| [Image: Figure 1] | | [Image: Figure 2] | | [Image: Figure 3] | | [Image: Figure 4] | | [Image: Panicles] | | [Image: Blight Symptoms] | | [Image: Lafitte Plant] | | [Image: Figure 5] |
M.C. "Chuck" Rush, Q.M. Shao, Shuli Zhang, A.K.M. Shahjahan, Kathy O'Reilly, Ding Shih, Donald Groth and Steven D. Linscombe
Diseases are a major constraint to rice production in Louisiana and the other Gulf of Mexico rice-producing states. They cause millions of dollars in direct losses and losses related to the use of control measures. The most damaging diseases are sheath blight and rice blast, caused by fungal pathogens, and bacterial panicle blight and sheath rot. Several fungicides are available to help control sheath blight and blast, but they are expensive and require superior management to obtain economic control. No pesticides are available for controlling bacterial panicle blight.
Biotechnology-related research is becoming increasingly important in the development of measures to supplement pesticides and conventional breeding efforts for disease control. In a coordinated effort involving the rice pathology laboratory in the Department of Plant Pathology and Crop Physiology, the Department of Biological Sciences, the School of Veterinary Medicine and the Rice Research Station, research is being conducted in the laboratory and field to develop new technologies for disease control.
Improving Conventional Breeding Through ‘Clonal Variation’
Sheath blight is the leading cause of disease loss in Louisiana rice. No source of complete resistance has been identified throughout the world for Rhizoctonia solani, the cause of this disease, but some rice varieties have partial resistance. For many years we have been locating sources of partial resistance and incorporating them into our breeding efforts.
In our search for better sources of partial resistance, we have exploited a technology called clonal variation. Clonal variation comes from the increased rate of mutations found in plants regenerated through tissue culture. Tissue culture is the process of creating new plants from cells in a test tube.
Using tissue culture technology we have developed a new source of high-level partial resistance from a susceptible variety. Two lines having this new resistance were registered as the “elite lines” LSBR-5 and LSBR-33. These lines have been used for several years, along with other natural, high-level sources of partial resistance identified in our program, to generate thousands of breeding lines with sheath blight resistance.
Each year new lines are screened for disease resistance, yielding ability, grain shape, and quality and cooking characteristics. The best materials are advanced to the rice breeding program at the Rice Research Station in Crowley, La.
Transformation Using Foreign Genes
Transformation is a technology where foreign genes, or genes from other plants, animals and microorganisms, can be transferred to a crop plant to introduce a new trait. We are interested in transferring “PR” genes, or genes involved in plant disease resistance, from other plants to rice. The genes are cloned from the source organism and put into plasmid DNA, which is a type of bacterial DNA that can be transferred into the target plant. The procedure we use involves coating small particles of gold with plasmid DNA containing the foreign gene and shooting them with an air-pressure device into cultured rice cells.
Our laboratory began working with rice transformation in the early 1990s. We first transformed rice with the gene for resistance to the antibiotic hygromycin (hpt gene). Since then we have transformed many varieties with a gene for resistance to Liberty herbicide, including the commercial varieties Drew and Cypress (Figure 1).
The PR genes with which we are working include a thionin gene from barley, a chitinase gene from soybean and a beta glucanase gene from tobacco. All of these genes are involved in defense against diseases in the plant from which they were derived. We have transformed rice with the thionin gene and found in preliminary greenhouse tests that it greatly increased resistance to the bacterial panicle blight and sheath rot disease.
We also used a process called cotransformation to transform rice plants with different combinations of the chitinase and beta glucanase genes, which plants use to defend against fungal pathogens, along with the gene for resistance to Liberty herbicide. These plants show a high level of resistance to sheath blight in greenhouse and field tests (Figures 2, 3 and 4). They also show resistance to Liberty herbicide. We still have to cross this material with nontransformed plants to determine whether the sheath blight resistance is stable and if disease resistance is linked to Liberty resistance.
Several years ago, we transformed Taipei 309 and Nipponbare rice with the gene for resistance to Liberty herbicide. These plants were selected because they were easy to use in tissue culture and gave stable resistance. They are not U.S. varieties. For commercial use of transformed plants, single transformation events must be registered by the federal government in a process similar to that used for new pesticides. These registered transformation events can then be used to develop new varieties by conventional breeding. The original transformant would not be released as a variety.
We used our Liberty-resistant transformants in extensive testing to determine how many generations of crossing and backcrossing it would take to transfer the transgene from a single transformation event into a commercial variety. Four to five backcrosses over two and a half years were required to recover stable transformed lines with the characteristics of the variety. These characteristics included appearance, height, grain shape and quality, time-to-maturity and yield potential. These transformed lines are available to the breeding program at the Rice Research Station.
Other research with transformed plants showed that when many transformation events (individual transformed plants with the same transgene) were crossed, insertion was not completely random. This suggests that certain chromosome sites were more likely to have the transgene inserted. By identifying these sites and the factors controlling insertion, we may be able to dictate insertion sites when developing trans-genic plants. This would allow us to avoid the transformation problems associated with insertion in unfavorable locations and benefit from insertion into pre-selected sites that favor gene expression.
Using Biotechnology with Pesticides
Using Cypress rice transformed with the bar gene, which is the gene that creates resistance to Liberty herbicide, we have conducted field tests for several years to determine if Liberty herbicide could be used as a pesticide to control rice diseases. Liberty herbicide is an antibiotic as well as a herbicide. As transgenic rice varieties with Liberty resistance become available to rice growers, it is important to know the effects of Liberty on rice diseases as well as weeds. Also, there is potential to greatly increase the use of Liberty in rice.
We determined the rates and timings for use of Liberty as a fungicide in the field. In comparative field tests conducted for three years, Liberty applied at the correct rate and timing provided significantly better control of sheath blight than Moncut fungicide and was equal to or better than Quadris fungicide. These are the fungicides now used to control sheath blight in Louisiana rice.
Our research demonstrates that Liberty used as a fungicide on transgenic rice will have dramatic effects on diseases. In preliminary field studies Liberty also appeared to reduce bacterial panicle blight. In laboratory tests, the compound was highly inhibitory to the sheath blight, blast and bacterial panicle blight pathogens, as well as several other rice pathogens common in Louisiana rice. The compound has the potential to be a broad spectrum pesticide for controlling both weeds and diseases in rice.
Rice Panicle Blight Pathogen
Rice produced in the U.S. Gulf Coast area has a long history of loss to panicle blighting. The damage in Louisiana was severe in 1995 and 1998, years with record high temperatures. Yield losses in some fields were estimated as high as 40 percent.
In 1996, the cause of this panicle blighting was identified by our laboratory as the seedborne bacterial plant pathogen Burkholderia glumae. Further studies have implicated B. gladioli, B. plantarii and B. cepacia as other potential causal agents of panicle blighting in rice. The relationship of these plant pathogens to bacterial panicle blighting and sheath rot in rice must be determined to develop procedures for identifying commercial seed infected with the causal bacteria.
Rice crops planted with infected seeds will suffer severe losses during summers with unusually high nighttime temperatures. The symptoms of bacterial panicle blight include seedling blight, sheath rot and panicle blight (Figure 5). Research in this project is directed toward developing a method for identifying infected certified seed with high levels of the causal bacterial pathogens. Damage can be avoided by not planting seed with high levels of bacterial infection or by treating infected seed with antibacterial compounds. This will greatly reduce the potential for loss by producers in Louisiana and other Southern rice-producing states.
The goals of our research are to clarify the causal relationships of the Burkholderia species associated with blighted rice, to develop procedures for extracting pathogenic bacteria from infected seeds, to develop monoclonal and polyclonal antibodies specific for B. glumae and B. gladioli and to develop an enzyme-linked immunosorbant assay (ELISA) system for identifying the bacterial pathogens from rice seeds. The sensitivity and specificity of the assay are being determined using an existing collection of rice-field isolates of B. glumae, B. gladioli, B. plantarii and B. cepacia. Development of this technology will help increase rice yields and minimize some of the economic uncertainty related to rice production.
(This article appeared in the fall 2003 issue of Louisiana Agriculture.)
|