Mixed infection of two mandariviruses identified by high-throughput sequencing in Kinnow mandarin and development of their specific detection using duplex RT-PCR.

In the current study, high-throughput sequencing (HTS) was used to identify viruses associated with the Kinnow mandarin (Citrus reticulata) plants exhibiting yellow vein clearing, mottling, and chlorosis symptoms at experimental farm of ICAR-Indian Agricultural Research Institute, New Delhi, India. During November 2022, leaf samples of symptomatic and asymptomatic Kinnow mandarin trees were collected, subjected to HTS and one of the representative symptomatic samples was subjected to leaf-dip electron microscopy (EM). In the EM results, flexuous virus particles typical of mandarivirus were observed. Ribosomal RNA was depleted from total RNA of pooled samples and RNA sequencing was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs, which were annotated through BLASTn using database of plant viruses/viroids reference genomes (NCBI). Results of assembled contigs revealed near-complete genomes of two mandariviruses, i.e., citrus yellow vein clearing virus (CYVCV) and citrus yellow mottle-associated virus (CiYMaV). The values of fragments per kilo base transcript length per million fragments mapped estimation indicated the dominance of CYVCV in HTS data and it was also confirmed through krona plot distribution of viruses in the pooled samples. A rapid and reliable duplex RT-PCR assay was also developed and standardized for the simultaneous detection of both CYVCV and CiYMaV in a pooled Kinnow mandarin sample. The developed duplex RT-PCR was then validated for the presence of these viruses in individual Kinnow mandarin samples. The specificity and sensitivity results confirmed that primers were highly specific to their targets and able to detect viruses up to 10-2 dilutions of RNA in standard and duplex RT-PCR. Therefore, the developed rapid duplex RT-PCR can be used for virus indexing and production of virus-free Kinnow mandarin plants for certification programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04011-9.

Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus.

Citrus is an economically important fruit crop, belongs to family Rutaceae, cultivated commercially in over 130 countries, which holds a leading profitable position in the international market. The most important citrus varieties are mandarins, oranges, lemons, sweet limes, grapefruits and pomelos. Citrus yellow vein clearing virus (CYVCV) is an important graft transmissible plant pathogen known to reduce productivity of citrus fruits due to its predominant association and widespread occurrence. Requirement of fast, reliable, efficient & economical CYVCV indexing assay is a prerequisite for production of healthy planting material. Currently, nucleic acid isolation and thermal cycler-based assay available for CYVCV indexing is a cumbersome lab intensive method. The present study was undertaken to develop and validate reverse transcription-recombinase polymerase amplification (RT-RPA) assay requiring no tedious RNA isolation, separate cDNA synthesis and costlier instrument like thermo-cycler. Optimized RT-RPA assay was able to amplify CYVCV up to 10-7 dilution (equivalent to 0.1 pg/µl) with the prepared templates of both RNA and crude saps and showed higher sensitivity in detection of CYVCV infection in field samples as compared to the conventional RT-PCR. Developed RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus tristeza virus, citrus exocortis viroid and huanglongbing). RT-RPA using crude leaf sap as template is quite simple, robust, highly sensitive, time and cost effective; therefore, it can be used in resource constrained laboratories as screening tool, for field surveys and on-site testing programs in farms, nurseries and biosecurity. Present study, first time reports the development, optimization and validation of crude sap-based RT-RPA assay for the detection of CYVCV infection in citrus plants namely; Kinnow mandarin, Mosambi and Grape fruit.

Development and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars.

Geminiviruses are known to infect several fields and horticultural crops around the globe. Grapevine geminivirus A (GGVA) was reported in the United States in 2017, and since then, it has been reported in several countries. The complete genome recovered through high-throughput sequencing (HTS)-based virome analysis in Indian grapevine cultivars had all of the six open reading frames (ORFs) and a conserved nonanucleotide sequence 5'-TAATATTAC-3' similar to all other geminiviruses. Recombinase polymerase amplification (RPA), an isothermal amplification technique, was developed for the detection of GGVA in grapevine samples employing crude sap lysed in 0.5 M NaOH solution and compared with purified DNA/cDNA as a template. One of the key advantages of this assay is that it does not require any purification or isolation of the viral DNA and can be performed in a wide range of temperatures (18°C-46°C) and periods (10-40 min), which makes it a rapid and cost-effective method for the detection of GGVA in grapevine. The developed assay has a sensitivity up to 0.1 fg µl-1 using crude plant sap as a template and detected GGVA in several grapevine cultivars of a major grapevine-growing area. Because of its simplicity and rapidity, it can be replicated for other DNA viruses infecting grapevine and will be a very useful technique for certification and surveillance in different grapevine-growing regions of the country.

Complete Genomic RNA Sequence of Tuberose Mild Mosaic Virus and Tuberose Mild Mottle Virus Acquired by High-Throughput Sequencing.

Tuberose (Polianthes tuberosa) is an ornamental flowering crop of the Amaryllidaceae family. Tuberose mild mosaic virus (TuMMV) and tuberose mild mottle virus (TuMMoV), members of the genus Potyvirus, are ubiquitously distributed in most tuberose growing countries worldwide with low biological incidence. Here, we report the first coding-complete genomic RNA of TuMMV and TuMMoV obtained through high-throughput sequencing (HTS) and further, the presence of both the viruses were confirmed using virus-specific primers in RT-PCR assays. Excluding the poly (A) tail, the coding-complete genomic RNA of TuMMV and TuMMoV was 9485 and 9462 nucleotides (nts) in length, respectively, and contained a single large open reading frame (ORF). Polyprotein encoded by both the viral genomes contained nine putative cleavage sites. BLASTn analysis of TuMMV and TuMMoV genomes showed 72.40-76.80% and 67.95-77% nucleotide sequence similarities, respectively, with the existing potyviral sequences. Phylogenetic analysis based on genome sequences showed that TuMMV and TuMMoV clustered in a distinct clade to other potyviruses. Further studies are required to understand the mechanism of symptom development, distribution, genetic variability, and their possible threat to tuberose production in India.

High-throughput RNA sequencing and genetic structure studies of turnip mosaic virus infecting black and yellow mustard revealing emergence of world-B3 pathotype in India.

The RNA viruses are marked by high genetic diversity, which allows them to quickly adapt to new and resistant hosts. The pathogenic turnip mosaic virus (TuMV) infects Brassicaceae plant species all over the world. AIM: To study the evolution and host expansion of a TuMV for the first time in India using molecular population genetic framework. MATERIALS AND RESULTS: Here, we decipher the complete genome sequences of two TuMV world-B3 strains infecting yellow and black mustard in India through high-throughput RNA sequencing subjecting ribosomal RNA depleted mRNA isolated from leaves exhibiting puckering and mosaic symptoms with 100% incidence and high severity in the experimental field. The viral genomes of the two isolates were 9817 and 9829 nucleotides long. They featured two open reading frames (ORFs), one of which encoded a polyprotein comprised of 3164 amino acids and the other of which encoded a PIPO protein of 62 amino acids. CONCLUSIONS: The two TuMV strains from New Delhi region shared identity with the world-B pathotype and subpathotype world B3 showcasing its emergence first time in South Asia. In contrast, other isolates reported previously from South Asia were all Asian-BR pathotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: According to our knowledge, this is the first instance of TuMV association with black mustard naturally. Their geographical prevalence justifies a lower degree of genetic differentiation and higher rate of gene flow calculated between the world-B and Asian-BR pathotypes. This study provides insights on population structuring, expansions and evolution, level of genetic heterogeneity and variability of worldwide prevalent isolates of TuMV which will further aid in understanding virus epidemiology and help prevent losses.

Dwarf polish wheat hosts a novel closterovirus: Revelation by transcriptome data-mining.

losteroviruses are positive sense single-stranded RNA genome-containing plant viruses with narrow natural host range and wide distribution. In the present study, a putative novel closterovirus, Triticum polonicum closterovirus (TriPCV) was identified in the transcriptome assembled contigs of dwarf polish wheat available in public domain. The genome of TriPCV (15.36 kb; TPA Acc. No.: BK059767) contained nine open reading frames (ORFs) that encode for proteins involved in viral replication, cell-to-cell movement, encapsidation and suppression of host RNA silencing. Phylogenetic analysis revealed that TriPCV was distantly related to other members of the genus Closterovirus. Based on genome organization, sequence similarities in BLAST analysis, predicted motifs and phylogeny, TriPCV can be regarded as a putative novel member of the genus Closterovirus Keywords: Closterovirus; Triticum polonicum; transcriptome; public domain.

Transcriptomic Changes of Bemisia tabaci Asia II 1 Induced by Chilli Leaf Curl Virus Trigger Infection and Circulation in Its Vector.

Bemisia tabaci (Hemiptera: Aleyrodidae) is a highly efficient vector in the spread of chilli leaf curl virus (ChiLCV, Begomovirus) which is a major constraint in the production of chilli in South Asia. Transcriptome analysis of B. tabaci post-6 h acquisition of ChiLCV showed differential expression of 80 (29 upregulated and 51 downregulated) genes. The maximum number of DEGs are categorized under the biological processes category followed by cellular components and molecular functions. KEGG analysis of DEGs showed that the genes are involved in the functions like metabolism, signaling pathways, cellular processes, and organismal systems. The expression of highly expressed 20 genes post-ChiLCV acquisition was validated in RT-qPCR. DEGs such as cytosolic carboxypeptidase 3, dual-specificity protein phosphatase 10, 15, dynein axonemal heavy chain 17, fasciclin 2, inhibin beta chain, replication factor A protein 1, and Tob1 were found enriched and favored the virus infection and circulation in B. tabaci. The present study provides an improved understanding of the networks of molecular interactions between B. tabaci and ChiLCV. The candidate genes of B. tabaci involved in ChiLCV transmission would be novel targets for the management of the B. tabaci-begomovirus complex.

Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi.

Thrips cause considerable economic losses to a wide range of food, feed, and forest crops. They also transmit several plant viruses. Being cryptic, it is often difficult to distinguish thrips species in crops and large consignments by conventional methods. Melon thrips (Thrips palmi Karny, Thysanoptera: Thripidae) is an invasive insect pest of vegetables, legumes, and ornamentals besides being vector to several viruses. It poses a threat to domestic and international plant biosecurity and can invade and establish in new areas. Here, we report a polymerase spiral reaction (PSR)-based isothermal assay for rapid, sensitive, specific, low-cost, and on-site detection of T. palmi. To the best of our knowledge, this is the first application of PSR in the identification of any insect species. A primer pair designed based on 3'-polymorphism of mtCOIII region can specifically identify T. palmi without any cross-reactivity with predominant thrips species. The assay uses crude lysate of a single thrips saving time and reagents involved in nucleic acid extraction. The presence of T. palmi is visualized by the appearance of bright fluorescence under ultraviolet light or a change in reaction color thus avoiding gel electrophoresis steps. The entire process can be completed in 70 min on-site using only an ordinary water bath. The assay is sensitive to detecting as little as 50 attograms of T. palmi template. The assay was validated with known thrips specimens and found to be efficient in diagnosing T. palmi under natural conditions. The described method will be useful for non-expert personnel to detect an early infestation, accidental introduction to a new area, restrict the spread of diseases and formulate appropriate management strategies.

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi.

Thrips palmi (Thysanoptera: Thripidae) is the predominant tospovirus vector in Asia-Pacific region. It transmits economically damaging groundnut bud necrosis virus (GBNV, family Tospoviridae) in a persistent propagative manner. Thrips serve as the alternate host, and virus reservoirs making tospovirus management very challenging. Insecticides and host plant resistance remain ineffective in managing thrips-tospoviruses. Recent genomic approaches have led to understanding the molecular interactions of thrips-tospoviruses and identifying novel genetic targets. However, most of the studies are limited to Frankliniella species and tomato spotted wilt virus (TSWV). Amidst the limited information available on T. palmi-tospovirus relationships, the present study is the first report of the transcriptome-wide response of T. palmi associated with GBNV infection. The differential expression analyses of the triplicate transcriptome of viruliferous vs. nonviruliferous adult T. palmi identified a total of 2,363 (1,383 upregulated and 980 downregulated) significant transcripts. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed the abundance of differentially expressed genes (DEGs) involved in innate immune response, endocytosis, cuticle development, and receptor binding and signaling that mediate the virus invasion and multiplication in the vector system. Also, the gene regulatory network (GRN) of most significant DEGs showed the genes like ABC transporter, cytochrome P450, endocuticle structural glycoprotein, gamma-aminobutyric acid (GABA) receptor, heat shock protein 70, larval and pupal cuticle proteins, nephrin, proline-rich protein, sperm-associated antigen, UHRF1-binding protein, serpin, tyrosine-protein kinase receptor, etc., were enriched with higher degrees of interactions. Further, the expression of the candidate genes in response to GBNV infection was validated in reverse transcriptase-quantitative real-time PCR (RT-qPCR). This study leads to an understanding of molecular interactions between T. palmi and GBNV and suggests potential genetic targets for generic pest control.

Identification and Characterization of a Garlic Virus E Genome in Garlic (Allium sativum L.) Using High-Throughput Sequencing from India.

Garlic (Allium sativum L.) plants exhibiting mosaics, deformation, and yellow stripes symptoms were identified in Meerut City, Uttar Pradesh, India. To investigate the viruses in the garlic samples, the method of high-throughput sequencing (HTS) was used. Complete genome of the garlic virus E (GarV-E) isolate (NCBI accession No. MW925710) was retrieved. The virus complete genome comprises 8450 nucleotides (nts), excluding the poly (A) tail at the 3' terminus, with 5' and 3' untranslated regions (UTRs) of 99 and 384 nts, respectively, and ORFs encoding replicase with a conserved motif for RNA-dependent RNA polymerase (RdRP), TGB1, TGB2, TGB3, serine-rich protein, coat protein, and nucleic acid binding protein (NABP). The sequence homology shared 83.49-90.40% and 87.48-92.87% with those of GarV-E isolates available in NCBI at the nucleotide and amino acid levels, respectively. Phylogenetic analysis showed a close relationship of this isolate from India (MW925710) with GarV-E isolate YH (AJ292230) from Zhejiang, China. The presence of GarV-E was also confirmed by RT-PCR. The present study is the first report of GarV-E in garlic cultivar Yamuna Safed-3 grown in northern India. However, further studies are needed to confirm its role in symptom development, nationwide distribution, genetic diversity, and potential yield loss to the garlic in India.

A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis.

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150-450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.

Development of polyclonal antibodies using bacterially expressed recombinant coat protein for the detection of Onion yellow dwarf virus (OYDV) and identification of virus free onion genotypes.

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774 bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.

Molecular characterization of chickpea chlorotic dwarf virus and peanut witches' broom phytoplasma associated with chickpea stunt disease and identification of new host crops and leafhopper vectors in India.

An investigation was carried out to identify and characterize the phytoplasma and viruses associated with the chickpea varieties showing severe stunting, leaf reddening, yellowing and phyllody symptoms during the summer season of 2018-2019 and 2019-2020 in eight states of India. The average disease incidence was recorded from 3 to 32% in different states. The presence of chickpea chlorotic dwarf virus (CpCDV) was confirmed in thirty-seven chickpea samples by amplification of CpCDV coat protein gene and sequence comparison analysis. No record of association of luteovirus, polerovirus and cucumovirus could be detected in any of the symptomatic chickpea samples by RT-PCR assay. Brassica nigra, B. juncea, Lens culinaris, two weeds (Heteropogan contartus, Aeschynomene virginica) and one leafhopper (Amarasca biguttula) were identified as new putative hosts for CpCDV. Association of peanut witches' broom phytoplasma was confirmed in twenty-eight chickpea samples, Sesamum indicum, five weeds hosts and two leafhopper species (Exitianus indicus, Empoasca motti) using nested PCR assays with primer pairs P1/P7 and R16F2n/R16Rn. The results of phytoplasma association in plants and leafhopper samples were further validated by using five multilocus genes (secA, rp, imp, tuf and secY) specific primers. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene and five multilocus genes confirmed the identity of association of 16SrII-C and 16SrII-D subgroups of phytoplasmas strain with chickpea samples collected from Andhra Pradesh (AP), Telangana, Karnataka, Madhya Pradesh, Uttar Pradesh and New Delhi. Mixed infection of phytoplasma (16SrII-D) and CpCDV was also detected in symptomatic chickpea samples from AP and Telangana. The reports of association of 16SrII-C subgroup phytoplasma in chickpea and 16SrII-D subgroup phytoplasma in C. sparsiflora and C. roseus are the new host records in world and from India, respectively.

Candidatus Liberibacter asiaticus manipulates the expression of vitellogenin, cytoskeleton, and endocytotic pathway-related genes to become circulative in its vector, Diaphorina citri (Hemiptera: Psyllidae).

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 upregulated, and 1715 downregulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins, such as vitellogenins, extensin, laminin, tropomyosin, troponin C, and flightin. Majority of the differentially expressed genes were categorized under molecular functions followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide, and energy metabolic pathways, the endocytotic pathway, and the defense-related pathways. Differential regulation of genes associated with the key pathways might favour CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02641-x.

Genetic Variability Based on Tandem Repeat Numbers in a Genomic Locus of 'Candidatus Liberibacter asiaticus' Prevalent in North East India.

The genetic variability of 'Candidatus Liberibacter asiaticus' (CLas) population associated with huanglongbing (HLB) disease of citrus in North Eastern (NE) region of India, a geographically locked region, and home for the diversity of many citrus species was analyzed on the basis of tandem repeat numbers (TRN) in variable CLIBASIA_01645 genomic loci. Fifty-five CLas strains sampled from different groves of NE Hill (NEH) region of India were in single amplicon group, but there was remarkable genetic variability in TRNs. The TRN in HLB-associated CLas strains varied from 0-21 and two novel repeat motifs were also identified. Among the NE population of CLas, TRN5 and TRN9 were most frequent (total frequency of 36.36%) followed by TRN4 (14.55%) and TRN6, TNR7 with a frequency of 12.73% each. Class II type CLas genotypes (5 < TRN ≤ 10) had highest prevalence (frequency of 60.00%) in the samples characterized in present study. Class I (TRN ≤ 5) genotypes were second highest prevalent (29.09%) in the NEH region. Further analysis of genetic diversity parameters using Nei's measure (H value) indicated wide genetic diversity in the CLas strains of NE India (H value of 0.58-0.86). Manipur CLas strains had highest genetic variability (0.86) as compared to Eastern, Southern and Central India. The R10 values (TRN ≤ 10/TRN > 10) of NE CLas population was 10.43 (73/7), higher from other regions of India. Present study conclusively reported the occurrence of high genetic variability in TRN of CLas population in North East Indian citrus groves which have evolved to adapt to the specific ecological niche.

Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein.

Citrus yellow mosaic virus (CYMV) is a member of genus Badnavirus of the family Caulimoviridae. It is the causal agent of citrus yellow mosaic disease in citrus and causes reduction in yield. As the virus is vegetative propagated by grafting, development of high-throughput diagnosis methods based on serological techniques is a prerequisite for production of healthy virus-free planting material. The current study describes the development of polyclonal antibodies raised in rabbits against purified recombinant virion-associated protein (rVAP) encoded by ORF-II of CYMV. The specificity of developed antiserum was evaluated in immunosorbent electron microscopy (ISEM), antigen-coated plate-enzyme linked immunosorbent assay (ACP-ELISA) and immunocapture PCR (IC-PCR). The antiserum specifically reacted up to a dilution of 1:2000 in ACP-ELISA for detection of CYMV-infected plants. The antiserum was validated by screening CYMV-infected plants maintained in the glass house through ACP-ELISA. To the best for our knowledge, this is the first report on production of polyclonal antiserum using recombinant virion-associated protein as fusion protein, which could be used for screening CYMV-infected plants by ELISA and IC-PCR. These immunodiagnostic methods can be effectively employed in routine indexing of citrus and in quarantine process.

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.

Subpopulation level variation of banana streak viruses in India and common evolution of banana and sugarcane badnaviruses.

Genome sequences of three episomal Banana streak MY virus (BSMYV) isolates sampled from triploid banana hybrids (Chini Champa: AAB; Malbhog: AAB and Monthan: ABB), grown in North-East and South India are reported in this study by sequence-independent improved rolling circle amplification (RCA). RCA coupled with restriction fragment length polymorphism revealed diverse restriction profiles of five BSMYV isolates. Nucleotide substitution rates of BSMYV subpopulation and Banana streak OL virus subpopulation was 7.13 × 10(-3) to 1.59 × 10(-2) and 2.65 × 10(-3) to 5.49 × 10(-3), respectively, for the different coding regions. Analysis of the genetic diversity of banana and sugarcane badnaviruses revealed a total of 32 unique recombination events among banana and sugarcane badnaviruses (inter BSV-SCBV), in addition to the extensive recombination with in banana streak viruses and sugarcane bacilliform viruses (intra-BSV and intra-SCBV). Many unique fragments were shown to contain similar ruminant sequence fragments which indicated the possibility that the two groups of badnaviruses or their ancestors to colonise same host before making the host shift. The distribution of recombination events, hot-spots (intergenic region and C-terminal of ORF3) as well as cold-spots (distributed in ORF3) displayed the mirroring of recombination traces in both group of badnaviruses. These results support the hypothesis of relatedness of banana and sugarcane badnaviruses and the host and geographical shifts that followed the fixation of the species complex appear to be a recent event.

Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein.

A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.