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.     

The transmission of banana bunchy-top virus in micropropagated bananas

Plants were established in vitro from banana bunchy0top virus (BBTV) infeeted plants. Explants containing either vegetative shoot apices or terminal floral apices were used to initiate cultures. Plants multiplied in culture were indistinguishable from non-infected (control) plants and lacked characteristic symptoms of BBTV infection. After 16 months in culture plants were established in the glasshouse and after 1 month in pots some plants started to show symptoms of the disease. After a further 5 months, 73% of the plants showed characteristic symptoms of the disease while 27% were symptomless and similar in appearance to control plants. These plants have been grown to maturity in the field without showing recognizable symptoms. This study demonstrates that BBTV can be transmitted in an apparently symptomless condition in culture and has important consequences for the dissemination of banana germplasm within Australia and internationally.     

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.     

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.     

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。     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

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。     

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.     

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。     

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.     

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