Marker-Assisted Selection of Xa21 Conferring Resistance to Bacterial Leaf Blight in indica Rice Cultivar LT2

Bacterial leaf blight of rice (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive diseases in Asian rice fields. A high-quality rice variety, LT2, was used as the recipient parent. IRBB21, which carries the Xa21 gene, was used as the donor parent. The resistance gene Xa21 was introduced into LT2 by marker-assisted backcrossing. Three Xoo races were used to inoculate the improved lines following the clipping method. Eleven BC₃F₃ lines carrying Xa21 were obtained based on molecular markers and agronomic performance. The 11 lines were then inoculated with the three Xoo races. All the 11 improved lines showed better resistance to BLB than the recipient parent LT2. Based on the level of resistance to BLB and their agronomic performance, five lines (BC₃F₃ 5.1.5.1, BC₃F₃ 5.1.5.12, BC₃F₃ 8.5.6.44, BC₃F₃ 9.5.4.1 and BC₃F₃ 9.5.4.23) were selected as the most promising for commercial release. These improved lines could contribute to rice production in terms of food security.     

Rhizosphere Bacteria for Biocontrol of Bacterial Blight and Growth Promotion of Rice

Several bacterial strains were isolated from different rhizospheres. Among these, strain PDY7 exhibited strong antibacterial activity against the rice bacterial blight (BB) pathogen Xanthomonas oryzae pv. oryzae (Xoo) by the laboratory dual plate assays. The antibacterial property of the strain PDY7 was further investigated for the production of 2,4-diacetylphloroglucinol (DAPG), which amplified a characteristic of 629-bp DNA fragment by PCR-based screening method using phlD primers. The application of phlD positive strains was carefully evaluated for disease control and growth promotion of rice plants under field conditions. The selected strain PDY7 suppressed the rice BB by 58.83% and 51.88% under glass house and field conditions, respectively. In addition, the strain PDY7 showed significant two-fold increase in root length (18.08 cm), shoot length (29.81 cm), and grain yield (96.07 g). Strain PDY7 promoted the growth of rice plants by production of indole-3-acetic acid (IAA), which was determined by high performance liquid chromatography (HPLC) analysis. Our findings suggest that PDY7 belongs to the P. fluorescens group and can serve as potential biocontrol of BB as well as biofertilizer agent for growth promotion of rice.     

Identification of QTLs underlying resistance to a virulent strain of Xanthomonas oryzae pv. oryzae in rice cultivar DV85

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice in China. A strongly virulent Xoo strain, designated Z-173, is widely distributed across China and Southeast Asia. Indica rice DV85 is known to carry the two resistance genes, xa5 and Xa7. However, their effectiveness against Z-173 is unknown. Using a recombinant inbred line (RIL) population derived from a cross between DV85 and the susceptible cultivar Kinmaze, we have identified the quantitative trait loci (QTLs) responsible for the resistance of DV85 to Z-173. Following 2 years of phenotyping, three QTLs associated with the resistance were detected. These were linked to RFLP markers X362, X292 and G1091 on chromosomes 3, 5, and 6, respectively. Qxa-5 and Qxa-6 probably correspond to xa5 and Xa7, respectively. Both the xa5 and Xa7 resistances are stable over different years, and act independently of one another in determining resistance. The effect of xa5 was larger than that of Xa7. Efficient ways to improve the resistance to Z-173 are discussed.     

PCR detection ofclavibacter michiganensis subsp.sepedonicus- infected tuber samples in a plate capture assay

The speed and sensitivity of PCR-based assays allow shorter turnaround times for the detection of pathogens for which culture and serological methods are difficult or unavailable. PCR was performed with primer sets Cms50 and Cms72, designed previously by Millset al. (1997) through subtractive hybridization to detectClavibacter michiganensis subspeciessepedonicus (Cms). In bacterial suspensions, fewer than three cells/10 ul reaction were detected after PCR amplicons were hybridized with specific DIG-labeled DNA probes in an enzyme-linked oligonucleosorbent assay (ELOSA). In naturally infected tuber samples representing three cultivars of potato, the diagnostic sensitivity of PCR/ ELOSA was 96%, while the specificity exceeded 99%. PCR/ELOSA detectedCms in infected tuber samples with equal sensitivity regardless of colony morphology, potato cultivar, or primer sets.     

Resistance responses of rice to rice blast fungus after seed treatment with the endophytic Achromobacter xylosoxidans AUM54 strains

In the present investigation, we studied resistance imparted by seed treatment with an endophytic strain of Achromobacter xylosoxidans, AUM54, against rice blast caused by Magnaporthe oryzae. In vitro studies showed that A. xylosoxidans AUM54 was able to inhibit mycelial growth of M. oryzae by 11% and was able to increase rice germination and seedling vigor index of rice by 31 and 114%, respectively. AUM54 also showed better survivability in the spermosphere and spermoplane and was able to move systemically through the roots and stem. Among the evaluated carriers, liquid formulation amended with 2% glycerol sustained the maximum bacterial population (7.4 log cfu ml⁻¹) after six-months-storage at room temperature. Plants treated with A. xylosoxidans AUM54 followed by inoculation with M. oryzae showed a significant increase in the activities of defense related enzymes such as polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and chitinase. A. xylosoxidans AUM54 treatment was able to reduce blast disease incidence by 39% in treated rice plants. Additionally, inoculation with A. xylosoxidans AUM54 significantly enhanced the growth (3–13% plant height), and yield (11–31%) of inoculated rice plants under no-disease and disease conditions in the greenhouse experiments.     

Detection and characterization of bismerthiazol-resistance of Xanthomonas oryzae pv. oryzae

The resistance to bismerthiazol in the Xanthomonas oryzae pv. oryzae population in China has not been clearly determined. Three bismerthiazol-resistant mutants (1-1-1, 2-1-1, 4-1-1) of X. oryzae pv. oryzae were induced on rice plants and the characteristics were compared with their parental strain ZJ173. After 10 subcultures on fresh bismerthiazol-free nutrient agar plates and after being inoculation onto and re-isolation from untreated rice plants two times, the mutants remained pathogenicity on rice plants treated with 300 μg/ml bismerthiazol, indicating that the in vivo-induced bismerthiazol resistance was stable in vivo. The in vitro characteristics of the bismerthiazol-resistant mutants and a wild-type bismerthiazol-sensitive strain (ZJ173) were also compared. The three mutants grew faster than ZJ173 during the logarithmic growth phase but at the same rate during the decline phase. Bismerthiazol had protective and curative activity against strain ZJ173 but not against the three mutants. To evaluate the current degree of bismerthiazol resistance in field populations of X. oryzae pv. oryzae in China, 781 isolates of X. oryzae pv. oryzae were obtained from 1419 leaf samples collected from Yunnan, Sichuan, Jiangsu, Anhui, Hubei, Guangdong, Hainan, and Hunan Provinces in China from 2007 to 2009. The in vivo sensitivity of 505 of the 781 isolates to bismerthiazol was determined by applying them to rice plants treated with 300 μg/ml bismerthiazol and evaluating blast severity. The tested strains were considered resistant, if they showed <70% inhibition relative to strain ZJ173. Of the 505 isolates, 62 were resistant to bismerthiazol. The resistance frequencies among field population ranged from 0 to 21.1%, differed among the provinces, and was 11.2, 20.0 and 10.4% in 2007, 2008 and 2009, respectively. Resistance determined in vivo was not related to resistance determined in vitro. The in vivo-induced bismerthiazol resistance identified in this study will serve as a base line for further studying bismerthiazol resistance in fields, and the current degree of bismerthiazol resistance in field populations of X. oryzae pv. oryzae in China determined would be useful for the rice growers for control of rice bacterial blight.     

Improvement of Bacterial Blight Resistance in Rice Cultivars Jyothi and IR50 via Marker-Assisted Backcross Breeding

Abstract

In pathogen population analysis of 208 Xanthomonas oryzae pv. oryzae(Xoo) strains that were assembled from different parts of India, 21 pathotypes were identified on the basis of disease reactions on near-isogenic lines (NILs) and 13 pathotypes, on rice differentials. Rice cultivars, Jyothi and IR50, which are high yielding but highly prone to bacterial blight (BB) caused by pathogen populations of Xanthomonas oryzae pv. oryzae in India, were chosen. To improve the BB resistance of these two varieties, a pyramid line, NH56, containing four R-genes, Xa4, xa5, xa13, and Xa21, was selected as the R-donor based on resistance to existing pathogen population. The four R-genes were successfully transferred to cultivars through a traditional backcross method and their presence was documented with marker-aided selection (MAS). Thirty BC₄F₂ plants derived from JxNH56 (cv. Jyothi) and 45 BC₄F₂ plants derived from IR50xNH56 (cv. IR50) had all four resistance genes (Xa4, xa5, xa13, and Xa21), which should be useful resistance donors for breeding other BB-resistant elite indica varieties.     

Development ofNew InDel Marker to Detect Genotypes of Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility inRice简

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility (CMS) in japonica rice, a functional marker InDel-Rf-1a based on the difference of nucleotide sequence in Rf-1a locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-1arf-1a showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-1a locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F₂ segregation population, and three genotypes in Rf-1a locus could be distinguished distinctly. Therefore, the marker InDel-Rf-1a could be widely used for genetic identification and marker-assisted selection (MAS) in breeding japonica restorer lines.     

水稻白叶枯病为害损失测定与防治指标探讨

水稻白叶枯病是浙江稻区常发流行性病害,调查研究其为害与损失,科学制订防治指标,对于有效预测和综合防控病害,确保水稻丰产和农民增收具有重要意义。通过对浙江桐庐、余杭等地几年来水稻白叶枯病发生为害损失调查测定,探明了病害发生为害与损失的关系,分别建立了水稻分蘖、圆秆拔节和孕穗破口期株、叶发病率与产量损失率关系的6个模型,考查了侵染发病对主要经济性状的影响,受害稻株株高下降、实粒数减少、空秕率增加是导致减产的主要原因。调查分析了田间用药和防治成本,测定了经济允许水平,首次提出了该病防治指标为水稻株发病率5%、叶发病率3%。该指标经浙江桐庐、温岭、温州等地大田验证与示范应用,与实际发生为害情况相符,药治面积压缩1/3,省药、节本、增效,取得了显著的经济、社会和生态效益。     

Phylogenomic Relationships of Rice Oxalate Oxidases to the Cupin Superfamily and Their Association with Disease Resistance QTL

Rice oxalate oxidase genes (OXO) may play a role in resistance to Magnaporthe oryzae. Genome analyses showed four tandemly duplicated OXO genes, OsOXO1–OsOXO4, which mapped to a blast resistance QTL in chromosome 3. These genes have >90% nucleotide and amino acid identity, but they have unique gene structures, conserved motifs, and phylogeny compared to the 70 other members of the cupin superfamily in the Nipponbare genome, which were divided into several classes. In resistant and susceptible Vandana/Moroberekan advanced backcross lines, only OsOXO4 was expressed during rice–M. oryzae interactions, and its expression increased earlier in resistant than susceptible lines. The earlier expression of OsOXO4 in resistant lines correlated with a 26-bp promoter insertion containing an additional copy of the bacterial responsive nodulation cis-element. Our results showed that OsOXO1–4 are in a separate class of rice cupin genes and supports a role for the promoter variant of OsOXO4 in resistance to M. oryzae.     

The latex agglutination test as a rapid serological assay forCorynebacterium sepedonicum

The latex agglutination test was evaluated for detection of the potato bacterial ring rot pathogen,Corynebacterium sepedonicum. The bacterium was detected in infected potato stems and tubers tested and in inoculated eggplant stems. Test reliability was confirmed using blind tests. A minimum of 10⁶ bacterial cells/ml was requisite for observable agglutination. Nonspecific reactions were not observed from healthy extracts of plant tissues. Nonspecific reactions with other bacteria maintained in pure culture were eliminated by addition of bovine serum albumin to sensitized latex preparations.     

Species-specific PCR primers for detection of Xanthomonas vesicatoria

This study presents the development of a PCR method for specific detection of Xanthomonas vesicatoria, causal agent of bacterial spot of tomato and pepper. Primer pair XV1F and XV1R was designed based on partial DNA sequence of the catalytic subunit of the ATP synthase gene atpD and tested using a collection of various bacterial pathogens mainly for tomato and pepper. After optimization of the PCR conditions, the 365 bp long fragment was only produced in X. vesicatoria samples. Therefore, the primers and protocol allow rapid and species-specific detection of X. vesicatoria.     

Genetic control of processing quality in a bread wheat mapping population grown in water-limited environments

End-use quality of bread wheat (Triticum aestivum L.) is affected by both genetic and environmental variation. Current understanding of the genetic control of wheat quality traits is mainly based on genetic experiments conducted using grain produced in favourable conditions. The objective of this research was to extend the genetic analysis of these traits by using grain produced in water-limited environments. Grain samples harvested from a mapping population grown in field experiments at two locations in Australia were used to assess characteristics of the grain, flour, dough and bread. Quantitative trait loci (QTLs) were mapped. The parents of the population, RAC875 and Kukri, differ at several loci that are known to affect grain quality or plant phenology. Of these, a high-molecular-weight glutenin locus (Glu-B1) affected dough properties, the puroindoline-encoding Ha locus affected grain hardness, flour and loaf properties and a photoperiod response locus (Ppd-D1) affected flour extraction and protein content. Similarly, several previously reported quantitative trait loci (not associated with specific genes) also had effects in the stress environments used here. In addition, novel loci were detected for bread wheat quality traits; their effects may be specific to materials grown in water-limited environments.     

Improvements in the qualities of gluten-free bread after using a protease obtained from Aspergillus oryzae

From a nutritional perspective, rice flour is one of the most valuable flours and it is suitable for preparing food for people suffering from wheat allergy. However, bread made from rice flour is very difficult to bake because it lacks gluten. We found that pre-fermenting of rice flour using Aspergillus oryzae facilitated a better formulation of gluten-free rice bread. Bread swelling was remarkably improved with a longer pre-fermenting period at 55 °C. The specific loaf volume (SLV) without polymeric thickeners after a 12 h fermentation was approximately 2.2-fold (2.98 ml/g) higher than that after 0 h (1.36 ml/g). An enzymatic assay of the batter indicated that protease activity during the pre-fermentation period increased from 0.38 to 1.44 U/ml and this activity correlated with bread swelling. Furthermore, a commercial protease from A. oryzae also produced similar results with an adequate SLV of 3.03 ml/g. Rheological analysis showed that batter treated with protease had an increased batter viscosity and decreased flour settling behavior because of the aggregation of flour particle after partial cleavage of storage proteins. These results indicated that the improved SLV was mainly because of an A. oryzae protease, which affected the batter rheology thereby improving gas retention before baking.     

Genetic Improvement of Rice for Bacterial Blight Resistance: Present Status and Future Prospects

The production and productivity of rice has been challenged due to biotic and abiotic factors. Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae, is one of the important biotic stress factors, which reduces rice production by 20%–50%. The deployment of host plant resistance is the most preferred strategy for management of BB disease, and breeding disease resistant varieties remains a very economical and effective option. However, it is difficult to develop rice varieties with durable broad-spectrum resistance against BB using conventional approaches alone. Modern biotechnological tools, particularly the deployment of molecular markers, have facilitated the cloning, characterization and introgression of BB resistance genes into elite varieties. At least 46 BB resistance genes have been identified and mapped from diverse sources till date. Among these, 11 genes have been cloned and characterized. Marker-assisted breeding remains the most efficient approach to improve BB resistance by introducing two or more resistance genes into target varieties. Among the identified genes, xa5, xa13 and Xa21 are being widely used in marker-assisted breeding and more than 70 rice varieties or hybrid rice parental lines have been improved for their BB resistance alone or in combination with genes/QTLs conferring tolerance to other stress. We review the developments related to identification and utilization of various resistance genes to develop BB resistant rice varieties through marker-assisted breeding.     

白叶枯病菌xopXoo基因在致病性中的作用

辣椒斑点病菌(Xanthomonas campestris pv. vesicatoria,Xcv)在非寄主烟草上的过敏反应与xopX基因有关,但在白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)基因组中并未标注xopX同源基因的功能。为研究白叶枯病菌中xopX同源基因在致病性中的作用,对白叶枯病菌的xopXoo基因进行了克隆和突变,并对xopXoo突变体在非寄主烟草上的过敏反应、水稻苗期的水浸症状以及成株期的致病性和病菌生长能力进行了分析。测定结果显示,xopXoo基因突变并不改变白叶枯病菌在烟草上激发过敏反应和在水稻上产生水浸症状能力,但突变菌株在成株期水稻上的致病性和菌体生长能力显著下降。遗传互补能够恢复xopXoo突变体的致病性和菌体生长能力,表明xopXoo是白叶枯病菌的致病性基因。还对白叶枯病菌激发非寄主烟草产生过敏反应的可能基因进行了讨论。     

Biocontrol of rice blast by the phenaminomethylacetic acid producer of Bacillus methylotrophicus strain BC79

Strain BC79, isolated from primeval forest soil in Qinling, Mountains, China, was identified as Bacillus methylotrophicus based on morphological, biochemical, physiological and chemotaxonomic analyses as well as phylogenetic 16S rDNA sequencing data. This strain was able to suppress mycelial growth and conidial germination of numerous plant pathogenic fungi in dual cultures on solid media. For exploring potential biocontrol activity, we assessed fermentation conditions for studying B. meth1ylotrophicus BC79. The active substance of BC79, phenaminomethylacetic acid, was extracted by TLC and HPLC, and identified as the strongest inhibitory substance described in B. methylotrophicus. Experiments in a greenhouse showed that application of BC79 culture filtrates 24 h before inoculation of Magnaporthe oryzae, the causal agent of rice blast, had 89.87% biocontrol efficiency. B. methylotrophicus BC79 colonized rice plant tissues and at 10 days after filtrate application, its population in leaves (1.65 × 10⁸ CFU/g) was much larger than in stems (6.78 × 10⁷ CFU/g) or roots (3.56 × 10⁷ CFU/g). Field trials indicated that BC79 culture filtrate (4000 g/667 m²) showed the highest efficiency for M. oryzae, with 84.8% biocontrol effect, followed by of 15% phenaminomethylacetic acid extract (75.5%) and 20% tricyclazole (76.1%). Seedling and post-transplant stages were the best periods to apply BC79 for control of rice blast. The B. methylotrophicus BC79 strain hence has enormous potential as an agricultural agent for biocontrol of rice blast.