Twng Wah Mew

Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease

Abstract A 1.1-kb DNA fragment containing the coding region of a thaumatin-like protein (TLP-D34), a member of the PR-5 group, was cloned into the rice transformation vector pGL2, under the control of the CaMV 35S promoter. The Indica rice cultivars, ‘Chinsurah Boro II’, ‘IR72’, and ‘IR51500’ were transformed with the tlp gene construct by PEG-mediated direct gene transfer to protoplasts and by biolistic transformation using immature embryos. The presence of the chimeric gene in T0, T1, and T2 transgenic plants was detected by Southern blot analysis. The presence of the expected 23-kDa TLP in transgenic plants was confirmed by Western blot analysis and by staining with Coomassie Brilliant Blue. Bioassays of transgenic plants challenged with the sheath blight pathogen, Rhizoctonia solani, indicated that over-expression of TLP resulted in enhanced resistance compared to control plants.

Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars.

Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

Tagging and combining bacterial blight resistance genes in rice using RAPD and RFLP markers

Four genes of rice,Oryza sativa L., conditioning resistance to the bacterial blight pathogenXanthomonas oryzae pv.oryzae (X. o. pv.oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed betweenxa-5 and RFLP marker lociRZ390, RG556 orRG207 on chromosome 5.Xa-3 andXa-4 were linked to RFLP locusXNpb181 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker toXa-10, also located on chromosome 11, was the RAPD locusO072000 at a distance of 5.3 cM. From this study, the conventional map [19, 28] and two RFLP linkage maps of chromosome 11 [14, 26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes,Xa-4 +xa-5 andXa-4 +Xa-10. Lines carryingXa-4 +xa-5 andXa-4 +Xa-10 were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs of genes were resistant to more of the isolates than their single-gene parental lines. Lines carryingXa-4 +xa-5 were more resistant to isolates of race 4 than were either of the parental lines (‘quantitative complementation’). No such effects were seen forXa-4 +Xa-10. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.

Transgenic rice variety 'IR72' with Xa21 is resistant to bacterial blight

Abstract An elite indica rice variety, ‘IR72’, was transformed with a cloned gene, Xa21, through particle bombardment. Molecular analysis of transgenic plants revealed the presence of a 3.8-kb EcoRV-digested DNA fragment corresponding to most of the Xa21 coding region and its complete intron sequence, indicating the integration of Xa21 into the genome of ‘IR72’. In the T1 generation, the transgene was inherited and segregated in a 3:1 ratio. After inoculation with the prevalent races 4 and 6 of Xanthomonas oryzae pv. oryzae (Xoo), T1 plants positive for the transgene were found to be resistant to bacterial blight (BB). We also observed that the level of resistance to race 4 of Xoo was higher due to the pyramiding of Xa21 and Xa4 present in ‘IR72’. Since the inactivation of the transgene Xa21 occurred in the two transgenic T1 plants, a larger progeny should be obtained for selecting homozygous line with a consistently higher level of resistance to the BB pathogen.

Using Genetic Diversity to Achieve Sustainable Rice Disease Management

Host plant resistance is an important tool for rice disease control and has played a key role in sustaining rice productivity, especially in tropical Asia. Deploying resistant varieties as a means of disease control is attractive because it requires no additional cost to farmers and is environmentally safe (62). Furthermore, resistant varieties can be easily disseminated as seeds, leading to wide adoption (12). These are important considerations, because for resource-poor rice farmers in developing countries, the options for managing diseases are few. For example, during the 1970s and 1980s, when epidemics of rice tungro were frequent in the Philippines and Indonesia, farmers expressed more confidence in using resistant varieties than in other control measures. Disease control using chemicals is more common in the temperate or subtropical production environments where farmers apply fungicides for controlling blast (caused by Pyricularia grisea) and sheath blight (caused by Rhizoctonia solani). Despite regional differences in control measures, planting resistant varieties is considered most effective by rice farmers. Hence, breeding for disease resistance has been a major objective in rice improvement programs conducted at international agricultural research centers, such as the International Rice Research Institute (IRRI), and at the national agricultural research systems (NARS) of developing countries. There are limitations, however, in using resistant varieties alone to manage rice diseases. Most varieties are resistant only to a few major diseases that are the subjects of intensive breeding efforts. The rice production environments, particularly in the tropics, are habitats of many rice pathogens causing varying degrees of damage. Even the “minor” diseases collectively could pose a significant threat to production (63). Thus, pathologists and breeders have to deal with yield loss caused by diseases of epidemic and endemic nature. Epidemic loss is dramatic but less frequent, whereas endemic loss is less obvious but pervasive in each cropping season. Recent surveys indicated that an estimated annual yield loss from 1 to 10% was due to a combination of different diseases (80). Thus, resistance against a few targeted diseases offers only a partial solution to rice disease problems. To those diseases caused by nonspecialized pathogens, such as sheath blight and false smut (caused by Ustilaginoidea virens), no useful source of resistance has been identified to improve the resistance of rice varieties. To achieve sustainability of rice production in Asia, we need a rice production system built upon effective resistant varieties with broad resilience to a range of diseases and insect pests. Broad-spectrum resistance at the genotypic level and sustainability at the cropping systems level are therefore complementary approaches in managing rice diseases. Although considerable progress has been made over the past decades, much more can be done to integrate these two approaches to achieve results in farmers’ fields. Modern agricultural development has transformed the diverse, traditional rice production system into a monoculture system that relies only on a few fertilizer-responsive and high-yielding varieties. Farmers’ preference to high yield has led to wide adoption of modern rice varieties cultivated in millions of hectares of rice land. Although most modern varieties have built-in resistance against multiple diseases, genetic uniformity inevitably predisposes the system to disease epidemics, and under certain circumstances can lead to serious yield loss caused by diseases and insect pests (43). Varieties carrying a few resistance genes in a uniform genetic background are vulnerable to rapid adaptation of pathogens and pose uncertainty to farmers. For instance, emergence of new pathogen races caused several blast epidemics in Korea in the 1970s, leading to yield losses of 30 to 40% (38). In the 1980s, other disease outbreaks on a regional scale included epidemics of bacterial blight in northern India and Southeast Asia, tungro in Southeast Asia, and bacterial blight and blast in Japan (38,61,89). Another impact of the monoculture system is the gradual decline in the diversity of varieties grown by farmers. As modern high-yielding varieties expand to millions of hectares, they also replace the traditional varieties. Although useful genes from these traditional varieties are being used in breeding for modern varieties, many unique attributes and gene combinations resulting from years of selection are difficult to reconstitute. To achieve the productivity needed, it is not possible to revert to planting diverse traditional varieties that are poor yielding. However, it is within our capacity to work toward disease management methods that sustain productivity yet maintain adequate diversity and resilience in the production systems. In the past two decades, IRRI has moved toward increasing genetic diversity of modern rice varieties through resistance breeding (12,39,43) and deployment of different resistance genes based on an unCorresponding author: Twng Wah Mew, Entomology and Plant Pathology Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines; E-mail: T.Mew@cgiar.org

Looking Ahead in Rice Disease Research and Management

Rice production is subject to increasing environmental and social constraints. Agricultural labor and water, which are key resources for rice production, illustrate this point. Nearly all rice-producing countries face reduced availability of agricultural water and shortage of farm labor. Plant pathologists should be concerned with such large-scale evolutions because these global drivers have an impact on not only the rice production system but also on the individual field and single-rice-plant levels. These concerns are closely associated with the long-term sustainability and environmental consequences of the intensification of agricultural systems brought about by problems of feeding a rapidly growing human population. Furthermore, genetic diversity in rice production has been reduced, thus inducing frequent disease epidemics and pest outbreaks. Looking ahead, we need to realize the need to maintain the diversity and yet retain the high productivity of the system. Natural resources, including genetic resources, are not infinitely abundant. We have to be efficient in utilizing genetic resources to develop durable resistance to rice diseases. Developing resistance is an important first step in tackling the disease problem, but it is not the only step available to achieve durability. Deployment of resistance must be considered in conjunction with development of host plant resistance. To attain durability, we need a better understanding of the coevolution process between the pathogen and the host resistance gene. Our target is an integrated gene management approach for better disease control and more effective utilization of genetic resources. Plant pathology, as an applied science, derives its strengths from various disciplines. To do the job right, we need a better understanding of the pathosystems, the epidemiology, and the coevolution process between the pathogen and the host resistance gene. The challenge, as pointed out by pioneers in our profession, is to prove the usefulness and the relevance of our research. Thus, we need to strike a balance between mission-oriented and fundamental research and make sure that our profession is (still) useful in the information technology and genomic era. We believe that a gene-based and a resource-based disease management approach should allow us to incorporate these new scientific developments. However, we do need to incorporate the new science for fundamental research to solve practical problems of rice production.

Panicle Blast and Canopy Moisture in Rice Cultivar Mixtures

ABSTRACT Glutinous rice cultivars were sown after every fourth row of a nonglutinous, hybrid cultivar in an additive design. The glutinous cultivars were 35 to 40 cm taller and substantially more susceptible to blast than was the nonglutinous cultivar. Interplanting of glutinous and nonglutinous rice reduced the incidence and severity of panicle blast on the glutinous cultivars by >90%, and on the nonglutinous cultivar by 30 to 40%. Mixing increased the per unit area yield of glutinous rice by 80 to 90% relative to pure stand, whereas yield of the nonglutinous cultivar was essentially unaffected by mixing. To determine whether the different plant heights and canopy structures may contribute to a microclimate that is less favorable to blast infection, we monitored the moisture status of the glutinous cultivars in pure stand and mixture at 0800 h by measuring relative humidity at the height of the glutinous panicles using a swing psychrometer and by visually estimating the percentage of leaf area covered by dew. Averaged over the two seasons, the number of days of 100% humidity at 0800 h was 20.0 and 2.2 for pure stands and mixtures, respectively. The mean percentage of glutinous leaf area covered by dewwas 84 and 36% for the pure stands and mixtures, respectively. Although other mechanisms also were operative, reduced leaf wetness was likely a substantial contributor to panicle blast control in the mixtures.

Bacterial diseases of rice. II. Characterization of pathogenic bacteria associated with sheath rot complex and grain discoloration of rice in the Philippines

From over 5,600 bacteria isolated from rice plants with sheath rot complex and grain discoloration syndrome, and two batches of 1 kg of rice seed (cultivars IR54 and IR8866), 204 pathogens were initially characterized by phenotypic tests, serology, and growth on selective media, and further distinguished by API 20NE, Biolog, and cellular fatty acid methyl esterfingerprints. The best differentiation was obtained by the Biolog system. The nonfluorescent pathogens were represented by clusters D1 (Burkholderia glumae, formerly Pseudomonas glumae) and E (Acidovorax avenae subsp. avenae, formerly Pseudomonas avenae). Seven clusters were distinguished among the fluorescent strains associated with sheath rot complex and grain discoloration. Cluster A5 was identified as Pseudomonas aeruginosa, and cluster B1 as P. fuscovaginae. Cluster B2 is related to Pseudomonas aureofaciens, P. corrugata, P. fluorescens, and P. marginalis. Clusters B1 and B2 were only slightly different. The strains identified as P. fuscovaginae were different from the type strains in 2-ketogluconate production.

Applying Rice Seed-Associated Antagonistic Bacteria to Manage Rice Sheath Blight in Developing Countries

One promising area of disease management for resource-poor farmers that emerged in recent years in developing countries is the potential of biological control. Biological control agents (BCAs) were found to be ubiquitous in the rice ecosystem. Seed bacterization with BCAs appeared to promote plant growth. BCAs showed efficacy on sheath blight (Rhizoctonia solani AG 1) but produced inconsistent results over time in the field using it alone. The control efficiency ranged from 50 to over 90%, with a high variance. To improve the efficacy, a half-dose of a commonly used fungicide, like Jingangmycin in China and Validamycin in Vietnam, was introduced to mix with BCAs and was found to be effective, and it reduced the variance of the field performance tests. To scale up the BCA technology for resource-poor farmers, a participatory approach, engaging the farmers to evaluate the product, was initiated in China and Vietnam. The BCA strain that is indigenous at a locality is mass produced at the research institution based on the total area required for application, as relayed by the farmers to extension workers. The demand by farmers would serve as the basis for the amount to be produced and for delivery to the rice farmers who were participating in the trials and, later, to those farmers who ordered the product. This process alleviates BCA storage and shelf-life problems. Data from the field performance trials also were used by the researchers to apply for registration for commercial use of BCAs. Scaling up to extend the BCA technology to more rice farmers as an integral part of their pest management scheme, in particular, and crop management practices, in general, is foreseen in the near future.

Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control.

Aims:  To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria.