Molecular Characterization of Segments S7 to S10 of a Southern Rice Black-streaked Dwarf Virus Isolate from Maize in Northern China

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China,and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV,JNi4,was obtained from naturally infected maize plants from Ji'ning,Shandong province,in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%,72.3%-73%,73.9%-74.5% and 77.3%-79%,respectively,with corresponding segments of Rice black-streaked dwarf virus isolates,and identities of 99.7%,99.1%-99.7%,98.9%-99.5%,and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.

The Efficiency of Southern rice black-streaked dwarf virus Transmission by the Vector Sogatella furcifera to Different Host Plant Species

Southern rice black-streaked dwarf disease is a new rice disease that severely affects rice production in South China.To understand transmission capacity of the vector Sogatella furcifera to Southern rice black-streaked dwarf virus（SRBSDV） among different host plant species,potential host plants of SRBSDV collected from the diseased rice field and/or adjacent to the field in Hunan Province,China,were determined by RT-PCR,and the transmission rates of SRBSDV by S.furcifera among different host plant species were investigated.The results showed that host plants of SRBSDV in the rice fields were five of family Gramineae（Oryza sativa,Echinochloa crusgalli,Zea mays,Paspalum distichum,Alopecurus aequali） and two of family Cyperaceae（Juncellus serotinus and Cyperus difformis）.S.furcifera could not transmit SRBSDV between gramineous plants and cyperaceous plants,and could not transmit SRBSDV between the gramineous plants,J.serotinus and C.difformis as well.However,SRBSDV could be transmitted by S.furcifera within gramineous plants.S.furcifera could transmit SRBSDV between interspecies among three species plants（O.sativa,E.crusgalli and Z.mays）,and between P.distichum and A.aequali.At 15,20,25,30,and 35°C,both macropterous and brachypterous adult of S.furcifera could transmit SRBSDV from the plants（e.g.,E.crusgalli,Z.mays and O.sativa） infected with SRBSDV to rice seedlings.The transmission rates were first increased and then decreased with the increase of temperature.Macropterous adults transmitted SRBSDV from the viruliferous E.crusgalli,Z.may and rice plants to the healthy rice seedlings,and the infected rates of rice seedlings were 26.2,18.8 and 23.7% at 15°C,56.6,64.6 and 53.6% at 25°C,and was 11.2,10.2 and 7.3% at 35°C,respectively.Transmission capacity of brachypterous adults was significantly higher than that of macropterous adults at 15,20 and 25°C（P0.05）,while transmission capacity of brachypterous adults was relatively lower compared with that of macropterous ones at 35°C.These results offer evidence on the transmission of SRBSDV via the vector S.furcifer among different host plants,which can be helpful to control Southern rice black-streaked dwarf disease by the appropriate cultural measures in South China.

The Control Effect of Prevention and Control Technology Combination against Southern Rice Black-Streaked Dwarf Disease

Objective] This study aimed to explore a better prevention and control technology combination against southern rice black-streaked dwarf disease. [Method] The control effects of seed dressing, antiviral agents and fly net along with 25%pymetrozine against southern rice black streaked dwarf disease were determined. [Result] When the seeds were dressed with 60% imidacloprid （FS, 2 g a.i./kg）, and the rice seedlings were sprayed once with 25% pymetrozine （WP, 360 g/hm2） 10 d before the transplanting and sprayed twice with 30% Dufulin （WP, 800×） at three-leaf stage and 7 d after the transplanting respectively, the control efficiency reached 88.05%. When fly net and pesticides were applied simultaneously, the fly net was lifted and 25% pymetrozine （WP） was spayed once on the rice seedlings at six-leaf stage and the seedlings were hardened for 3 d and sprayed once with 25%pymetrozine （WP） 10 d after the transplanting, the control efficiency reached 80.50%. [Conclusion] Seed dressing or applying antiviral agents alone can not better control diseases. The southern rice black-streaked dwarf disease can be better con-trol ed by seed dressing, along with spraying of planthopper-kil ing agents at two-leaf stage, 3 d before the transplanting and 7 d after the transplanting, respectively. If condition al ow, fly net can be instal ed to achieve better control effect.

Transmission of Rice Black-Streaked Dwarf Virus from Frozen Infected Leaves to Healthy Rice Plants by Small Brown Planthopper (Laodelphax striatellus)

In order to preserve virus for identifying the resistance of rice varieties against rice black-streaked dwarf disease, a simple and reliable method was developed, through which virus-free small brown planthopper （SBPH） acquired rice black-streaked dwarf virus （RBSDV） from frozen infected leaves and the virus was transmitted to healthy rice plants. The experimental results showed that SBPH could obtain RBSDV from frozen infected rice leaves and the virus could be transmitted to a susceptible rice variety. For the ability to acquire RBSDV and transmit the virus to healthy plants by SBPH, there was no significant difference between frozen infected leaves and in vitro infected leaves. The novel method could be applied to identification of rice variety resistance to rice black-streaked dwarf disease, facilitating the breeding process for rice black-streaked dwarf disease resistance.

Resistance of Major Rice Cultivars in Hangzhou Area of China against Southern Rice Black-streaked Dwarf Virus

Resistance of 17 major rice cultivars in Hangzhou region of China against southern rice black-streaked dwarf virus was tested in the paper. The results showed that the incidence rate of southern rice black-streWed dwarf virus had certain positive correlation with quantity of white-backed planthopper （Sogatellafurci-fera） and resistance among various cultivars was significant. Two varieties, Yueyou 9113 and Tianyouhuazhan, were most susceptible ; Yongyou 8, Jiayou 2 and Xi-ushui 134 had best resistance and could be used as disease-resistant varieties to prevent the disease.

Prevalence Characteristics of Rice Black-streaked Dwarf Virus Disease and Continuous Control Strategies

Through summarizing the prevalence characteristics of rice black-streaked dwarf virus disease(RBSDVD)in Linyi City of Shandong Province,this paper analyzed its prevalence is related to changes in farming and cultivation systems,the increase in the population of venomous Laodelphax striatellus Fallén and its own migration and spread,the poor disease resistance of cultivated varieties,and inadequate time of prevention and control.Besides,based on the practice of local control,it came up with some comprehensive control measures including strengthening monitoring,early warning and forecasting,planting resistant(tolerant)rice varieties according to local conditions,appropriately delaying the sowing(planting)period,supplemented by insect nets to cover seedlings,and making scientific use of chemical control.It is expected to provide a reference for the prevention and control of RBSDVD.

Occurrence and Chemical Control Techniques of Rice Black-streaked Dwarf Disease in Rongshui County

The chemical control technologies of southern rice black-streaked dwarf disease in hybrid rice were studied in Rongshui County,Guangxi,through a combination of investigation,observation and experiments.Three treatments were set up,namely,no water,shallow water and deep water,and each of which had a treatment area of 67 m^(2).Each treatment was sprayed once with 40 g of 200 million/g Bacillus subtilis wettable powder and 20-30 g of 25%pymetrozine wettable powder per 667 m^(2) in a mixture with 30 kg of water.On the day of spraying,100 diseased rice hills in each treatment were marked for field observation.The test results showed that the control of southern rice black-streaked dwarf disease should be done by draining the field water and spraying the mixture of B.subtilis and pymetrozine evenly,and a better effect can be achieved by keeping the field moist for 6-7 d after spraying.

Southern rice black-streaked dwarf virus:A new proposed Fijivirus species in the family Reoviridae

For the past several years, a novel dwarf disease has been observed on rice (Oryza sativa) in some regions of Guangdong Province and Hainan Province, southern China. Infected plants showed stunting, dark leaf and small enations on stem and leaf back. Typical Fijivirus viroplasma containing crystalline arrayed spherical virons approximately 70―75 nm in diameter and tubular structures were detected in ultrathin sections by an electron microscope in parenchyma phloem cells of the infected plants. The virus was transmitted to rice seedlings by white-backed planthoppers, Sogatella furcifera (Hemiptera: Delphacidae), collected in the diseased fields. Analysis of dsRNA extracts from infected plants revealed ten linear segments, which were similar to the electrophoretic profile of Rice black-streaked dwarf virus (RBSDV). RT-PCR with a single primer which matched to a linker sequence ligated to both 3′ ends of the viral genomic dsRNAs resulted in amplification of genome segments 9 (S9) and 10 (S10) cDNA products. The complete nucleotide sequences of S9 and S10 were obtained from clones of the RT-PCR amplicon exhibited characteristic properties of Fijivirus including low GC content (34.5% and 35.6%), genus conserved 5′ and 3′ termini sequences and similar genome organization. Blast searches indicated that the sequences of S9 and S10 shared 68.8%―74.9% and 67.1%―77.4% nucleotide identities with those of viruses in the Fijivirus group 2, respectively. These values were similar to those among other viruses in the Fijivirus group 2 and considerably lower than those among RBSDV isolates. Phylogenetic trees based on S9 and S10 nucleotide sequences and their putative amino acid sequences showed that this virus represented a separate branch among other Fijiviruses. The virus was also detected by a nested RT-PCR assay in corn (Zea mays), barnyard grass (Echinochloa crusgalli), Juncellus serotinus and flaccidgrass (Pennisetum flaccidum) in and/or adjacent to the infected rice fields. It is proposed that this virus be considered as a new species, Southern rice black-streaked dwarf virus, in the group 2 of the genus Fijivirus in the family Reoviridae.

Detection,Occurrence,and Survey of Rice Stripe and BlackStreaked Dwarf Diseases in Zhejiang Province,China

The major viral diseases that occur on rice plants in Zhejiang Province, eastern China, are stripe and rice black-streaked dwarf diseases. Rice stripe disease is only caused by rice stripe tenuivirus （RSV）, while rice black-streaked dwarf disease can be caused by rice black-streaked dwarf fijivirus （RBSDV） and/or southem rice black-streaked dwarf fijivirus （SRBSDV）. Here we review the characterization of these viruses, methods for their detection, and extensive surveys showing their occurrence and spread in the province.

A Rapid and Simple Method for the Detection of Rice Dwarf Virus by Aqueous Extract

Rice dwarf disease caused by rice dwarf virus （RDV） is one of the major rice virus diseases in China,which widely distributes in the rice area of China.A simple and rapid method for detection of RDV in rice plants and Nephotettix cincticeps was investigated in this study,and the whole detection only lasted for 20 min.After diseased leaves of rice （10 mg） and leaves of Nephotettix cincticeps （10 mg） both infected by RDV were ground by sterile water （100 μl）,the supernatant was analyzed by 1% agarose gel electrophoresis to steadily detect five specific electrophoretic bands with 1 000 bp to 5 000 bp.However,there was no electrophoretic band in the healthy rice leaves and aqueous extract of non-viruliferous N.cincticeps.Therefore,this method was more rapid and simple to detect RDV in rice plants and Nephotettix cincticeps,which avoided expensive reagents and tedious steps of conventional serological testing and molecular detection （RT-PCR and Western-blot）.

Screening of Rice Genes Interacting with p5b of Rice BlackStreaked Dwarf Virus

Rice black-streaked dwarf virus （RBSDV） is a recognized member of the genus Fijivirus, family Reoviridae. Its genome has ten double-stranded RNA （dsRNA） segments （$1-$10）, in which the fifth genome segment （$5） contains two open reading frames （ORFs） with a partially overlapping region. The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function. To reveal the function of p5b, its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay. The bait protein p5b was detected in yeast by western blot, and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast. Then the bait protein p5b was used for screening the cDNA expression libraries of rice. Gene fragments of some pivotal enzymes involved in photosynthesis, respiration and other important metabolic processes, were identified to interact with p5b in yeast, suggesting that these interactions may play roles in symptom development in infected plants.

Morphological Analysis and Gene Mapping of a Rice Dwarf Mutant

[Objective] This study aimed to conduct morphological analysis and gene mapping of a rice dwarf mutant. [Method] A dwarf mutant （Xiaoxiang＇ai） was used as test material for morphological observation. Xiaoxiang＇ai was used as female parent and semi-dwarf material Xiangzao143 was used as male parent to construct populations for genetic analysis. Gene mapping was conducted by using micro- satellite markers. [Result] The average height of Xiaoxiang＇ai was 55 cm; genetic analysis results showed that plant height of F1 individuals was similar with the male parent, while semi-dwarf plants and dwarf plants were observed in F2 populations and the segregation ratio was nearly 3：1, indicating that plant height trait of Xiaoxi- ang＇ai is mainly controlled by one pair of recessive gene located on rice chromo- some 5 and in the upstream of RM249, with a genetic distance of 8.4 cM. [Conclu- sion] rRH is a new dwarf gene in rice.

Genetic analysis and fine-mapping of a dwarfing with withered leaf-tip mutant in rice

A dwarf mutant of rice （Oryza sativa L.） by mutagenesis of ethylene methylsulfonate （EMS） treatment from Nipponbare was identified. The mutant exhibited phenotypes of dwarfism and withered leaf tip （dwll）. Based on the intemode length of dwll, this mutant belongs to the dm type of dwarfing. Analysis of elongation of the second sheath and m-amylase activity in endosperm showed that the phenotype caused by dwll was insensitive to gibberellin acid treatment. Using a large F2 population derived from a cross between the dwll and an indica rice variety, TN1, the DWL1 gene was mapped to the terminal region of the long arm of chromosome 3. Fine-mapping delimited it into a 46 kb physical distance between two STS markers, HL921 and HL944, where 6 open reading frames were predicted. Cloning of DWL1 will contribute to dissecting molecular mechanism that regulates plant height in rice, which will be beneficial to molecular assisted selection of this important trait.

Ecological Fitness of Non-vector Planthopper Sogatella furcifera on Rice Plants Infected with Rice Black Streaked Dwarf Virus

We evaluated the effects of rice black streak dwarf virus （RBSDV）-infested rice plants on the ecological parameters and its relevant defensive and detoxification enzymes of white-backed planthopper （WBPH） in laboratory for exploring the relationship between RBSDV and the non-vector planthopper. The results showed that nymph survival rate, female adult weight and fecundity, and egg hatchability of WBPH fed on RBSDV-infested rice plants did not markedly differ from those on healthy plants, whereas the female adult longevity and egg duration significantly shortened on diseased plants. Furthermore, significantly higher activities of defensive enzymes （dismutase, catalase and peroxidase） and detoxification enzymes （acetylcholinesterase, carboxylesterase and glutathione S-transferase） were found in WBPH adults fed on infected plants. Results implied that infestation by RBSDV increased the ecological fitness of non-vector planthopper population.

Growth and Photosynthesis Responses of a Super Dwarf Rice Genotype to Shade and Nitrogen Supply

Specific aspects of plant cultivation require tests under fully controlled environmentalconditions with restricted energy supply, such as orbit-based space laboratories and low-light conditions.For these growing conditions, super dwarf plants have been developed as model crops, and a gibberellindeficientSuper Dwarf Rice genotype was proposed as a model crop for space flight plant experiments.We tested this genotype in a climate chamber experiment under different illumination and nitrogen supplylevels to assess its suitability under scenarios with limited resource availability. A 25% reduction inillumination led to a 75% reduction in yield, mainly due to a 60% reduction in formed tillers and 20%reduction in grain weight, and a 80% reduction in illumination caused total yield loss. Leaf area underreduced illumination was significantly lower, and only marginal changes in the dimensions of leaves wereobserved. Plant photosynthesis was not significantly different between control and 75% illumination. Thiswas explained by a higher photochemical efficiency under lower light conditions and a reduced mesophyllresistance. Therefore, we concluded that this genotype is well-suited for plant experiments under spaceand light-limited conditions since it kept its small stature and showed no shade avoidance mechanisms,such as leaf elongation, which would complicate experiments under low-light conditions. Nitrogenconcentrations of 2.8 and 1.4 mmol/L led to no differences in plant growth. We concluded that a nitrogenconcentration of 1.4 mmol/L is sufficient for this genotype under the light intensities.

Cloning and Expression of Gene Responsible for High-Tillering Dwarf Phenotype in Indica Rice Mutant gsor23

High-tillering dwarf mutant gsor23 was generated from an indica rice variety Indica9 radiatied by y-ray. Genetic analysis showed that this phenotype was controlled by one single recessive gene, which was mapped within a physical distance of 386 kb between two insertion-deletion （InDel） markers CI-WT2 and C1-WT4 on the long arm of chromosome 1. There is a known gene DIO within this region, the mutation of which causes high-tillering in rice. Sequence analysis of the DIO allele in gsor23 revealed that the base cytosine （C） at the 404th position in the coding region was deleted, which would cause frameshift mutation after the 134th amino acids. The mutation site and indica background of gsor23 were different from the previously reported japonica mutants d10-1 and d10-2. Therefore, gsor23 is a novel allelic mutant of D10 which encodes the carotenoid-cleaving dioxygenase 8 （CCD8）, a key enzyme involved in the biosynthesis of the new plant hormone strigolactones （SLs）. After treatment with GR24, a synthetic analogue of SLs, the high-tillering phenotype of gsor23 was restored to normal. Real-time RT-PCR analysis showed that D10 expression was high in roots, but low in leaves. Compared with the wild type Indica9, the expression of the SL biosynthesis gene DIO was upregulated, while genes likely involved in the SL signal transduction pathway such as D3 and D14 were down-regulated in the gsor23 mutant.

Phenotypic analysis of a dwarf and deformed flower3 (ddf3) mutant in rice (Oryza sativa L.) and characterization of candidate genes

Dwarf mutants are the crucial resources for molecular biology research and rice breeding. Here, a rice mutant, dwarf and deformed flower3（ddf3）, was identified in tissue culture of Oryza sativa cv. Dongjin. Compared with wild type, the ddf3 mutant exhibited severe dwarfism, a greater number of tillers and significantly decreased fertility. In addition, leaf length, panicle length, and grain length, were significantly shorter. All internodes of ddf3 were shorter than those of wild type, and histological analysis revealed that internode cell elongation was significantly inhibited in ddf3. In the ddf3 mutant, pollen activity was significantly decreased, and the development of most stigmas was abnormal. Genetic analysis indicated that the ddf3 mutant phenotypes are controlled by a single or tightly linked nuclear genes. Using an F2 mapping population generated from a cross between ddf3 and Yangdao 6（9311）, the DDF3 gene was mapped to a 45.21-kb region between insertion-deletion（In Del） markers M15 and M16 on the long arm of chromosome 7. Sequencing revealed a 13.98-kbdeletion in this region in the ddf3 mutant genome that resulted in the complete or partial deletion of ZF（DHHC type zinc finger protein）, EP（expressed protein）, and FH2（actin-binding FH2 domain-containing protein） genes. Quantitative RT-PCR analyses revealed that in wild type, the transcript levels of FH2 were almost the same in all organs, while ZF was mainly expressed in the panicle, and no expression of EP was detected in any organ. Based on these results, ZF and FH2 could be potential DDF3 candidate genes involved in the regulation of rice morphology and flower organ development. Our work has laid the foundation for future functional analysis of these candidate genes and has provided a profitable gene resource for rice breeding for increased fertility in the future.

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.

Recent amplification of Osr4 LTR-retrotransposon caused rice D1 gene mutation and dwarf phenotype

A novel rice d1 mutant was identified using map-based cloning and comparative analysis of known dl mutants.The mutant[d1-a) shows a mild dwarf trait,which differs only slightly from the wildtype in plant height at the tillering stage.The dl-a mutant is different from other dl mutants.We found that it was interrupted by an Osr4 long terminal repeat(LTR)-retrotransposon,which resulted in the loss of exon 7 in the mutant D1 mRNA.A paralog of the D1 gene.D1-like,was revealed.D1-like is a truncated gene that might have resulted from recombination between retrotransposons.We identified 65 Osr4LTR-retrotransposons in Nipponbare,and found more LTR variants in contrast to coding DNA sequence(CDS) in the retrotransposons.We also identified five possible regulatory motifs in LTRs which may control the expression of the retrotransposons.In addition,we predicted six putative functional Osr4 retrotransposons that contain complete CDSs and all important elements.Osr4 retrotransposons were classified into 4 groups,and this type of retrotransposon only appears to be present in monocots.Members of group 1-1,which included all putative functional retrotransposons,showed a high similarity with each other.The retrotransposons were expressed in all tissues,at especially higher levels in some leaves and seeds.These findings imply that transpositions of group 1-1 members might have occurred frequently and recently.

Genetic Identification of a New Small Grain Dwarf Gene in Rice

The plant material used in the study was rice line 162d, a new small grain dwarf mutant. Polymorphic analysis of 221 SSR loci demonstrated that 162d derived from a semidwarf variety Shuhui 162 through mutation, and 162d and Shuhui 162 were just a pair of near isogenic lines. Genetic analysis of F_1 and F_2 populations suggested that dwarfism in 162d was controlled by a single recessive gene. Phenotypic characteristics of the mutant gene were that plant height was about a quarter of normal height, grain size about a quarter of normal size, leaf was short and broad, and seed setting rate was very low, compared with the near isogenic line Shuhui 162. The mutant gene was sensitive to gibberellin (GA_3) treatment and did not located on the region near the centromere of rice chromosome 5, where dl gene located. Therefore, it was concluded that the mutant gene of 162d was a new small grain dwarf gene in rice.