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.     

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.     

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.     

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.     

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.     

Population distribution and density of Pentalonia nigronervosa (Hemiptera: Aphididae) within banana mats: influence of plant age and height on sampling and management

The banana aphid, Pentalonia nigronervosa Coquerel (Hemiptera: Aphididae), is the most economically important pest of banana (Musa spp.) fields in Hawaii. Recently, there has been a concerted effort in Hawaii to learn more about the biology and ecology of this pest. However, limited work has been directed at determining the distribution of P. nigronervosa in banana fields and developing an integrated pest management plan. Therefore, a survey was conducted in banana fields throughout the Hawaiian Islands to determine the distribution and density of P. nigronervosa within banana mats from plants of different stages. Another aim was to determine whether the presence of ants on banana plants could be used as a reliable indicator of aphid infestations. Results of the survey showed that plants < or = 1.5 m (small sucker) in height contain the highest aphid populations per meter in plant height and that mother plants (> or = 2.5 m) had the lowest aphid counts and rate of infestation compared with small and intermediate suckers (> 1.5 < 2.5 m). More specifically, aphid population was reduced by approximately 12 aphids for every meter increase in plant height and that aphids are rarely found > or = 2.5 m within the plant canopy. Although there was an increase likelihood of finding ants on banana plants with higher aphid densities, results suggest that ants would be present on plants in the absence of aphids. Implications of these and other findings with respect to sampling and managing P. nigronervosa and associated Banana bunchy top virus are discussed.     

Cucurbit aphid‐borne yellows virus (CABYV) modifies the alighting,settling and probing behaviour of its vector Aphis gossypii favouring its own spread

Plant pathogens are able to influence the behaviour and fitness of their vectors in such a way that changes in plant–pathogen–vector interactions can affect their transmission. Such influence can be direct or indirect, depending on whether it is mediated by the presence of the pathogen in the vector's body or by host changes as a consequence of pathogen infection. We report the effect that the persistently aphid‐transmitted Cucurbit aphid‐borne yellows virus (CABYV, Polerovirus) can induce on the alighting, settling and probing behaviour activities of its vector, the cotton aphid Aphis gossypii. Only minor direct changes on aphid feeding behaviour were observed when viruliferous aphids fed on non‐infected plants. However, the feeding behaviour of non‐viruliferous aphids was very different on CABYV‐infected than on non‐infected plants. Non‐viruliferous aphids spent longer time feeding from the phloem in CABYV‐infected plants compared to non‐infected plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. Viruliferous aphids showed a clear preference for non‐infected over CABYV‐infected plants at short and long time, while such behaviour was not observed for non‐viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimise the transmission and spread of this phloem‐limited virus.     

Relations of carrot red leaf and carrot mottle viruses with their aphid vector, Cavariella aegopodii

Studies with Scottish isolates of carrot red leaf (CRLV) and carrot mottle (CMotV) viruses confirmed the dependency of CMotV on CRLV for transmission by the aphid Cavariella aegopodii. CMotV was transmitted by aphids only when the two viruses were present in the same source plant, and its transmission was not assisted by anthriscus yellows virus, which acts as a helper for parsnip yellow fleck virus. Some test plants became infected with CRLV alone, and a few with CMotV alone. In winter, aphid transmission of CRLV and CMotV was greatly increased when the source plants received supplementary lighting whereas the CMotV infectivity of sap was not increased. C. aegopodii acquired CRLV and CMotV after minimum acquisition access times of 30 min and inoculated them after minimum inoculation access times of 2 min. There was a minimum latent period of 7–18 h. The viruses were retained by the aphid after moulting and are therefore circulative in the vector, but were not transmitted to progeny insects. Aphids allowed 24 h to acquire the viruses continued to transmit them for at least 12 days, but some aphids allowed 6 h or less for virus acquisition ceased to transmit after 3 or 4 days. CRLV is considered a tentative member of the luteovirus group.     

Cytopathology, Mode of Aphid Transmission and Search for the Casual Agent of Cotton Bunchy Top Disease

The cytopathological effects of cotton bunchy top (CBT) disease and its mode of transmission by Aphis gossypii Glover (cotton aphid), were studied. CBT infection affected the leaf epidermal layer producing a loose, ruptured and rough surface morphology with many stomata closed and misshapen. Roots of CBT‐infected plants showed reduced growth, small knots and a dark brown appearance. A single aphid per plant was capable of transmitting CBT at 5%, whereas three aphids per plant transmitted CBT to 50% of the cotton seedlings and 20 aphids per plant transmitted the disease agent to 80% of the cotton seedlings. Aphis gossypii acquired CBT after a minimum acquisition access period of 5 min and transmitted the agent after a minimum inoculation access period of 1 h. Both alate and apterous aphids and nymph instars 2, 3 and 4 of A. gossypii transmitted CBT. This preliminary data suggest that A. gossypii transmits CBT in a semi‐persistent manner. Myzus persicae Sulz (green peach aphid) was unable to transmit CBT. A comprehensive attempt to isolate the CBT agent, using a range of virological techniques including double‐stranded RNA extraction, two‐dimensional gel electrophoresis for viroid, circular DNA test, nanovirus polymerase chain reaction (PCR), luteovirus PCR and enzyme‐linked immunosorbent assay, phytoplasma test, nucleoprotein purification and electron microscopy, was unsuccessful, raising the possibility that CBT may be caused by a unique new pathogen.     

Relations of the semi-persistent viruses, parsnip yellow fleck and anthriscus yellows, with their vector, Cav arietta aegopodii

Studies were made of the relations of parsnip yellow fleck virus (PYFV) and its helper virus, anthriscus yellows (AYV), with their aphid vector, Cavariella aegopodii. Apterous insects were more efficient vectors than alates; apterous nymphs were as efficient as apterous adults. C. aegopodii never transmitted PYFV in the absence of AYV, but aphids carrying both viruses infected some test plants with one or other virus alone. C. aegopodii that fed first on a source of AYV and then on a source of PYFV transmitted both viruses to test plants, but aphids that fed on the sources in the reverse order transmitted only AYV. Test plants receiving some aphids from a source of AYV, and others from a source of PYFV, became infected only with AYV. C. aegopodii acquired AYV or the AYV/PYFV complex from plants in a minimum acquisition access time (AAT) of 10–15 mm and inoculated the viruses to test plants in a minimum inoculation access time (IAT) of 2 min. Increasing either AAT or IAT, or both, to 1 h or longer increased the frequency of transmission of each virus. Starving the insects before the acquisition feed on AYV or AYV/PFYV sources did not affect transmission. Aphids already carrying AYV acquired PYFV from plants in a minimum AAT of only 2 min; they acquired and inoculated PYFV in a minimum total time of 12 min. The data suggest that AYV is confined to deeply lying tissues whereas PYFV is distributed throughout the leaf. C. aegopodii transmitted both PYFV and AYV in a semi-persistent manner: the aphids retained both viruses for up to 4 days but lost them on moulting. Neither virus was passed to progeny of viruliferous adults. Earlier results suggesting that AYV is a persistent virus may have been caused by contamination of the AYV culture with carrot red leaf virus.     

Molecular comparative analysis of component 1 (DNA-R) of an Egyptian isolate of banana bunchy top nanovirus isolated from banana aphid (Pentalonia nigronervosa)

We are reporting a molecular comparative analysis of component 1 BBTV-DNA-R of an Egyptian isolate of (BBTV) and 30 different geographical isolates. DNA was extracted from BBTV-infected adult banana aphids collected from El-Qalubia Governorate, Egypt. Using specific primers the BBTV-DNA-R was amplified, cloned into a prokaryote vector, sequenced and a molecular comparative analysis of BBTV-DNA-R of this study and some overseas isolates of BBTV-infected banana plants was determined. Results showed that the component 1 consists of 1108 nts and contains a sequence of 69 nts representing the CR-SL of 31 nts. A CR-M (90 nts) at the position (972–1062) characterized with GC-rich sequence from nts 76 to 90 (average of 80% G + C) was found. Alignment results of BBTV DNA-R confirmed the presence of a number of conserved regions in all isolates. Large ORF of 861 nts at position 102 to 962 in the virion sense were detected. The predicted protein of this ORF consisted of 286 amino acids and had a molecular weight of 33.8 kDa. The DNA-phylogenetic analysis showed a percent identity of 98.0 and 97.9 between BBTV DNA-R and isolates of Pakistan (isolate TJ1) and Australia (isolate V1), respectively. The similarities between the gene product of Egyptian BBTV DNA-R and the 30 overseas isolates ranged from 93.7 to 99.0%. Differences in phylogenetic trees based on the entire sequence of BBTV DNA-R, CR-M and amino acid sequences confirmed the existence of two taxonomic groups of BBTV and the Egyptian isolate belongs to the south pacific group.     

香蕉束顶病毒nsp酵母双杂交诱饵质粒的构建及自激活验证

香蕉束顶病毒(Banana bunchy top virus,BBTV)DNA6编码的核穿梭蛋白(nuclear shuttle protein,NSP)在病毒的侵染、复制、运输中起重要作用。为了利用酵母双杂交系统研究BBTV DNA6与寄主香蕉蛋白的互作,本实验利用两对引物的PCR扩增得到BBTV -nsp片段,混合各自PCR产物进行熔化退火可得到1/4两端含有EcoRⅠ和BamHⅠ的酶切位点序列的DNA产物,将目的片段连接到酵母双杂交系统的pGBKT7诱饵载体中,成功获得pGBKT7-nsp,并将重组质粒pGBKT7-nsp转化入Y2H Gold酵母菌株中进行毒性检测和自激活验证。结果表明,pGBKT7-nsp没有自激活活性,同时对酵母细胞也无毒性,符合酵母双杂交诱饵质粒的要求,可用于下一步的蛋白互作实验。     

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.     

The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii

Transmission of parsnip yellow fleck virus (PYFV) by the aphid Cavariella aegopodii occurs only when the aphids are also carrying the helper virus, anthriscus yellows (AYV). None of five other viruses tested was able to act as helper. In experiments in which aphids were allowed to feed through membranes on crude or treated extracts from infected plants, aphids already carrying AYV acquired PYFV, but virus-free aphids failed to acquire either AYV or PYFV. PYFV was not transmitted by insects injected with haemolymph from aphids carrying both viruses, or with purified preparations of PYFV. PYFV was transmitted when AYV-carrying aphids, except those whose stylets had been removed, were contaminated externally with PYFV preparations. Ultraviolet irradiation of infected leaves did not prevent aphids from acquiring AYV, presumably because it is confined to deeply-lying tissues. AYV-carrying aphids could acquire PYFV from u.v.-irradiated leaves after acquisition access times of 2 h but not after feeds of only 2 or 15 min (which are adequate on unirradiated leaves), suggesting that PYFV is present in all parts of the leaf. No ‘helper agent’ distinct from AYV itself was detected in these experiments or in experiments on minimum acquisition feeding time or maximum period of persistence in the aphid. U.v.-inactivated PYFV competed with infective PYFV for retention sites in AYV-carrying aphids, whereas AYV apparently did not. It is suggested that there is no helper agent for PYFV, other than AYV particles. The possibility that there is one for AYV is not excluded.     

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.     

Aphid behavioral responses to virus‐infected plants are similar despite divergent fitness effects

We compared the settling preferences and reproductive potential of an oligophagous herbivore, the pea aphid, Acyrthosiphon pisum Harris (Hemiptera: Aphididae), in response to pea plants, Pisum sativum L. cv. ‘Aragorn’ (Fabaceae), infected with two persistently transmitted viruses, Pea enation mosaic virus (PEMV) and Bean leaf roll virus (BLRV), that differ in their distribution within an infected plant. Aphids preferentially oriented toward and settled on plants infected with PEMV or BLRV in comparison with sham‐inoculated plants (plants exposed to herbivory by uninfected aphids), but aphids did not discriminate between plants infected with the two viruses. Analysis of plant volatiles indicated that plants inoculated with either virus had significantly higher green leaf volatile‐to‐monoterpene ratios. Time until reproductive maturity was marginally influenced by plant infection status, with a trend toward earlier nymph production on infected plants. There were consistent age‐specific effects of plant infection status on aphid fecundity: reproduction was significantly enhanced for aphids on BLRV‐infected plants across most time intervals, though mean aphid fecundity did not differ between sham and PEMV‐infected plants. There was no clear pattern of age‐specific survivorship; however, mean aphid lifespan was reduced on plants infected with PEMV. Our results are consistent with predictions of the host manipulation hypothesis, extended to include plant viruses: non‐viruliferous A. pisum preferentially orient to virus‐infected host plants, potentially facilitating pathogen transmission. These studies extend the scope of the host manipulation hypothesis by demonstrating that divergent fitness effects on vectors arise relative to the mode of virus transmission.