7 and P10 do not form part of RRSV particle. This is further supported by observed sequence homology (though very weak) of predicted RRSV P7 and P10 with those of rice dwarf virus (RDV) non-structural proteins Pns6 and Pns9, respectively.