Youyong Zhu

Genetic diversity and disease control in rice

Crop heterogeneity is a possible solution to the vulnerability of monocultured crops to disease. Both theory and observation indicate that genetic heterogeneity provides greater disease suppression when used over large areas, though experimental data are lacking. Here we report a unique cooperation among farmers, researchers and extension personnel in Yunnan Province, China—genetically diversified rice crops were planted in all the rice fields in five townships in 1998 and ten townships in 1999. Control plots of monocultured crops allowed us to calculate the effect of diversity on the severity of rice blast, the major disease of rice. Disease-susceptible rice varieties planted in mixtures with resistant varieties had 89% greater yield and blast was 94% less severe than when they were grown in monoculture. The experiment was so successful that fungicidal sprays were no longer applied by the end of the two-year programme. Our results support the view that intraspecific crop diversification provides an ecological approach to disease control that can be highly effective over a large area and contribute to the sustainability of crop production.

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

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.

Crop diversity for yield increase.

Traditional farming practices suggest that cultivation of a mixture of crop species in the same field through temporal and spatial management may be advantageous in boosting yields and preventing disease, but evidence from large-scale field testing is limited. Increasing crop diversity through intercropping addresses the problem of increasing land utilization and crop productivity. In collaboration with farmers and extension personnel, we tested intercropping of tobacco, maize, sugarcane, potato, wheat and broad bean – either by relay cropping or by mixing crop species based on differences in their heights, and practiced these patterns on 15,302 hectares in ten counties in Yunnan Province, China. The results of observation plots within these areas showed that some combinations increased crop yields for the same season between 33.2 and 84.7% and reached a land equivalent ratio (LER) of between 1.31 and 1.84. This approach can be easily applied in developing countries, which is crucial in face of dwindling arable land and increasing food demand.

Abundant Within-varietal Genetic Diversity in Rice Germplasm from Yunnan Province of China Revealed by SSR Fingerprints

In order to estimate genetic diversity of rice (Oryza sativa L.) germplasm in Yunnan Province of China, 60 varieties from different regions were analyzed by microsatellite (SSR) fingerprints. Nine selected SSR primer pairs amplified a total of 55 alleles from these varieties, and high genetic diversity (0.706) was found, although it was not evenly distributed across the regions. Marked genetic variation was detected within the traditional varieties. A UPGMA dendrogram based on SSR polymorphism indicated a great variation among the rice varieties, with coefficients ranging between 0.229 and 1.000. The formation of the rice diversity pattern in Yunnan is associated with natural conditions and especially with diverse cultural demands and farming styles. Strategic conservation of rice germplasm in Yunnan is important, and this could be implemented by collecting varieties across geographic regions with sufficient individuals within the same varieties. Effective rice conservation should also consider cultural aspects during collection.

Autotoxic ginsenosides in the rhizosphere contribute to the replant failure of Panax notoginseng.

Background and Aims Sanqi ginseng (Panax notoginseng) growth is often hampered by replant failure. In this study, we aimed to examine the role of autotoxicity in Sanqi replant failures and assess the role of ginsenosides in autotoxicity. Methods The autotoxicities were measured using seedling emergence bioassays and root cell vigor staining. The ginsenosides in the roots, soils, and root exudates were identified with HPLC-MS. Results The seedling emergence and survival rate decreased significantly with the continuous number of planting years from one to three years. The root exudates, root extracts, and extracts from consecutively cultivated soils also showed significant autotoxicity against seedling emergence and growth. Ginsenosides, including R1, Rg1, Re, Rb1, Rb3, Rg2, and Rd, were identified in the roots and consecutively cultivated soil. The ginsenosides, Rg1, Re, Rg2, and Rd, were identified in the root exudates. Furthermore, the ginsenosides, R1, Rg1, Re, Rg2, and Rd, caused autotoxicity against seedling emergence and growth and root cell vigor at a concentration of 1.0 µg/mL. Conclusion Our results demonstrated that autotoxicity results in replant failure of Sanqi ginseng. While Sanqi ginseng consecutively cultivated, some ginsenosides can accumulate in rhizosphere soils through root exudates or root decomposition, which impedes seedling emergence and growth.

Genome-wide analysis of microsatellite sequence in seven filamentous fungi

Abundance of microsatellites with repeated unit lengths of 1-6 base pairs in seven fungi: Aspergillus nidulans, Coprinus cinereus, Cryptococcus neoformans (serotype A), Fusarium graminearum, Magnaporthe grisea, Neurospora crassa and Ustilago maydis were investigated on genomic scale. The results showed that each species has its specific profile for different types and different motifs of SSR loci. Ascomycetes fungi M. grisea, N. crassa and basidiomycete fungus U. maydis adopt much more microsatellites than other fungi examined. Total amount of 15,751, 14,788 and 6,854 SSR loci were observed respectively, average density is 406, 389 and 347 per Mbp sequence; overall length of SSR sequence was 0.82%, 0.95% and 0.79% of genomic sequence respectively. While ascomycetes fungus F. graminearum and A. nidulans contains the least SSRs in the genomic DNA, only 4,679 and 4,837 tracts were observed in 36 Mb and 30 Mb genomic sequence respectively. Microsatellite repeats in protein coding regions are investigated in Aspergillus nidulans, Magnaporthe grisea, and Neurospora crassa also, the results show that the difference of different types and motifs among three fungi is very little than that in whole genomic sequence. For trinucleotide repeats, overrepresent (comparing to the total base pair of protein coding region) of AGC, GGC, AGG, ACG and ACC was observed in coding region, frequencies of AAC and AAG were not difference between coding and non-coding region, AAT, AGT and ATG were underrepresent in coding region excepted for A. nidulans, in which ATG was overrepresentative.

The Bsister MADS gene FST determines ovule patterning and development of the zygotic embryo and endosperm.

Many homeotic MADS-box genes have been identified as controllers of the floral transition and floral development. However, information regarding Bsister (Bs)-function genes in monocots is still limited. Here, we describe the functional characterization of a Bs-group MADS-box gene FEMALE-STERILE (FST), whose frame-shift mutation (fst) results in abnormal ovules and the complete abortion of zygotic embryos and endosperms in rice. Anatomical analysis showed that the defective development in the fst mutant exclusively occurred in sporophytic tissues including integuments, fertilized proembryos and endosperms. Analyses of the spatio-temporal expression pattern revealed that the prominent FST gene products accumulated in the inner integument, nucellar cell of the micropylar side, apical and base of the proembryos and free endosperm nuclei. Microarray and gene ontology analysis unraveled substantial changes in the expression level of many genes in the fst mutant ovules and seeds, with a subset of genes involved in several developmental and hormonal pathways appearing to be down-regulated. Using both forward and reverse genetics approaches, we demonstrated that rice FST plays indispensable roles and multiple functions during ovule and early seed development. These findings support a novel function for the Bs-group MADS-box genes in plants.

First report of pomegranate wilt caused by Ceratocystis fimbriata in Yunnan, China.

Pomegranate (Punica granatum Linn.) is an important fruit crop in Yunnan Province, China. Recently, older pomegranate bushes in Mengzi County, Yunnan began dying. Initial symptoms were yellowing and wilting of leaves on one to several branches, followed by sudden death of the bush within 3 to 4 weeks. Roots of diseased bushes appeared brown to black, and irregularly shaped lesions were observed when the bark was removed. A species of Ceratocystis was consistently isolated from discolored roots, stem, and branch tissues from wilted bushes on potato dextrose agar (PDA) and was identified as Ceratocystis fimbriata Ellis & Halst, based on the morphology of perithecia, ascospores, conidia, and conidiophores. Perithecia were black with a globose base (130 to 300 μm) and a long neck (450 to 800 μm). Ascospores exuded from the apex of the perithecium neck in a long coil and were small, hyaline, and hat-shaped (3.8 to 5.0 μm long × 2.3 to 4.0 μm wide). Conidiophores were septate and hyaline to dark greenish brown. Hyaline conidia, 8 to 17 μm long × 6 to 15 μm wide, were usually produced in chains of 10 or more. Thick-walled endoconidia were globose to oval, olive brown, and 8 to 20 μm in diameter. Because of the increasing occurrence of the disease, surveys of the main pomegranate production areas in Mengzi County, including Xinan, Duofale, Caoba, Hongzhai, and Shilipu townships, were conducted from 10 to 20 August 2002. The disease was detected in 17 of 50 plantings surveyed. Disease was more severe in older plantings than in younger plantings. Disease incidence was 1% in 1- to 5-year-old bushes, 3.6% in 6- to 10-year-old bushes, and 6% in bushes more than 10 years old. Scolytid beetles were occasionally found on bushes, but we were unable to isolate the fungus from them as has been reported (1). Inoculations with an isolate of C. fimbrata were made by inserting mycelium with perithecia from 12-day-old cultures growing on PDA into root wounds made with a sterile scalpel on five pomegranate plants and then covering the wounds with Parafilm. Sterile medium was placed in an equal number of wounded bushes to serve as controls. Fourteen days later, symptoms began to appear in two bushes, and 5 days later, all bushes exhibited symptoms. No symptoms were observed on control bushes. The first visible symptom was a small area of blackened tissue near the point of inoculation. Lesions expanded slowly, but they expanded more rapidly upward than downward. The fungus was reisolated on PDA from roots of all artificially inoculated bushes. C. fimbriata has been previously reported as the cause of pomegranate wilt in India (2); however, to our knowledge, this is the first report of C. fimbriata on pomegranate in China. Because environmental conditions which favor the pathogen (temperatures ranging from 18 to 30°C and frequent rains) typically occur in many areas during late spring and summer, the disease has the potential to seriously impact pomegranate production in China. References: (1) Y. M. Somasekhara. Plant Dis. 83:400, 1999. (2) Y. M. Somasekhara, et al. Res. Crops 1(1):63, 2000.

Structure and evolution of full-length LTR retrotransposons in rice genome

The long terminal repeat (LTR) retrotransposons are the most abundant class of transposable elements in plant genomes and play important roles in genome divergence and evolution. Their accumulation is the main factor influencing genome size increase in plants. Rice (Oryza sativa L.) is a model monocot and is the focus of much biological research due to its economic importance. We conducted a comprehensive survey of full-length LTR retrotransposons based on the completed genome of japonica rice variety Nipponbare (TIGR Release 5), with the newly published tool LTR-FINDER. The elements could be categorized into 29 structural domain categories (SDCs), and their total copy number identified was estimated at >6,000. Most of them were relatively young: more than 90% were less than 10 My. There existed a high level of activity among them as a whole at 0–1 Mya, but different categories possessed distinct amplification patterns. Most recently inserted elements were specific to the rice genome, while a few were conserved across species. This study provides new insights into the structure and evolutionary history of the full-length retroelements in the rice genome.