香蕉束顶病毒复制酶基因克隆及转基因表达

以广州市郊获得的香蕉束项病毒(BBTV)的DNA为模板,进行PCR扩增得到香蕉束项病毒复制酶基因的1.1 kb DNA.所获得的DNA序列与澳大利亚的BBTV序列的同源性达90%,这部分序列编码香蕉束顶病毒复制酶基因的羧基端.将改造的BBTV复制酶基因克隆到pBll21的CaMV 35S和NOS终止序列之间,构建表达载体,并采用基因枪轰击香蕉试管苗生长点组织的方法,经PCR检测和Westem blot分析,获得4株具有BBTV复制酶基因整合表达的To代转基因香蕉.转基因植株的抗病性正在检测之中.     

Within-plant distribution and binomial sampling of Pentalonia nigronervosa (Hemiptera: Aphididae) on banana

The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), infests banana (Musa spp.) worldwide. Pentalonia nigronervosa is the vector of Banana bunchy top virus (family Nanoviridae, genus Babuvirus) the etiological agent of Banana bunchy top disease (BBTD). BBTD is currently the most serious problem affecting banana in Hawaii. Despite the importance of this vector species, little is known about its biology or ecology. There are also no sampling plans available for P. nigronervosa. We conducted field surveys to develop a sampling plan for this pest. Ten plots were surveyed on seven commercial banana farms on the island of Oahu, HI, for the presence of P. nigronervosa on banana plantlets. We found aphids more frequently near the base of plants, followed by the newest unfurled leaf at the top of the plant. Aphids were least likely to be located on leaves in between the top and bottom of the plant. Aphid infestation on surveyed plots ranged from 8 to 95%. We developed a sequential binomial sampling plan based on our surveys. We also discovered that the within-plant distribution of P. nigronervosa is an important factor to consider when sampling for this pest. Our sampling plan will assist in the development of sustainable management practices for banana production.     

Bioinformatic Analysis of BBTV Satellite DNA in Hainan

Banana bunchy top virus (BBTV),family Nanaviridae,genus Babuvirus,is a single stranded DNA virus (ssDNA) that causes banana bunchy top disease (BBTD) in banana plants.It is the most common and most destructive of all viruses in these plants and is widespread throughout the Asia-Pacific region.In this study we isolated,cloned and sequenced a BBTV sample from Hainan Island,China.The results from sequencing and bioinformatics analysis indicate this isolate represents a satellite DNA component with 12 DNA seque...     

香蕉束顶病毒研究进展

香蕉束顶病毒(Bananabunchytopvirus,BBTV)是引起香蕉束顶病害(Bananabunchytopdisease,BBTD)的病毒,它严重地危害了香蕉的生产。综述了近年来香蕉束顶病毒的分离提纯方法,株系划分以及分类地位,较为全面的介绍了BBTV病毒基因组分结构和各组分编码蛋白的功能等,并提出了目前需要进一步澄清的问题。     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

Differential expression of pathogenesis-related proteins and defense enzymes in banana: interaction between endophytic bacteria,Banana bunchy top virus and Pentalonia nigronervosa

Banana bunchy top disease caused by Banana bunchy top virus is the most serious viral disease of banana and plantain worldwide. The virus is transmitted by the aphid vector Pentalonia nigronervosa in a persistent manner. This paper deals with the effect of the interaction between plant growth promoting endophytic bacteria, Banana bunchy top virus, and the banana aphid Pentalonia nigronervosa in the expression of Pathogenesis-related proteins (PR-proteins) and defense enzymes in banana. The existence of virus in the aphids was confirmed by ELISA, DIBA and PCR. PCR could amplify 1100-bp replicase gene of BBTV from viruliferous aphids. A significant increase in the enzymatic activity of all measured PR proteins and defense enzymes, as compared to control plants, was seen in the plants inoculated with endophytic bacteria and challenged with viruliferous aphids. Native gel electrophoresis revealed expression of more isoforms of PR proteins viz., peroxidase and chitinase in the banana plants challenged with mixtures of plant growth promoting endophytic bacteria and viruliferous aphids. Enhanced activity of a PR-2 protein viz., β-1,3-glucanase was also noticed in the viruliferous aphids infested plants. Some of the defense-related enzymes viz., Polyphenol oxidase and Phenylalanine ammonia lyase and phenolic compounds were also upregulated, up to 5 days after aphid infestation and thereafter there was a reduction in the enzymatic activity. Thus, there exist a differential accumulation of PR proteins and defense-related enzymes, when there is tri-tropic interaction between endophytic bacteria, virus, and insect and the role of the endophytic bacteria in the defense mechanisms against insect pests needs to be elucidated.     

Bioinformatic analysis of BBTV satellite DNA in Hainan

Banana bunchy top virus (BBTV),family Nanaviridae,genus Babuvirus,is a single stranded DNA virus (ssDNA) that causes banana bunchy top disease (BBTD) in banana plants.It is the most common and most destructive of all viruses in these plants and is widespread throughout the Asia-Pacific region.In this study we isolated,cloned and sequenced a BBTV sample from Hainan Island,China.The results from sequencing and bioinformatics analysis indicate this isolate represents a satellite DNA component with 12 DNA sequences motifs.We also predicted the physical and chemical properties,structure,signal peptide,phosphorylation,secondary structure,tertiary structure and functional domains of its encoding protein,and compare them with the corresponding quantities in the replication initiation protein of BBTV DNA1.     

Effect of banana bunchy top virus infection on morphology and growth characteristics of banana

Field experiments were conducted in Oahu, Hawaii, to investigate the effects of banana bunchy top virus (BBTV) infection on growth and morphology of banana ( Musa acuminata ). The time interval between aphid inoculation of BBTV and the initial appearance of disease symptoms (i.e. incubation period) was also determined. Plants infected with BBTV showed a significant reduction in petiole size (i.e. length and distance), plant canopy and height, leaf area, pseudostem diameter and chlorophyll content compared with control plants. Growth differences between virus-infected and control plants were not observed until 40–50 days after the plants were inoculated with viruliferous aphids. Other growth parameters such as petiole width and leaf production were not statistically different between infected and control plants. The incubation period of banana bunchy top disease or appearance of symptoms ranged from 25 to 85 days after aphid inoculation. However, PCR assays provided earlier detection of BBTV in banana plants compared with visual symptoms.     

A PCR-based method, with internal control, for the detection of Banana bunchy top virus in banana

Banana bunchy top disease is a major constraint to banana production in most regions where this crop is grown. The disease is caused by Banana bunchy top virus (BBTV), a multicomponent, single-stranded DNA virus of the family Nanoviridae. We have designed primers to a conserved region of the master replication-associated protein that are useful for the polymerase chain reaction (PCR)-mediated detection of BBTV. In addition, primers to banana genomic sequence are used as an internal control, overcoming the uncertainty (owing to false-negatives) inherent in PCR diagnostics. Together these primer sets are a valuable tool in the effort to control BBTV, particularly in screening micropropagated banana plantlets for the absence of virus before release to farmers.     

特异扩增BBTV基因组Ⅲ、Ⅳ、Ⅰ编码区部分序列及其在检测中的应用

香蕉束顶病(BBTD)是香蕉植株的一种毁灭性病害,正在世界(包括中国)的许多香蕉种植区蔓延[1～7].其病原物为香蕉束顶病毒(Banana Bunchy Top Virus,BBTV),被列为我国第三类检疫对象.目前生产上主要采用培育脱毒组培苗来防治BBTD的发生,因此建立一种能快速、灵敏、特异地检测BBTV的方法就显得很重要.国内现在大多采用ELISA方法,但其灵敏度不够高,且需要制备特异性强的抗血清,否则较易出现假阳性.     

Cleavage of the Babuvirus Movement Protein B4 into Functional Peptides Capable of Host Factor Conjugation is Required for Virulence

Banana bunchy top virus(BBTV) poses a serious danger to banana crops worldwide. BBTV-encoded protein B4 is a determinant of pathogenicity. However, the relevant molecular mechanisms underlying its effects remain unknown. In this study, we found that a functional peptide could be liberated from protein B4, likely via proteolytic processing. Site-directed mutagenesis indicated that the functional processing of protein B4 is required for its pathogenic effects, including dwarfism and sterility, in plants. The released protein fragment targets host proteins, such as the large subunit of RuBisCO(RbcL)and elongation factor 2(EF2), involved in protein synthesis. Therefore, the peptide released from B4(also a precursor) may act as a non-canonical modifier to influence host–pathogen interactions involving BBTV and plants.     

Biology of Pentalonia nigronervosa (Hemiptera, Aphididae) on banana using different rearing methods

The banana aphid, Pentalonia nigronervosa Coquerel, is present worldwide where banana (Musa spp.) is grown. It is the vector of Banana bunchy top virus (Nanoviridae, Babuvirus), the etiological agent of banana bunchy top disease, currently the most important constraint for banana producers in Hawaii. P. nigronervosa is not well studied, and effects of temperature on its growth and reproduction are unknown. We studied the longevity and fecundity of one clone of banana aphid on different types of plant materials to determine an effective method to study the insect in the laboratory. We found that insects performed better unconfined on plantlets, followed by leaf midrib cuttings. We also conducted complete life table studies with P. nigronervosa on banana leaf midrib cuttings at 20, 25, and 30 degrees C, with a photoperiod of 12:12. Intrinsic rate of increase (r), net reproductive rate (R0), doubling time (DT), nymphal mortality, and mean offspring per female all showed maximal rates at 25 degrees C. Population growth was studied on whole banana plantlets as well, and growth rates were also highest at 25 degrees C. We found r to be greater when aphids were reared on intact banana plantlets than on cuttings. Our results show the importance of comparing insect rearing methods for studies such as life tables.     

香蕉束顶病毒的纯化及理化特性

从具有典型香蕉束顶病(BBTD)症状的香蕉病组织中提纯了香蕉束顶病毒(Banana bunchy top virus,BBTV)。电镜下可观察到直径为18nm的球形病毒颗粒。最高紫外吸收在255nm,最低紫外吸收在240nm,A_(260)/A_(280)为1.30。用标准BBTV抗体通过ECL-Western转印法测定其外壳蛋白分子量为21kDa。其核酸经DNaseI、RNaseA和Mung Bean Nuclease分析,表明是约1kb的ssDNA。结果与国外文献报道一致。     

香蕉束顶病和花叶心腐病的快速测定技术研究

种植无病蕉苗是防治香蕉束顶病和花叶心腐病的根本措施。筛选无病蕉苗的快速测定技术。除可引用2,3,5-氯化三苯基四(氨)唑浸渍徒手切片;在36℃条件下。24小时后:束顶病株叶脉切片的维管束呈现红色,其它组织为红褐色;花叶心腐病株叶脉切片(含维管束)全部呈现黑褐色;而健株叶脉切片由原绿色变成红色,最后褪成无色。此外。也可取叶片主脉的徒手切片,用1％曙红B水溶液染色。健株切片呈桔红色,病株切片呈砖红色,通过镜检:束顶病株叶片切片的表皮下,可观察到围绕在维管束周围成堆的分布有畸形叶绿体细胞团;花叶心腐病的畸形叶绿体细胞团,多分布在薄壁细胞中,且多散生;而健株的切片没有发现这种畸形的叶绿体细胞团,两种方法同样可靠。     

香蕉束顶病毒研究新进展

介绍香蕉束顶病毒从发现到诊断和检测,从分子生物学研究到抗病毒基因工程,探究香蕉束顶病毒100多年的研究历程,为香蕉束顶病毒的深入研究和有效防治奠定了坚实的基础。     

香蕉束顶病毒DNA组分2、3的启动子区的组织特异性分析

香蕉束顶病毒（BBTV）基因组至少由6个大小约为1.0-1.1kb的单链环状DNA组分所组成,每一个DNA组分包含编码区与非编码区。本文在前人的研究基础上进一步了解BBTV DNA组分启动子的功能。首先根据BBTV 海南分离物的全序列,通过常规PCR扩增出长为540bp的 BBTV DNA3组分启动子序列BV3.1,同时通过重叠PCR扩增出646bp的DNA2与DNA3组分非编码区拼接的重组启动子序列BV23,分别替代pBI121 35S启动子序列与gus基因进行融合,构建植物表达载体pBIBV3.1、pBIBV23。农杆菌介导转化获得的pBIBV3.1转基因烟草经GUS化学组织染色后,在其叶片的叶脉处检测到微弱的GUS活性,证实了DNA3组分的韧皮部特异表达活性;而pBIBV23转基因烟草,其叶片经GUS组织化学染色后,在叶肉、叶缘及一些叶脉上检测到弱GUS活性,这表明由BV23驱动的gus基因在烟草中类似于组成型表达,则DNA2组分转录方式可能有异于DNA3组分。     

Potential implications of biopriming in banana (Musa spp) plantlets against banana bunchy top virus (BBTV)

Abstract

Transplant media as a means for the introduction of biological agents is currently being investigated in a variety of crops. This study aimed to investigate the impact of microbial inoculation in micropropagated banana plantlets to enhance their resistance against Banana bunchy top virus (BBTV). Virus indexed micropropagated plantlets of banana were subjected to root colonization followed by foliar spraying with bacterial strains Pseudomonas fluorescens Pf1, CHA0 and Bacillus subtilis EPB22 during primary and secondary hardening stage in the nursery, at the time of repotting and 3 months after planting in the pot. Microbe inoculated plantlets showed enhanced PR proteins and defense enzymes besides reducing banana bunchy top disease incidence under glasshouse condition. The results indicated the effective use of beneficial microbes in reducing the disease incidence of BBTV in tissue culture banana plantlets. In addition, the molecular characterization of endophytes isolated from banana plantlets, using SDS-PAGE and RAPD-PCR revealed that endophytes were categorized into two distinct groups. These results emphasize the significance of microorganisms in protection of young plantlets from transplanting stresses in field. Further, the use of beneficial microorganisms instead of chemicals sustains an ecological niche in the agricultural ecosystem.     

Spread of an introduced vector-borne banana virus in Hawaii

Emerging diseases are increasing in incidence; therefore, understanding how pathogens are introduced into new regions and cause epidemics is of importance for the development of strategies that may hinder their spread. We used molecular data to study how a vector-borne banana virus, Banana bunchy top virus (BBTV), spread in Hawaii after it was first detected in 1989. Our analyses suggest that BBTV was introduced once into Hawaii, on the island of Oahu. All other islands were infected with isolates originating from Oahu, suggesting that movement of contaminated plant material was the main driving factor responsible for interisland spread of BBTV. The rate of mutation inferred by the phylogenetic analysis (1.4 × 10⁻⁴ bp/year) was similar to that obtained in an experimental evolution study under greenhouse conditions (3.9 × 10⁻⁴ bp/year). We used these values to estimate the number of infections occurring under field conditions per year. Our results suggest that strict and enforced regulations limiting the movement of banana plant material among Hawaiian islands could have reduced interisland spread of this pathogen.