INVESTIGATOR: Rush, M. C.

PERFORMING INSTITUTION:
PLANT PATHOLOGY & CROP PHYSIOL
LOUISIANA STATE UNIVERSITY
BATON ROUGE, LOUISIANA 70893

BREEDING, ETIOLOGICAL, EPIDEMIOLOGICAL, AND BIOTECHNOLOGICAL RESEARCH FOR CONTROLLING RICE DISEASES

CLASSIFICATION HEADINGS: R202 . Plant Genetic Resources; S1530 . Rice; F1080 . Genetics; F1160 . Pathology; F1170 . Epidemiology

NON-TECHNICAL SUMMARY: Rice diseases are a major constraint on rice production. The most economic control of rice diseases is disease resistance. Resistance must be found or created and transferred to commercial rice varieties. Rice germplasm with new disease resistances will be provided to the rice breeding program. This project will obtain or develop resistance to major rice diseases and transfer this resistance to germplasm useful to the rice breeding program in the LSU Agricultural Center. It will also develop the elements of etiology and epidemiology needed for utilization of this resistance for disease control in rice.

OBJECTIVES: The general objective of this project is to develop the biological, etiological, epidemiological, genetic, and biotechnological information necessary to control losses due to rice diseases. This objective will be met by identifying or developing new sources of disease resistance, by transfer of resistance to breeding lines for use in the LSU Agricultural Center rice breeding program, by elucidation of mechanisms of resistance, through investigation of the transfer and inheritance of resistance mechanisms, and by developing a better understanding of host-pathogen interactions and other factors impacting on the successful use of disease resistance and other control measures in rice production. Specific objectives are to develop and/or transfer new sources of disease resistance in rice through the use of cell culture, somaculture, in vitro cell selection, rice organ culture, and transformation; development of in vitro screening methods for selecting resistance to pathogens, pathogen toxins, and other toxic constraints to rice; modification of rice for resistance to disease, pathogen toxins, or other biological constraints by transformation from plasmids with known gene structure or using DNA from plants with non-host resistance to these constraints; locating and maintaining sources of natural disease resistance in Oryza sativa L., Oryza species, and related graminaceous species for use in rice improvement; identification of mechanisms of disease resistance in rice to major pathogens, to develop information on mode-of-action of mechanisms, and to study the inheritance of genes controlling resistance mechanisms; development of rice lines with desirable levels of disease resistance using conventional breeding methods and to combine this resistance in germplasm with desirable plant type and yield characteristics for use as elite lines in the Louisiana Agricultural Experiment Station rice breeding program conducted at the LSU Agricultural Center Rice Research Station at Crowley, LA.; and development of biotechnological methods for identifying and quantifying pathogens in diseased tissues and seeds.

APPROACH: New sources of disease resistance will be developed and transferred to rice lines through the use of mutation, cell culture, somaculture, in vitro cell selection, and transformation. Sources of natural resistance will be located, conserved, and transferred to germplasm for use by the rice breeding program. Tissue culture, transformation, and in vitro selection techniques will be developed as necessary to carry out this research. Mechanisms of disease resistance in rice will be elaborated as necessary to increase the efficiency of transfer of these resistances to rice. Biotechnological methods for identifying pathogens in diseased rice tissues and seeds will be developed and applied as required to enhance these research efforts. These methods, such as PCR, Real-Time PCR, and ELISA, will be further developed for use by commercial rice growers.

KEYWORDS: rice diseases; rice transformation; disease control in rice; rice tissue culture; clonal variation; rice breeding; mutations for disease control; disease diagnosis in rice; elisa; pcr and real-time pcr

PROGRESS: 2007/01 TO 2007/12
OUTPUTS: A total of 4600 progeny rows from our crosses and control rows were planted and inoculated with the rice pathogens Rhizoctonia solani and/or Burkholderia glumae in 2007 for various disease resistance evaluations; including rice blast, sheath blight (SB), and bacterial panicle blight (BPB). Eighty six advanced SB resistant lines selected in 2006 were planted in harvest-size plots to obtain grain quality and yield data in the joint breeding/pathology yield test in comparison to five commercial varieties Bengal, Cocodrie, Trenasse, Jupiter and Wells. The experimental lines were grown in yield plots using standard rice production practices and a split plot design with two replicates. In each replicate, one plot (split) was inoculated with R. solani and one plot was not inoculated (healthy rice) for determination of the effect of SB infection on yield. The lines and varieties were rated for SB development at maturity using the standard 0-9 rating scale and agronomic characteristics were evaluated. Three hundred and three progeny rows in the F5 generation, selected from crosses among and between lines and varieties discovered by our laboratory to have high levels of partial resistance to BPB and the susceptible variety Cocodrie, were grown in the field in 2007, inoculated with B. glumae, and reselected for disease resistance (BPB and sheath blight) and desirable agronomic characteristics. Studies were initiated in 2007 to develop a method for treating the water used to pre-sprout rice seeds before water-seeding with chemicals that would kill the seedborne B. glumae and B. gladioli pathogens on the seed. Antibacterial compounds, such as, Starner, acids (acetic acid, lactic acid), and copper compounds (Kocide 2000, Copper chloride, Copper sulfate) were used at various concentrations in laboratory and field tests to determine treatment rates that would not be phytotoxic to rice seedlings and which would kill the bacterial pathogens in seeds. In 2007 we began a study of gene expression analysis in the variety Jupiter, which we have shown to have a high level of partial resistance to BPB. This research was to identify resistance-related genes at the molecular level using microarray, with the rice Affymetrix GeneChip, to contrast partially resistant and susceptible rice cultivars before and after inoculation with the BPB pathogen B. glumae. Also, pathogenic B. glumae strains produce a toxin, toxoflavin, which is the main pathogenicity factor for this pathogen. The intent of another project in 2007 was to develop toxoflavin minus mutants of B. glumae and test them for their ability to cause disease under greenhouse and field conditions. This was to determine if other pathogenicity systems can operate when toxoflavin is not produced by the pathogen. Results from our studies are published in refereed scientific journals, trade journals, experiment station reports and publications, and newspapers. PARTICIPANTS: Participants in this project included the Principal Investigator/Project Leader Dr. Milton C. Rush, Professor; Postdoctoral Researcher Dr. R. Nandakumar, and Project Technician Patricia Bollich. Dr. Donald E. Groth, Professor; was our cooperator and Field Coordinator at the Louisiana State University Agricultural Center Rice Research Station in Crowley, Louisiana. The project also had two student workers hired throughout 2007. The Partner Organization on this project was the LSU Agricultural Center Rice Research Station in Crowley, Louisiana. All field research was conducted at this Station in 2007. Funding for this project was provided by two grants from the Louisiana Rice Research Board, which is funded by the Louisiana rice industry. TARGET AUDIENCES: Target audiences for this project include rice producers in Louisiana and throughout the southern United States, students and faculty at Louisiana State University, and rice producers and scientists throughout the world.

IMPACT: 2007/01 TO 2007/12
Sixteen experimental rice lines yielded as high as or higher than the control varieties and these lines had agronomic characteristics similar to commercial long-grain varieties as well as SB resistance. Seventy SB progeny rows were harvested for a yield test in 2008. Rows with a high level of partial resistance to BPB and low SB ratings ranging were harvested for yield testing in 2008. These lines will be tested in yield-sized plots for disease resistance, yield potential, milling, grain quality characteristics, height, days-to-heading and other agronomic characteristics in 2008. The resistant parents were lines LR 2065 (MCR00-6145), AB647, LM-1, and the variety Nipponbare. In the BPB control test using pre-sprouting of seed, the Starner, acid, and copper treatments killed pathogenic bacteria in the seeds and increase yield over untreated seeds up to 1300lb/A. It was clear from the laboratory and two field tests in 2007 that it is possible to significantly decrease the amount of Burkholderia glumae in infected seeds by pre-sprouting seed in solutions of lactic and acetic acids, CloroxTM, and Copper solutions and to see these results reflected in increases in yield. This test indicated a high level of probability that the seedborne pathogens causing BPB can be controlled by pre-sprouting seed for water-seeding in water solutions of antimicrobial compounds. Disease resistance is due to the expression of certain genes during plant-plant pathogen interactions. Identification of these genes and their products, through use of genomic methods, will help elaborate the basis for high-level partial resistance either directly or indirectly. Results to date indicate that the medium grain rice variety Jupiter exhibits significant partial resistance with average disease ratings of 3.0 on a 0-9 scale, where susceptible varieties had ratings ranging from 6 to 9 in field trials. A method for extracting total RNA from rice panicles was standardized. Microarray analysis with panicle samples collected from Trenasse and Jupiter 2 days before and after inoculation with the BPB pathogen indicated that the seed development protein gene clusters (glutelin, prolamin and globulin), defense related genes, especially defensin, and several genes involved in the signal transduction process, were over-expressed several fold in Jupiter rice in response to inoculation with the pathogen. Production of the seed development genes was totally shut down in Trenasse leading to seed sterility or abortion. The four toxin minus mutants we developed were still capable of causing panicle blight symptoms (disease ratings 2 to 4 on 0-9 scale) under greenhouse and field conditions. However, the mutants were less virulent than wild type strain (ratings 8-9) in causing BPB symptoms. These results suggest that other pathogenicity systems in B. glumae can still cause disease in rice when the gene for toxoflavin is removed.

PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Nandakumar, R., Bollich, P.A., Groth, D.E and Rush, M.C. 2007. Confirmation of the partial resistance of Jupiter rice to bacterial panicle blight caused by Burkholderia glumae through reduced disease and yield loss in inoculated field tests. Phytopthology 97(S):82-83.
2. Rush, M.C., Nandakumar, R., Sha, X.Y., Groth, D.E. and S.D. Linscombe. 2007. Partial resistance to bacterial panicle blight in Jupiter rice. Louisiana Agriculture, Pp. 22-23.
3. Nandakumar, R. and Rush, M.C. 2007. Association of Burkholderia glumae and B. gladioli with panicle blight symptoms on rice in Panama. Plant Disease 91:767. Nandakumar, R., Yu,T.-W., and Rush, M.C. 2007. Burkholderia glumae toxoflavin biosynthesis gene cluster, complete sequence. Accession #: EF587764.
4. Nandakumar, R., Rush, M.C., Shahjahan, A.K.M. 2006. PCR detection of the bacterialpanicle blight pathogens. In. LSU-Agcenter-Rice Research Station-98th Annual Research Report. Pp.343-344.
5. Groth, D., Rush, M. C., and Lindberg, D. 2007. Blast in rice: researchers detect resistance to fungicides. Louisiana Agriculture, P. 21.

PROGRESS: 2006/01/01 TO 2006/12/31
Progeny rows (6000) were planted and inoculated with Rhizoctonia solani or Burkholderia glumae in 2006 for disease resistance evaluations; including rice blast, sheath blight (SB), and bacterial panicle blight (BPB). Field evaluations of germplasm for SB and blast resistance included 5100 F4-F9 progeny rows and 800 rows for evaluation of transgenic lines. Ninety-six advanced SB resistant lines were selected in 2005 to obtain data on grain quality, agronomic characteristics (height, days to maturity, lodging), and yield data in a in 2006 yield test that included the check varieties Bengal, Cheniere, Cocodrie, Francis, Pirogue, Trenasse, Jupiter and Wells. Lines were grown using a split plot design with two replicates, with one split in each replicate inoculated with R. solani. Twenty-five lines out-yielded all check varieties in the non-inoculated plots with yields up to 10,000 lb/A. Sixteen inoculated lines out-yielded all check varieties with yields up to 9,000 lb/A. Three lines will be recommended for inclusion by the breeding program in the 2007 Uniform Rice Yield Nursery. A second yield test included 36 lines, and seven check varieties. These lines yielded more in the 2003, 2004, and 2005 yield tests when inoculated than when non-inoculated and may be expressing tolerance to SB. The test had split plots, inoculated and non-inoculated, with three replicates. In this test several inoculated lines yielded the same as non-inoculated plots, or higher, and may be expressing true tolerance. Bacterial panicle blight is a continuing problem in rice in Louisiana. Three inoculated BPB yield loss tests were planted in 2006. Early and late plantings were made to determine the effect of panicle blight on yield potential of Bengal, Frances, Trenasse, Cocodrie and Jupiter rice under field conditions. The June planting of this test also provided tissue samples for a microarray study of genes contributing to partial resistance Among the varieties tested, Trenasse (rating = 8.8), Frances (rating = 8.5), Cocodrie (rating = 7.5) and Bengal (rating = 7.3) were very susceptible, whereas, Jupiter (rating = 3.3) was significantly resistant. There was a non-significant difference, 532 lb/A (8.0 %) loss, between the inoculated and non-inoculated plots of Jupiter, while B. glumae inoculation caused significant yield losses in Trenasse (45%), Cocodrie (35%), Frances (35%), and Bengal (34%). The second planting had similar results. A test was planted to determine the virulence of soilborne B. gladioli isolates when compared to B. glumae and B. gladioli strains from infected rice panicles. Plots were rated for BPB at maturity (0-9 scale). Four soil isolates of B. gladioli (S10, 3S5 and S15) caused significant panicle blighting when compared to non-inoculated plots while a seedborne B. gladioli strain and another soil isolate, 3S4, did not cause significant panicle blighting. None of the B. gladioli isolates caused infections as severe on Trenasse (rating = 4.0) or Bengal (rating = 4.0) as that caused by B. glumae (rating = 8.7). Inoculation with B. gladioli soil isolates did not give significant yield losses, but inoculation with B. glumae gave a significant loss.

IMPACT: 2006/01/01 TO 2006/12/31
Sheath blight (SB) is the most serious rice disease in Louisiana and throughout the southern rice producing areas because of its endemic, and occasionally epidemic, development each year resulting in significant yield loss. The use of SB resistant cultivars, if available, would be the best way to control this disease. Thus far no completely resistant cultivars have been identified for this disease worldwide. However, our program for breeding for sheath blight resistance has created and identified several hundred breeding lines with a useful level of partial resistance. Many of these lines have desirable agronomic characteristics coupled with high yield potential and the grain quality characteristics required by the rice industry. Every year the best of these lines are advanced to the LSU Agricultural Center Rice Breeding program conducted at the Rice Research Station in Crowley, LA. This research is leading to the development of SB resistant cultivars for Louisiana, which will mean millions of dollars saved in the rice industry through reduction in yield losses and reduced use of pesticides. A similar program has been carried out for developing resistance to bacterial panicle blight, one of the top three diseases in Louisiana rice. Resistance sources have been identified and lines with resistance are being developed. This resistance will be combined with the genes for partial resistance to SB with the goal of developing varieties resistant to both diseases.

PUBLICATIONS: 2006/01/01 TO 2006/12/31
1. Linscombe, S.D., X. Sha, J.A. Bond, K. Bearb, M.C. Rush, Q.R. Chu, D.E. Groth, L.M. White, and R.T. Dunand. 2006. Registration of Trenasse rice. Crop Science 46:2318.
2. Shao, Q.M., M.C. Rush, M.S. Wu, D.E. Groth, M.S. Kang, and S.D. Linscombe. 2006. Transgene transfer to United States commercial cultivars via conventional breeding techniques. Journ. Crop Improvement 16:49-65.
3. Shao, Q., M.C. Rush, S.D. Linscombe, D.E. Groth, and M.S. Kang. 2006. Allelic Analysis of bar Gene from Different Rice Transformation Events. Journal of Crop Improvement. 15:(1)33-49.
4. Chu, Q.R., S.D. Linscombe, M.C. Rush, D.E. Groth, J. Oard, X. Sha, and H.S. Utomo. 2006. Registration of a C/M Doubled Haploid Mapping Population of Rice. Crop Science 46:1417
5. Nandakumar, R., M. C. Rush, A.K.M. Shahjahan, K. O'Reilly and D.E, Groth. 2006. Detection methods for bacterial panicle blight of rice caused by Burkholderia glumae and B. gladioli. p.96. Proceedings 31st RTWG, The Woodlands, TX, Feb 26-Mar 1, 2006.
6. Rush, M.C., R. Nandakumar, S. Zhang and D.E, Groth. 2006. Status of our research on developing high levels of partial resistance to sheath blight in lines with high yield potential and good agronomic characteristics. p.91. Proceedings 31st RTWG, The Woodlands, TX, Feb 26-Mar 1, 2006.
7. Groth, D.E., M.C. Rush, and G.D. Lindberg. 2006 Possible development of resistance by Pyricularia gresea to fungicides. p.92. Proceedings 31st RTWG, The Woodlands, TX, Feb.26-Mar.1, 2006.
8. Groth, D.E., M.C. Rush, and A.K.M. Shahjahan. 2006. Strategies for screening multiple rice diseases. p.106. Proceedings 31st RTWG, The Woodlands, TX, Feb.26-Mar.1, 2006.
9. Nandakumar, R., M.C. Rush, and A.K.M. Shahjahan. 2006. Accession #: EF088208-EF088215 (2006). Soil borne Burkholderia gladioli strains (S10, S12, S9, 3S4, 3S5), both 16S ribosomal RNA gene and 16S-23S ribosomal RNA Intergenic spacer, partial sequence. Genbank.
10. Nandakumar, R., M.C. Rush, and A.K.M. Shahjahan. 2006. Accession #: DQ355164 - DQ355167 (2006). Burkholderia glumae strains (336gr-1, 398gr-3, 189gr-8, 99gr-4b) 16S ribosomal RNA gene, Partial sequence. Genbank.
11. Nandakumar, R., M.C. Rush, and A.K.M. Shahjahan. 2006. Accession #: DQ355168 - DQ355169 (2006). Burkholderia gladioli strains (223gr-1, 321gr-6) 16S ribosomal RNA gene, Partial sequence. Genbank.

PROJECT CONTACT:

Name: Rush, M. C.
Phone: 225-578-1393
Fax: 225-578-1415
Email: mrush@agcenter.lsu.edu