Cassava Breeding and Cultivation Challenges in Thailand: Past, Present, and Future Perspectives.

Cassava (Manihot esculenta Crantz) was introduced to Southeast Asia in the 16th-17th centuries and has since flourished as an industrial crop. Since the 1980s, Thailand has emerged as the leading producer and exporter of cassava products. This growth coincided with the initiation of cassava breeding programs in collaboration with the International Center for Tropical Agriculture (CIAT), focusing on root yield and starch production. The success of Thai cassava breeding programs can be attributed to the incorporation of valuable genetic diversity from international germplasm resources to cross with the local landraces, which has become the genetic foundation of many Thai commercial varieties. Effective evaluation under diverse environmental conditions has led to the release of varieties with high yield stability. A notable success is the development of Kasetsart 50. However, extreme climate change poses significant challenges, including abiotic and biotic stresses that threaten cassava root yield and starch content, leading to a potential decline in starch-based industries. Future directions for cassava breeding must include hybrid development, marker-assisted recurrent breeding, and gene editing, along with high-throughput phenotyping and flower induction. These strategies are essential to achieve breeding objectives focused on drought tolerance and disease resistance, especially for CMD and CBSD.

An xa5 Resistance Gene-Breaking Indian Strain of the Rice Bacterial Blight Pathogen Xanthomonas oryzae pv. oryzae Is Nearly Identical to a Thai Strain.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) constrains production in major rice growing countries of Asia. Xoo injects transcription activator-like effectors (TALEs) that bind to and activate host "susceptibility" (S) genes that are important for disease. The bacterial blight resistance gene xa5, which reduces TALE activity generally, has been widely deployed. However, strains defeating xa5 have been reported in India and recently also in Thailand. We completely sequenced and compared the genomes of one such strain from each country and examined the encoded TALEs. The two genomes are nearly identical, including the TALE genes, and belong to a previously identified, highly clonal lineage. Each strain harbors a TALE known to activate the major S gene SWEET11 strongly enough to be effective even when diminished by xa5. The findings suggest international migration of the xa5-compatible pathotype and highlight the utility of whole genome sequencing and TALE analysis for understanding and responding to breakdown of resistance.

A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Recent Thai Isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host "susceptibility" (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

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.

Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library.

Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V(kappa) and V(H) genes with newly designed primer sets. Initially, we amplified V(kappa) and V(H) genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V(kappa) and V(H) genes from the framework region 1 to the joining region. The V(kappa) and V(H) genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.

Pathotype and Avirulence Gene Diversity of Pyricularia grisea in Thailand as Determined by Rice Lines Near-Isogenic for Major Resistance Genes.

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.

Sexually Fertile Magnaporthe grisea Rice Pathogens in Thailand.

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.