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.     

Field evaluation of micropropagated bananas derived from plants containing Banana Bunchy-Top Virus

Micropropagated bananas derived from Banana Bunchy-Top Virus (BBTV) infected plants, but displaying no symptoms of the disease, were established in the field. They were grown for three years and produced a plant crop and ratoon crops. No disease symptoms were observed. There was uncertainty as to whether

Use of polymerase chain reaction (PCR) to study transmission of banana bunchy top virus by the banana aphid (Pentalonia nigronervosa)

Banana bunchy top virus (BBTV) is a ssDNA virus transmitted by the banana aphid, ( Pentalonia nigronervosa ). A polymerase chain reaction (PCR) assay was used to study BBTV transmission efficiency, to determine the minimum acquisition-access period, the minimum inoculation-access period, the retention time, and to examine the possibility of transovarial transmission in this vector. BBTV was acquired by banana aphids within 4 h and was transmitted within 15 min feeding. On average, more than 65% of single viruliferous adult aphids transmitted BBTV. The aphids retained BBTV for their adulthood of 15–20 days. None of the 131 offspring from adult aphids reared on infected bananas were BBTV positive. Aphid transmission experiments were conducted to determine if taro and gingers are hosts of BBTV. None of the 87 taro and ginger plants exposed to aphid inoculation were infected by BBTV. The BBTV-free status of these plants was verified by PCR assay for 6 months post-inoculation. In addition, none of the taro and ginger samples collected from fields adjacent to BBTV-infected banana plants tested positive for BBTV.     

Screening of banana bunchy top virus through multiplex PCR approach

Bunchy top disease caused by the banana bunchy top virus (BBTV) is a serious disease in hill banana. Detection of the BBTV infection in the planting material could help in the effective management of the disease. An attempt was made to develop a sensitive polymerase chain reaction (PCR) and multiplex PCR-based method for detection of BBTV in hill banana. DNA was isolated from the experimental plants at third and sixth months after planting. Multiplex PCR was done with Coat Protein (CP) and Replicase (Rep) gene-specific primer, and banana ethylene insensitive like protein (EISL) primer as internal control to identify failure in PCR reaction. This study revealed that multiplex PCR is effective for BBTV screening in hill banana with the advantage of overcoming the false positive in PCR amplification.     

Biopriming of micropropagated banana plants at pre- or post-BBTV inoculation stage with rhizosphere and endophytic bacteria determines their ability to induce systemic resistance against BBTV in cultivar Grand Naine

ABSTRACT

Rhizospheric and endophytic bacteria isolated from the roots and corms of banana were tested to find out their efficiency in controlling against banana bunchy top virus (BBTV). Bioformulations of mixtures of endophytic Bacillus pumilus and B. subtilis isolated from banana cv. Grand Naine and rhizobacterial isolate Pseudomonas fluorescens (Pf1) were found to be effective in increasing the growth and physiological parameters such as pseudostem girth and height, number of leaves, phyllochron, and leaf area in biohardened plants under greenhouse study. The consortia of bioformulation mixture of B. pumilus, B. subtilis, and P. fluorescens I showed 61.62% disease reduction over control. The defence enzymes such as peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and total phenol were induced to an elevated level in biohardened plants. The applications of bioformulations to plants led to delay the symptom expression for 63.75 to 70.50 days compared to control after challenge inoculation with the virus in 34–67% of plants that exhibited the symptoms till 150 DAI. However, biohardening of plants with the same combinations of bacteria three days after BBTV inoculation led to express the symptoms 29.16 to 36.71 days and there was a significant decrease in plant growth parameters. Biopriming prior to BBTV infection has attributed to the enhanced plant growth and resistance against BBTV whereas, the same treatments after virus inoculation did not induce resistance. This study has proved that the time of application of consortia of bio-inoculants determines their effect of induced resistance to BBTV in micropropagated plants.     

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.     

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.     

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。     

香蕉束顶病毒DNA组分6的克隆和序列分析

香蕉束顶病(banana bunchy top disease,BBTD)是香蕉生产上重要的病害之一,它威胁着世界约1/4香蕉产区的生产[1].到1998年7月,世界上报道发生该病害的国家和地区达20多个,遍及亚洲、南太平洋地区和少数非洲国家.     

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...     

感染BBTV香蕉叶片cDNA文库的构建及评价

为了研究香蕉束顶病毒与香蕉寄主致病的互作分子机制,本文报道利用Make Your Own“Mate＆Plate^TM”Library System试剂盒成功构建感染BBTV香蕉叶片的cDNA文库。通过改良CTAB法提取感染BBTV香蕉叶片的总RNA,采用SMART法反转录合成双链cDNA,经SfiI酶切并去除短片段之后,与经同样酶切的pGADT7-SfiI载体连接,利用电转法将重组载体转化到大肠杆菌宿主细胞中,获得初级cDNA文库,最后以初级文库100万克隆为基数扩增,得到扩增文库并提取质粒。结果得到库容量大于2.0×10⁶的初级文库,检测表明文库cDNA插入片段长度主要分布在700~2 000 bp,文库重组率为87.5％。结果表明,该文库质量较好,可用于后续酵母双杂交互作蛋白筛选试验,本研究为开展病毒与寄主互作的研究奠定基础。     

Induction of defense-related proteins by mixtures of plant growth promoting endophytic bacteria against Banana bunchy top virus

Rhizosphere and endophytic bacterial isolates from the roots and corms of banana were tested for their biocontrol efficiency against Banana bunchy top virus (BBTV). Molecular characterization using RAPD and microsatellite markers revealed genomic variability in the endophytic Pseudomonas and Bacillus isolates. Bio-formulations of mixtures of the rhizobacterial isolate Pseudomonas fluorescens (Pf1) and endophytic Bacillus spp. (EPB22) were effective in reducing the incidence of BBTV under green-house (80%) and field conditions (52%). Reduction in virus titer (0.64) was noticed in the plants treated with compatible mixtures of rhizobacterial and endophytic bacterial isolates as evidenced by ELISA, in comparison to control plants (1.69). In addition to disease control, a significant increase in the yield (53.33%) was noticed in the bacterized plants when compared to the control plants. Pathogenesis-related (PR) proteins, chitinase and β-1,3-glucanase and defense-related proteins, peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase and phenolic compounds were significantly activated in the bacterized plants, thus inducing resistance against bunchy top virus. Populations of endophytic bacteria also remained high and stable throughout the growing period. Thus, application of mixtures of rhizosphere and endophytic bacteria increases yield and has a potential role in inducing resistance against Banana bunchy top virus.     

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.     

Genetic transformation of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' via microprojectile bombardment

 An effective method has been developed for the stable transformation and regeneration of Cavendish banana (Musa spp. AAA group) cv 'Grand Nain' by microprojectile bombardment. Embryogenic cell suspensions were initiated using immature male flowers as the explant. Cells were co-bombarded with the neomycin phosphotransferase (nptII) selectable marker gene under the control of a banana bunchy top virus (BBTV) promoter or the CaMV 35S promoter, and either the β-glucuronidase (uidA) reporter gene or BBTV genes under the control of the maize polyubiquitin promoter. Plants were regenerated, under selection with kanamycin, that were co-transformed with nptII and either the uidA or BBTV genes. Molecular characterisation of transformants demonstrated that the transgenes had been stably integrated into the banana genome. Received: 22 June 1998 / Revision received: 29 March 1999 / Accepted 1 May 1999     

香蕉幼苗三类有机小分子溶质对尖孢镰刀菌侵染的生理响应

为阐明香蕉枯萎病发病机制,研究了尖孢镰刀菌侵染后,香蕉植株中几种对尖孢镰刀菌生长有显著作用的物质(氨基酸、有机羧酸、酚酸)种类和含量的变化。结果表明:(1)病原菌侵染后,伤害逐渐加剧,株高和生物量显著下降。(2)病原菌侵染后,叶片氨基酸总量显著升高,其中丝氨酸、缬氨酸、组氨酸、异亮氨酸和亮氨酸增幅较大,病原菌侵染16 d,其含量分别为侵染前的7.1、6.2、4.4、3.5和2.3倍;而根氨基酸总量开始显著降低,差异逐渐变小。(3)叶片有机羧酸酸含量在病原菌侵染后显著增加,而在根中显著降低。侵染植株叶片中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株叶片的2.6、1.6、1.9、1.8和2.3倍;根中草酸、柠檬酸、苹果酸、琥珀酸和延胡索酸含量分别是未侵染植株的81%、42%、44%、28%和59%。(4)病原菌侵染后,植株叶片和根中酚酸含量都显著升高。叶片中阿魏酸、肉桂酸和水杨酸含量分别是未侵染叶片的2.9、1.7和2.9倍;而根中对羟基苯甲酸和丁香酸含量分别是未侵染根的4.3和1.5倍。研究结果表明,尖孢镰刀菌侵染后,植物与病原菌的相互作用使得植物体内抑菌物质和促菌物质都会相应的增加,植株对病害有一定的抗性,但促菌物质种类和含量较高最终使得感病植株发病。     

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.