F. N. Lee

Screening Oryza Species Plants for Rice Sheath Blight Resistance

Rice wild relatives, Oryza species, are one possible source of sheath blight (Rhizoctonia solani) resistance genes. However, Oryza spp. cannot be screened in the field as is done for cultivated rice (O. sativa) because the plant canopy does not favor disease development and many plants drop mature seed. Thus, a growth chamber-greenhouse method of screening Oryza spp. and their early generation progeny is needed. Primary-secondary and ratoon tillers of rice cultivars-germplasm which ranged from moderately resistant to very susceptible were evaluated first for sheath blight susceptibility. Plants were inoculated by placing R. solani-colonized toothpicks at the leaf collar, then incubating plants in a growth chamber. After 7 days, plants were visually rated for sheath blight severity, and the lesion length of each leaf was measured. Ranking of cultivar-germplasm susceptibility by visual rating of primary-secondary tillers corresponded to the ranking from field ratings. Visual ratings correlated best with combined lesion length of the second and third leaves. For ratoon tillers, visual ratings correlated best with second-leaf lesion length. Next, this method was used with ratoon tillers to evaluate sheath blight susceptibility of 21 Oryza spp. accessions and F1 progeny from crosses between 17 accessions and cultivated rice. This method proved useful on a limited scale for screening germplasm that could not be evaluated under field conditions.

Confirming QTLs and Finding Additional Loci Responsible for Resistance to Rice Sheath Blight Disease

Rice sheath blight disease, caused by Rhizoctonia solani AG1-1A, is one of the most destructive rice diseases worldwide. Utilization of host resistance is the most economical and environmentally sound strategy in managing sheath blight (ShB). Ten ShB quantitative trait loci (QTLs) were previously mapped in a Lemont × Jasmine 85 recombinant inbred line (LJRIL) population using greenhouse inoculation methods at an early vegetative stage. However, confirmation of ShB-resistant QTLs under field conditions is critical for their utilization in marker-assisted selection (MAS) for improving ShB resistance in new cultivars. In the present study, we evaluated ShB resistance using 216 LJRILs under field conditions in Arkansas, Texas, and Louisiana during 2008 and 2009. We confirmed the presence of the major ShB-QTL qShB9-2 based on the field data and also identified one new ShB-QTL between markers RM221 and RM112 on chromosome 2 across all three locations. Based on the field verification of ShB evaluations, the microchamber and mist-chamber assays were simple, effective, and reliable methods to identify major ShB-QTLs like qShB9-2 in the greenhouse at early vegetative stages. The markers RM215 and RM245 were found to be closely linked to qShB9-2 in greenhouse and field assays, indicating that they will be useful for improving ShB resistance in rice breeding programs using MAS.

Determination of Resistance Spectra of the Pi-ta and Pi-k Genes to U.S. Races of Magnaporthe oryzae Causing Rice Blast in a Recombinant Inbred Line Population

Molecular tagged resistance (R) genes are useful for developing improved cultivar resistance using marker-assisted breeding. In the present study, R genes to common races of Magnaporthe oryzae, the causal agent of blast disease of rice (Oryza sativa), were mapped using an F10 recombinant inbred line (RIL) population derived from a cross of tropical japonica cv. Katy with breeding line RU9101001. Katy was resistant to 10 common U.S. races: IA-45, IB-1, IB-45, IB-49, IB-54, IC-17, ID-1, IE-1, IG-1, and IH-1 of M. oryzae. RU9101001 was resistant to races IA-45, IB-45, IB-54, IG-1, and IH-1. Katy and RU9101001 were susceptible to race IE-1k. Twenty-three polymorphic simple sequence repeat (SSR) markers were used to map R genes. Segregation ratios of 1:1 (resistant/susceptible) to races IB-1, IB-49, IC-17, ID-1, and IE-1 indicated the presence of a single dominant R gene in Katy. Ratios of 3:1 (resistant/susceptible) to races IA-45, IB-45, IG-1, and IH-1 indicated that a single R gene was present in Katy and a different R gene was present in RU9101001. Resistance to the abovementioned races was correlated with the presences of the Pi-ta gene and 11 Katy SSR alleles, suggesting that Pi-ta confers resistance to IA-45, IB-1, IB-45, IB-49, IC-17, IG-1, ID-1, IE-1, and IH-1. Katy, RU9101001, and all RILs were resistant to race IB-54, which was consistent with the presence of Pi-ks in Katy and Pi-kh in RU9101001. Resistance to IA-45, IB-45, IG-1, and IH-1 correlated with the presence of Pi-kh, suggesting that Pi-kh confers resistance to IA-45, IB-45, IG-1, and IH-1. These data suggest that Pi-ta and Pi-kh are effective R genes with overlapped resistance to the 10 common races of M. oryzae.

Genome-Wide Association of Rice Blast Disease Resistance and Yield-Related Components of Rice.

Robust disease resistance may require an expenditure of energy that may limit crop yield potential. In the present study, a subset of a United States Department of Agriculture rice core collection consisting of 151 accessions was selected using a major blast resistance (R) gene, Pi-ta, marker and was genotyped with 156 simple sequence repeat (SSR) markers. Disease reactions to Magnaporthe oryzae, the causal agent of rice blast disease, were evaluated under greenhouse and field conditions, and heading date, plant height, paddy and brown seed weight in two field environments were analyzed, using an association mapping approach. A total of 21 SSR markers distributed among rice chromosomes 2 to 12 were associated with blast resistance, and 16 SSR markers were associated with seed weight, heading date, and plant height. Most noticeably, shorter plants were significantly correlated with resistance to blast, rice genomes with Pi-ta were associated with lighter seed weights, and the susceptible alleles of RM171 and RM6544 were associated with heavier seed weight. These findings unraveled a complex relationship between disease resistance and yield-related components.

Development of monoclonal antibodies specific for Pyricularia grisea, the rice blast pathogen

The development of polyclonal antisera and monoclonal antibodies (MAbs) specific for Pyricularia grisea was explored. Polyclonal antisera and MAbs against germinating conidia cross-reacted with unrelated fungi and extracts of healthy rice tissue in ELISA. However, MAbs produced from crushed conidia showed various levels of specificity to the fungus. Fourteen hybridoma lines secreting antibodies positive for the immunogen and negative for healthy rice tissue were selected from three independent fusions of NS-1 myeloma cells with splenocytes from mice immunized with crushed conidial suspensions of P. grisea race IB-49. Four stable cell lines were recloned. MAbs secreted from cell lines 4G11, 8H1 and 3E4 reacted strongly with conidial antigen, and MAb 11C6 preferably with mycelial antigen in ELISA. MAb 4G11 bound to the surface of conidia whereas 8H1 and 3E4 bound on germ-tubes in the indirect immunofluorescence assay (IFA). In cross-reaction tests with ELISA MAb 4G11, an IgG1 antibody, reacted negatively with isolates representing 11 fungal genera and was defined as species-specific. Further tests against 17 isolates of P. grisea indicated that MAb 4G11 reacted positively with 11 and 12 of the isolates in ELISA and IFA, respectively. MAb 4G11 could detect homologous conidial antigen at 14–70 ng ml −1 , 10–20 conidia/well, and the fungal antigen in infected rice tissue in ELISA. It may have potential diagnostic value.

Small-Scale Induction of Postharvest Yellowing of Rice Endosperm

ABSTRACT Rice endosperm often develop a yellow discoloration during commercial storage in conditions of high temperature and moisture, thereby reducing the value of the grain. This postharvest yellowing (PHY) appears to be coincidental with fungal presence. To study the yellowing process in a controlled manner, we developed a technique to induce PHY on a small, laboratory scale. Milled rice kernels were rinsed with water and incubated in clear test tubes or microfuge tubes at 65–80°C. This allowed direct observation of the color change and measurement using a colorimeter. Every rice cultivar tested (long and medium grain japonicas and indicas) showed some level of PHY, which increased with temperature yielding a maximum color change at 79°C. Most color change occurred within one day. The moisture parameters required for yellowing to occur were measured. Using sterilization and culture techniques, we found no indications of direct fungal involvement in the yellowing process.

Field Resistance Expressed when the Pi-ta Gene is Compromised by Magnaporthe oryzae

The Pi-ta gene provided 14 years of durable resistance to contemporary field population of Magnaporthe oryzae in southern USA rice production areas before being overcome during 2004 in ‘Banks’, a Pi-ta-based cultivar, by race IE-1k of the blast pathogen. Previously detected in production fields in 1994, the rarely recovered race IE-1k appeared to be poorly adapted to local conditions. Although stable molecular variations were defined in field isolates from ‘Banks’, virulence bioassays do not distinguish between isolates from Banks and the type race IE-1k isolate. After 2004, blast epidemics were noted in other ‘Banks’ production fields but have not been observed in other cultivars containing Pi-ta including ‘Drew’, ‘Ahrent’ and ‘Cybonnet’. The Pi-ta allele in ‘Banks’ still confers resistance to all contemporary blast races except IE-1k and was determined to be molecularly identical to the Pi-ta allele in ‘Katy’. ‘Banks’ does not contain the minor blast resistance genes Pi-kh or Pi-ks that are present in ‘Drew’, ‘Ahrent’ and ‘Cybonnet’. An increase in leaf blast severity observed in moisture-stress tests using Pi-ta based cultivars suggests additional resistance genes, such the Pi-kh and Pi-ks, enhance Pi-ta gene efficacy against race IE-1k under field conditions. The data suggest that the Pi-ta gene functions as a partial resistance gene in ‘Katy’, ‘Ahrent’, ‘Cybonnet’, ‘Drew’, ‘Banks’, ‘Tetep’ and ‘Tadukan’ in regard to the broadly virulent blast pathogen races IE-1k and IB-33.