Banana Bunchy Top Virus Infection Research Articles

Biochemical changes in some banana cultivars infected by Banana bunchy top virus

Bunchy top is one of the major diseases on bananas that caused economic losses. This study aimed to determine changes in protein content, the concentration of phenolic compounds, the amount of chlorophyll, and sugar content in several Banana bunchy top virus (BBTV)-infected banana cultivars. The research involved the artificial inoculation, extraction, and biochemical analysis of banana seedlings from the cultivars Mas, Cavendish, Kepok, and Raja. Inoculation was performed using the vector Pentalonia sp.. A factorial complete randomized design (CRD) was used as the experimental design. Each experimental unit consisted of 5 repetitions. Protein, phenol, chlorophyll, and sugar content measurements were performed using a UV-Vis spectrophotometer. Test samples were in the form of fresh leaves from BBTV-infected and non-BBTV-infected plants. Regression analysis and tests for significance were performed to determine the biochemical changes that occurred after testing. The results showed that the average levels of phenols, chlorophyll, protein, and total sugars in BBTV-infected plants were significantly altered. The chlorophyll content decreased from 82–87% in Mas, Cavendish, and Raja cultivars. Total plant phenol in all BBTV-infected cultivars increased by 69.2 to 348.3 ppm. Specificity was evident in the change in protein concentration, which increased by 95 ppm and 84.5 ppm in the Mas and Cavendish cultivars, respectively. This contrasts with the Kepok and Raja cultivars, which decreased by 660.5 ppm and 113.6 ppm, respectively. Sugar levels increased from 3133.9 to 3298.6 ppm in all BBTV-infected breeds. According to the data, BBTV infection has been shown to cause physiological and biochemical changes in Mas, Cavendish, Kepok, and Raja.

Comparative RNA-seq analysis of resistant and susceptible banana genotypes reveals molecular mechanisms in response to banana bunchy top virus (BBTV) infection

Bananas hold significant economic importance as an agricultural commodity, serving as a primary livelihood source, a favorite fruit, and a staple crop in various regions across the world. However, Banana bunchy top disease (BBTD), which is caused by banana bunchy top virus (BBTV), poses a considerable threat to banana cultivation. To understand the resistance mechanism and the interplay of host suitability factors in the presence of BBTV, we conducted RNA-seq-based comparative transcriptomics analysis on mock-inoculated and BBTV-inoculated samples from resistant (wild Musa balbisiana) and susceptible (Musa acuminata ‘Lakatan’) genotypes. We observed common patterns of expression for 62 differentially expressed genes (DEGs) in both genotypes, which represent the typical defense response of bananas to BBTV. Furthermore, we identified 99 DEGs exclusive to the 'Lakatan' banana cultivar, offering insights into the host factors and susceptibility mechanisms that facilitate successful BBTV infection. In parallel, we identified 151 DEGs unique to the wild M. balbisiana, shedding light on the multifaceted mechanisms of BBTV resistance, involving processes such as secondary metabolite biosynthesis, cell wall modification, and pathogen perception. Notably, our validation efforts via RT-qPCR confirmed the up-regulation of the glucuronoxylan 4-O-methyltransferase gene (14.28 fold-change increase), implicated in xylan modification and degradation. Furthermore, our experiments highlighted the potential recruitment of host's substrate adaptor ADO (30.31 fold-change increase) by BBTV, which may play a role in enhancing banana susceptibility to the viral pathogen. The DEGs identified in this work can be used as basis in designing associated gene markers for the precise integration of resistance genes in marker-assisted breeding programs. Furthermore, the findings can be applied to develop genome-edited banana cultivars targeting the resistance and susceptibility genes, thus developing novel cultivars that are resilient to important diseases.

Phylogenetic analysis and molecular characterization of BBTV DNA-R of wild and cultivated banana isolates from East Java, Indonesia

Banana bunchy top virus (BBTV) molecular detection and understanding its origin are important issues for mitigating future spread. The aim of this study was to molecularly detect BBTV infection and analyze the characteristic also phylogenetic of banana isolates from East Java Indonesia. Two BBTV asymptomatic wild bananas and two BBTV symptomatic banana cultivars were examined. PCR amplifications were accomplished using BBTV DNA-R primers for master replication initiation protein. Sequences evaluations were conducted in SeqScanner. Sequences identification was performed in nucleotide BLAST. Translation of ORFs was determined using ORF Finder server tool. Protein identification was conducted in protein BLAST. Sequences polymorphisms were analyzed using DnaSP6. Phylogenetic analysis was employed using Neighbor-Joining algorithm with Kimura two-parameter (K2P) substitution model in MEGA7. Results showed that BBTV DNA-R components were detected in all isolates and confirmed as Rep protein. The sequences length were varied from 616 to 1,074 bp, low GC content (42.90%) and low conservation (56.47%). Asymptomatic wild bananas generated shorter length and more variable sequences, presumably related to the resistance mechanism. Phylogenetic analysis of BBTV DNA-R East Java with other 38 homolog sequences worldwide were found clustered in Asian Group, closely related to Vietnam, Thailand, and China. Hence, it presumably originated from China mainland via Malay Peninsula route.

The aphid Pentalonia nigronervosa (Hemiptera: Aphididae) takes advantage from the quality change in banana plant associated with Banana bunchy top virus infection.

Viral diseases can change plant metabolism, with potential impacts on the quality of the plant's food supply for insect pests, including virus vectors. The banana aphid, Pentalonia nigronervosa Coquerel, is the vector of the Banana bunchy top virus (BBTV), the causal agent of Banana bunchy top disease (BBTD), the most devastating viral disease of bananas in the world. The effect of BBTV on the life-history traits and population dynamics of P. nigronervosa remains poorly understood. We therefore studied the survival rate, longevity, daily fecundity per aphid, tibia length, population growth, and winged morph production of a P. nigronervosa clone grown on healthy or infected, dessert, or plantain banana plants. We found that daily fecundity was higher on infected banana than on healthy banana plants (plantain and dessert), and on plantain than on dessert banana plants (healthy and infected). Survival and longevity were lower on infected dessert bananas than on other types of bananas. In addition, virus infection resulted in a decrease in aphid hind tibia length on both plant genotypes. The survival and fecundity table revealed that the aphid net reproduction rate (Ro) was highest on plantains (especially infected plantain), and the intrinsic growth rate (r) was highest on infected plants. Finally, the increase of aphids and alate production was faster first on infected plantain, then on healthy plantain, and lower on dessert banana (infected and uninfected). Our results reinforce the idea of indirect and plant genotype-dependent manipulation of P. nigronervosa by the BBTV.

UJI KETAHANAN TIGA VARIETAS PISANG (Musa sp.) TERHADAP ISOLAST BANANA BUNCHY TOP VIRUS (BBTV)

Banana is a horticultural crop that has economic value and is widely cultivated in tropical countries. Banana Bunchy Top Virus (BBTV) is the cause of dwarf disease in banana plants (Musa. sp). The vector for the spread of this banana dwarf disease is the banana aphid (Pentalonia nigronervosa). BBTV has been reported in various regions of Indonesia, having been reported in Bengkulu in 2020. However, data on the resistance of BBTV infections in Indonesia are still very limited. This study aimed to evaluate the resistance of three types of bananas (Barangan, Kepok, and Ambon Hijau) to BBTV infection. In this study, two treatments were carried out, namely inoculated and without BBTV virus inoculation with three types of bananas. BBTV detection was carried out using the PCR method. BBTV transmission using aphids succeeded in infecting the test plants with a disease incidence of 12.5% ​​to 75.0% at 30 DAI. The most common symptom seen is, slightly yellowed leaf edges. the severity of BBTV on the Barangan banana plant was 20.83%, kepok was 4.17% and Ambon Hijau was 25.00%. Samples on the leaves of the test banana plant that showed symptoms of BBTV were taken for detection using Polymerase Chain Reaction (PCR) with a pair of related NSP primers in the BBTV genome with a size of ± 500 bp. The results of the detection of BBTV virus showed that the types of bananas that were resistant to BBTV were Kepok bananas, Barangan bananas and Ambon Hijau types which showed moderate resistance to BBTV

Identification of suitable internal control genes for gene expression analysis of banana in response to BBTV infection

Banana is one of the most abundant crops produced annually in the Philippines. The presence of banana bunchy top virus (BBTV) leading to banana bunchy top disease is one of the factors hindering the continuous production of the fruit crop. The use of an appropriate and stable internal control gene as reference in validation of differentially-expressed genes in an organism is important. This study aims to identify appropriate internal control genes for differential gene expression analysis in Musa balbisiana and Musa acuminata specific for BBTV infection. RNA extraction, complementary DNA (cDNA) synthesis and RT-qPCR (quantitative real time polymerase chain reaction) of BBTV-resistant and BBTV-susceptible Musa genotypes were performed. The RT-qPCR quantification data were then subjected to analysis on RefFinder software and geomean ranking values were calculated along with the four statistical algorithms (delta Cq, Genorm, BestKeeper and NormFinder). Based on the comprehensive ranking values in the software, L2 gene was the most suitable internal control gene for the differential expression analysis of both BBTV-resistant and BBTV-susceptible banana accessions. The internal control gene is recommended for the validation of selected candidate resistance and host factor genes in response to BBTV infection.

Response of banana germplasms to banana bunchy top virus

Banana bunchy top virus (BBTV) is one of the important viruses causing disease in bananas and its infection has the potential to cause yield loss. This study was conducted to evaluate the response of several commercial cultivars (Cavendish, Bebek, Goroho, Tanduk, and Barangan Merah) and wild accessions (Klutuk NTT, Halabanensis, SPn 001, LNT 001, and Microcarpa) of banana to BBTV infection. Transmission of BBTV was carried out through banana aphid Pentalonia nigronervosa, using 20 adult aphids per plant with an acquisition feeding period of 24 hours on BBTV-infected plants and an inoculation feeding period on healthy test plants for 48 hours. Observation on plant growth and disease intensity was conducted for 8 weeks after inoculation. At the end of the observation period, only 5 cultivars, i.e. Cavendish, Bebek, Goroho, Barangan Merah, and Halabanensis showed typical symptoms of BBTV with disease incidence reached 80%, 60%, 20%, 20%, and 20% respectively. Significant inhibition of plant height and leaves width occurred in Cavendish, Bebek, and Goroho i.e. 44.60%, 36.31%; 12.62%, 41.08%; and 25%, 10.13%, respectively. This paper discusses the need for banana germplasm exploration to find sources of resistance to BBTV.

Survey of Banana bunchy top virus on non-cultivated bananas in West Java

Banana is a horticulture crop originated from Southeast Asia. Therefore, the genetic diversity of bananas is presumably high in Indonesia. Banana bunchy top disease caused by Banana bunchy top virus (BBTV) has been reported occurred in several regions in Indonesia, especially in the banana cultivation. A survey was conducted in April 2019 in West Java to make documentation on the types of bananas that grow unattended and to monitor BBTV infection. Banana cultivars with various types of genomes was found during the survey, i.e. “Mas”, “Uli”, and “Rejang” (type AA); “Ambon”, “Muli”, and “Urang” (type AAA); “Kapas” and “Raja” (type AAB). Besides, wild bananas were also found, i.e.“Caukole” in Cianjur and “Tongka Langit” in Tasikmalaya with genome types AAw and T, respectively. Leaf samples from infected plants were collected and taken to the laboratory for detection of BBTV using polymerase chain reaction (PCR) method. Specific DNA band of BBTV around 240 bp was successfully amplified from all leaf samples using mRepF/mRepR specific primer pairs. The results of this survey indicated that BBTV has spread in the field and has the potential to be a source of disease inoculum if no immediate control efforts are made.

First Report of Banana Bunchy Top Disease on Banana in Bengkulu.

Banana is a horticulture crop that has economic value and is widely cultivated in tropical countries. Banana production in Bengkulu province reached 259,748 quintals, then durian (110,387 quintals), tangerines (94,396 quintals) (BPS 2015). Banana bunchy top disease caused by Banana bunchy top virus (BBTV) infection is considered the most crucial virus disease affecting yield losses of a banana plantation in Asia, Africa, and the South Pacific. However, the incidence and molecular characters of BBTV has never been reported in Bengkulu. This research aims to characterize symptom variations, disease incidence, and disease severity of BBTV infection in Bengkulu and virus detection using molecular methods by polymerase chain reaction (PCR). Disease incidence of BBTV was measured based on field symptoms. The disease survey was conducted in Bengkulu city, Bengkulu Utara district, and Rejang Lebong district. The study showed that the incidence of BBTV in Bengkulu City, Bengkulu Utara, and Rejang Lebong ranged from 0% to 100%. The most common symptoms observed in the field involved vein clearing, upturned leaf, chlorotic, and ragged margins, reducing petiole length, distance, lamina width, and stunting. Banana crops that are infected with BBTV in the vegetative phase will not produce fruit. In contrast, viral infection in the generative phase causes the formation of stunted fruit that is not suitable for harvesting. Thus, the potential loss of yield due to stunted disease can reach 100%. This study's results are the first reports of BBTV infection in banana crops in Bengkulu. Disease diagnosis will form the basis of disease control strategies in banana crops.

Virucidal activity of silver nanoparticles against Banana bunchy top virus (BBTV) in banana plants

BackgroundBanana bunchy top virus (BBTV) is a destructive viral disease in many countries including Egypt; it causes severe economic losses in banana crop. Recently, nanotechnology was used to generate resistance against plant viruses. The main purpose of this study was to use silver nanoparticles (AgNPs) as antiviral agents against BBTV. In this research, three different concentrations of AgNPs (40, 50 and 60 ppm) were applied by foliar spray post-BBTV inoculation. In addition, photopigments, oxidative enzymes, proline and phenolic compounds were determined. Besides, Random amplified polymorphic DNA (RAPD) and Sequence-related amplified polymorphism (SRAP) markers were used to evaluate the genotoxicity of AgNPs as antiviral factors against BBTV, compared with the control plants.ResultsIn the current study, it was observed that banana plants infected with BBTV and treated with 50 ppm AgNPs have not shown any external symptoms where the rate of infection was 36%. On the other hand, banana plants treated with 50 ppm AgNPs after viral infection gave a significant increase in dry weight and leaf area, compared with BBTV infected banana plants (viral control). Our study showed that 50 ppm AgNPs treatment post-virus inoculation induced non-significantly and significant changes in chlorophyll (a and b) and carotenoids, respectively, compared with healthy and nano-controls. In contrast, phenol, proline and oxidative enzymes were significantly increased in all plants treated with 50 ppm AgNPs post-virus inoculation, compared with the healthy control. Our findings observed that the banana plants sprayed with 50 ppm AgNPs after BBTV infection induced a few changes at the genomic DNA level in the banana plants, whereas both RAPD and SRAP markers scored nearly the same polymorphism 36.99 and 37.5%, respectively. So, genotoxicity induced by banana plants treated with 50 ppm AgNPs post-BBTV inoculation was low.ConclusionsIt is evident from the study results the role of AgNPs as a novel, safe and effective antiviral agent against BBTV. These results should be taken into consideration in future for the use of AgNPs for plant viruses management.

Independent modulation of individual genomic component transcription and a cis-acting element related to high transcriptional activity in a multipartite DNA virus

BackgroundThe genome of Banana bunchy top virus (BBTV) consists of at least six circular, single-stranded DNA components of ~ 1 kb in length. Some BBTV isolates may also carry satellite DNA molecules that are not essential for BBTV infection. The relation between multipartite DNA virus replication and their transcriptional levels and the underlying mechanism remain unclear.ResultsTo understand the coordinated replication and transcription of the multiple genomic components, the absolute amounts of each BBTV DNA component were measured by real-time PCR (qPCR), and their transcriptional levels were determined by RNAseq and reverse transcription-qPCR (qRT-PCR). Significant differences were found in the absolute amounts of individual BBTV genomic components. Transcriptional levels of each BBTV genomic component obtained from the RNAseq data matched closely to those obtained from qRT-PCR, but did not correspond to the absolute amount of each DNA component. The ratio of transcript over DNA copies ranged from 46.21 to 1059.44%, which was possibly regulated by the promoter region in the intergenic region of each component. To further determine this speculation, the promoter region of the DNA-S, −M or -N was constructed to the upstream of green fluorescent protein (GFP) gene for transient expression by agrobacterium-mediated transformation method. The qRT-PCR showed the highest transcriptional activity was promoted by DNA-N promoter, about 386.58% activity comparing with CaMV 35S promoter. Confocal microscopy observation showed that the intensity of green fluorescence was corresponding to that of qRT-PCR.ConclusionsOur data clearly showed that BBTV was able to control the transcriptional level of each DNA component independently by through the promoter sequences in the intergenic region. Moreover, a cis-acting element from DNA-N component had a high transcriptional activity.

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

ABSTRACTRhizospheric 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.

Virus titre determines the efficiency of Pentalonia nigronervosa (Aphididae: Hemiptera) to transmit banana bunchy top virus.

Banana bunchy top disease (BBTD) caused by banana bunchy top virus (BBTV) is one of the most serious viral diseases of banana and plantains. BBTV is transmitted by Pentalonia nigronervosa (Hemiptera, Aphididae) in a persistent circulative manner. Better knowledge of vector-virus-host relationship and the mechanism of transmission is essential for developing an effective control strategy. In this study, the viral copies in single to group of aphids with different acquisition access period (AAP) were quantified using SYBR green-based quantitative polymerase chain reaction (qPCR). The result indicated that a single aphid was able to acquire 861.04 copies of the virus after 24h of AAP from the infected banana plant and transmitted the virus to 16.6% tissue culture plants, whereas 50 viruliferous aphids (15,066.94 viral copies) were necessary to achieve 100% transmission in a shortest time of 21.6days. The number of viral copies acquired by the aphids were gradually increased with increased AAP. Hundred percent transmission was observed with 20 aphids in 48h of AAP or 30-50 aphids in 24h of AAP. The inoculated plants expressed typical bunchy top symptoms quickly when higher number of aphids (30 and above) were used with 24h of AAP. Further, we report that the tissue culture banana plants are highly prone or vulnerable to BBTV infection compared to sucker grown plants. We conclude that higher the number of viral copies in the vector, higher the percent transmission and quicker the symptom expression and the results will contribute to a better understanding of vector-BBTV interactions and useful for epidemiological studies.

Distribution, molecular characterization and diversity of banana bunchy top virus in Tripura, India.

Banana bunchy top virus (BBTV) is a serious threat to banana (Musa spp.) production in India. Generally, BBTV isolates within the country share very low genetic diversity. However, in India, relatively greater diversity has been observed between isolates from north-eastern (NE) region (Meghalaya) and rest of India. Tripura is situated in the south-west corner of NE India and shares international border with Bangladesh. During 2014-2015, diagnostic surveys were conducted in seven districts of Tripura and polymerase chain reaction based detection established that BBTD is widely prevalent in all parts of Tripura showing an average incidence of 22.02%. Among the cultivars, maximum BBTV infection (27.03%) was recorded in 'Chini Champa', followed by plantain (24.29%). A representative population (31 isolates) of BBTV from Tripura was characterized based on DNA R and DNA S. Phylogenetic analysis based on BBTV DNA R and DNA S generated two broad clusters of Pacific-Indian Oceans (PIO) and south-east Asian groups including all Tripura isolates within PIO cluster. The clustering pattern and genetic diversity of BBTV population from Tripura suggested monophyletic origin of majority of representative isolates from a common ancestor of PIO group. The exchange of vegetative propagules within and in between countries could have contributed to the geographical expansion of PIO isolates in Tripura. However, four variant BBTV isolates has been identified from North Tripura and Khowai districts possessing somewhat unique variability than that of distinct isolate (BBTV-Umiam) reported from NE India (Meghalaya).

Discovery of novel inhibitors targeting movement protein for controlling the transmission of banana bunchy top virus infection in plantain by structure-based virtual screening

Banana bunchy top virus (BBTV), the pathogen causing banana bunchy top disease (BBTD) belongs to the genus Babuvirus of the family Nanoviridae and produces significant yield loss. BBTD is the most destructive viral diseases affecting bananas worldwide causing infections that result in bunched leaves, stunted and fruitless plants. So far, there are no effective control measures for controlling and preventing this viral disease. The amino terminal region of the movement protein is responsible for cell-to-cell movement. The present study aims at inhibiting this target region by discovering novel inhibitors through virtual screening of small molecule libraries coupled with post-docking analysis of most potent inhibitors. Our study based on virtual screening of small molecule datasets determined 10 most potential inhibitors to be considered as lead compounds in controlling the spread of BBTV infection in plantain.

Discovery of novel inhibitors targeting movement protein for controlling the transmission of banana bunchy top virus infection in plantain by structure-based virtual screening

Banana bunchy top virus (BBTV), the pathogen causing banana bunchy top disease (BBTD) belongs to the genus Babuvirus of the family Nanoviridae and produces significant yield loss. BBTD is the most destructive viral diseases affecting bananas worldwide causing infections that result in bunched leaves, stunted and fruitless plants. So far, there are no effective control measures for controlling and preventing this viral disease. The amino terminal region of the movement protein is responsible for cell-to-cell movement. The present study aims at inhibiting this target region by discovering novel inhibitors through virtual screening of small molecule libraries coupled with post-docking analysis of most potent inhibitors. Our study based on virtual screening of small molecule datasets determined 10 most potential inhibitors to be considered as lead compounds in controlling the spread of BBTV infection in plantain.

Cloning of BBTV (Banana Bunchy Top Virus) components and screening of BBTV using functionalized gold nanoparticles.

Banana bunchy top virus (BBTV) affects all varieties of banana plants and causes heavy economic loss in most of the banana cultivating areas. The BBTV genome comprises of six DNA components; in this study, we have cloned the six BBTV-DNA components from one of the BBTV-infected plants (Tri-8) and were submitted to GenBank. Analysis of the BBTV DNA-R component showed that it belonged to south Pacific group. Resistance against BBTV has not been observed so far in banana plants and removal and killing of the infected plants has been routinely practiced. Hence, early detection of BBTV infection would be desirable and various detection methods routinely employed include enzyme linked immunosorbent assay (antigen-antibody based) and molecular-based methods such as polymerase chain reaction (PCR), qPCR, or LAMP PCR. Most of these methods require enzymes or antibodies for detection and hence are expensive. Here, we report a visual detection method (AuNP probe assay) using gold nanoparticles (AuNPs) functionalized with an ssDNA-thiolated probe (CR1). This method is based on the hybridization of the functionalized AuNPs with the target DNA (BBTV). In the AuNP probe assay, the functionalized AuNPs retains red colour when BBTV DNA is present, and in the absence of BBTV DNA, the colour of the functionalized AuNPs changes to purple when salt is added. The AuNP probe assay was compared with PCR for the detection of banana plants and it was found that AuNP probe assay was better than PCR in detecting BBTV infection (86.5% for AuNP probe assay and 65% for PCR). The AuNP probe assay was found to be highly specific to BBTV and was found to detect up to 1pg/μl of the plasmid (pTZBBTri 4, BBTV DNA) mixed with healthy banana DNA.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

Biochemical Characterization of Compatible Plant Virus Interaction: A Case Study with Bunchy Top Virus-Banana Host-Pathosystem

Environmental stresses of both biotic and abiotic nature produce characteristic changes in physiology and metabolic processes of higher plants (Miteva et al., 2005). Among these stresses, infection by the pathogens causes substantial alterations of biochemical changes leading to harmful effects on plant health. Banana cultivation is subjected to many natural calamities including pest and pathogen attacks which constitute the major production problem. Among plant diseases, viral diseases may cause considerable loss on production by retarding plant growth and reducing yield. Four viral diseases viz., Banana Bunchy Top Disease (BBTD) caused by Banana bunchy top virus (BBTV), Bract Mosaic Disease (BBrMD) caused by Banana bract mosaic virus (BBrMV), Banana Streak Disease (BSD) caused by different species of Banana streak virus (BSV) and banana mosaic or infectious chlorosis caused by Cucumber mosaic virus (CMV) occur in most of the banana growing regions of the world. Among banana viral diseases, Banana Bunchy Top Disease (BBTD) is one of the most destructive viral diseases in Tropical Asia, Pacific Indian Oceania (PIO) regions and Africa. Grand Nain, Virupakshi (Hill banana), Robusta, Nendran, Rasthali, Poovan, Ney Poovan, Monthan and Red Banana are severely affected by BBTD. There is no resistance gene source available in germplasm of bananas and plantains (Shekhawat et al., 2012). The BBTV affected plants show intermittent dark green dots, dash, streaks of variable length on leaf sheath, midrib, leaf veins and petioles of infected plants. Leaves produced are progressively shorter, brittle in texture, narrow and gives the appearance of bunchyness at the top leading to 100% yield loss (FAO., 2009). Plants have evolved various pre-existing physical and chemical barriers, as well as inducible defense responses that interfere with pathogen colonization (Jones and Dangl, 2006; Zhao et al., 2008; Vanitha et al., 2009). However, this requires comprehensive studies and understanding of the adaptive mechanisms and responses to BBTV infection in banana. Because viruses cannot be cultured in vitro, our knowledge about their interactions with host and changes on physiology, biochemistry and molecular biology of the host is limited. Moreover, a comprehensive report regarding biochemical alterations in banana plants infected by BBTV is still not clear. Therefore, the present investigation was conducted to find the quantitative estimations of physiological and biochemical parameters such as protein, pigment and carbohydrate contents, phenolic compounds, polyphenol oxidase (PPO), peroxidase (POX), catalase (CAT), ascorbate peroxidise (APX), guaiacol peroxidise (GPX) and superoxide dismutase (SOD) activities, indicating their role in BBTV inoculated and non-inoculated plants of Virupakshi (hill banana), a unique flavoured elite dessert banana cultivar and Grand Nain, which belongs to a Cavendish sub group occupying approximately 50% of area in the world.

Transgenic banana plants expressing small interfering RNAs targeted against viral replication initiation gene display high-level resistance to banana bunchy top virus infection

The banana aphid-transmitted Banana bunchy top virus (BBTV) is the most destructive viral pathogen of bananas and plantains worldwide. Lack of natural sources of resistance to BBTV has necessitated the exploitation of proven transgenic technologies for obtaining BBTV-resistant banana cultivars. In this study, we have explored the concept of using intron-hairpin-RNA (ihpRNA) transcripts corresponding to viral master replication initiation protein (Rep) to generate BBTV-resistant transgenic banana plants. Two ihpRNA constructs namely ihpRNA-Rep and ihpRNA-ProRep generated using Rep full coding sequence or Rep partial coding sequence together with its 5' upstream regulatory region, respectively, and castor bean catalase intron were successfully transformed into banana embryogenic cells. ihpRNA-Rep- and ihpRNA-ProRep-derived transgenic banana plants, selected based on preliminary screening for efficient reporter gene expression, were completely resistant to BBTV infection as indicated by the absence of disease symptoms after 6 months of viruliferous aphid inoculation. The resistance to BBTV infection was also evident by the inability to detect cDNAs coding for viral coat protein, movement protein and Rep protein by RT-PCR from inoculated transgenic leaf extracts. Southern analysis of the two groups of transgenics showed that ihpRNA transgene was stably integrated into the banana genome. The detection of small interfering RNAs (siRNAs) derived from the ihpRNA transgene sequence in transformed BBTV-resistant plants positively established RNA interference as the mechanism underlying the observed resistance to BBTV. Efficient screening of optimal transformants in this vegetatively propagated non-segregating fruit crop ensured that all the transgenic plants assayed were resistant to BBTV infection.