V. K. Baranwal

Sodium sulphite yields improved DNA of higher stability for PCR detection of Citrus yellow mosaic virus from citrus leaves.

Citrus yellow mosaic virus (CYMV), a non-enveloped bacilliform DNA virus causes a severe mosaic disease in sweet oranges in India. CYMV is weakly immunogenic, thus serodiagnosis is not a preferred method for its detection. As an alternative a rapid and reliable detection protocol by polymerase chain reaction (PCR) was developed. However, high levels of polyphenolics and tannins in citrus leaves generally interfered with obtaining good quality DNA, and thus affected the reliable detection of virus by PCR. Consequently, we evaluated the addition of sodium sulphite to a DNA extraction protocol used previously and compared the two methods with a commercially available plant DNeasy Kit (Qiagen). The addition of sodium sulphite improved the yield, quality and stability of DNA. The CYMV DNA was not only amplified at lower template DNA concentration, but also provided better DNA yields. In addition, the sodium sulphite extracted DNA survived at various temperatures much longer than those extracted without addition of sodium sulphite or with the commercial kit. The amplified product of CYMV DNA was cloned, sequenced and found to have 89% sequence identity with the only other sequenced Indian isolate of CYMV.

Applications of Next Generation High Throughput Sequencing Technologies in Characterization, Discovery and Molecular Interaction of Plant Viruses

Present era of molecular biology is witnessing revolutionary developments in sequencing technology. This advancement has considerably influenced plant virology in the field of diagnostics and host virus interaction. Next generation high-throughput sequencing technology has made it possible to directly detect, identify and discover novel viruses in several plants in an unbiased manner without antibodies or prior knowledge of the virus sequences. Entire viral genome could be sequenced from symptomatic or asymptomatic plants through next generation sequencing of total nucleic acids including small RNAs. It provides census of viral population in a particular ecosystem or cropping system. Viral genome variability, evolution within the host and virus defence mechanism in plants can also be easily understood by massive parallel sequencing. In this article, we provide an overview of the applications of next generation sequencing technology in characterization, discovery and molecular interaction of plant viruses.

Detection of Citrus exocortis viroid, Iresine viroid, and Tomato chlorotic dwarf viroid in New Ornamental Host Plants in India

Columnea latent viroid, originating from ornamental plants, is known to be harmful to crop plants (2). Despite the potential threat to crop plants, the importance of ornamental plants in viroid evolution is not fully appreciated. Availability of a Pospiviroid genus-specific primer pair (1) to detect the most prevalent viroids in ornamental plants and a simplified nucleic acid preparation protocol (3) for use in reverse-transcription polymerase chain reaction (RT-PCR) have facilitated surveys of ornamental plants for pospiviroids. Using the above protocol in India, leaf and shoot samples were collected randomly from roadside beds consisting of ground covers or creepers/trailing plants at the IARI campus, New Delhi. These were extracted in 50 mM NaOH + 2.5 mM EDTA solution, centrifuged to sediment the coarse debris from sap, and 10 μl of the supernatant was spotted on a nitrocellulose membrane. Individual spots were eluted with distilled sterile water (30 μl) and the eluates were used for RT-PCR detection of viroids (3). Amplified fragments or subsequently cloned plasmids were also purified using NaOH-EDTA membrane protocol. Cloning and sequencing of amplicons (195 to 224 bp) revealed a very high sequence identity with specific viroids from the viroid sequence database (NCBI). Among the 19 plant samples assayed, only three plant species were infected by viroids, although none of them exhibited any symptoms. The three plant species included: (i) moss verbena, Glandularia puchella (Verbenaceae, native to Argentina and Chile, now established in several regions of the world), infected with a viroid (Accession No. DQ846884) having 99% sequence identity to Citrus exocortis viroid (CEVd) (Accession No. S67446); (ii) trailing verbena, Verbena × hybrida (Verbenaceae, ornamental plant), doubly infected with a viroid (Accession No. DQ846885) having 95% sequence identity to CEVd (Accession No. DQ094297) and infected with another viroid (Accession No. DQ846883) having 98% sequence identity to Tomato chlorotic dwarf viroid (TCDVd) (Accession No. AF162131); and (iii) red joyweed, Alternanthera sessilis (Amaranthaceae, a perennial weed herb) infected with a viroid (Accession No. DQ846886) having 96% sequence identity to Iresine viroid (IrVd) (Accession No. DQ094293). CEVd and TCDVd were mechanically transferred to tomato seedlings causing reduced growth of plants, smaller leaves, and bunchy-top appearance of plants, symptoms similar to those typically observed with other isolates of these viroids. As expected from previous studies, IrVd was not transmitted to tomato plants. Natural infection of verbena with CEVd has been detected in North America (2) but this was a novel observation in India. Additional novel observations include: A. sessilis as a new host for IrVd; and TCDVd is the first crop viroid to be isolated from a naturally infected ornamental plant. The significance of these viroid findings in ground cover and widely grown ornamental plants may lie in their potential role in spreading the viroids to citrus plants in citrus-growing countries such as India. References: (1) H. Bostan et al. J. Virol. Methods 116:189, 2004, (2) R. P. Singh and J. A. Teixeira da Silva. Floriculture, Ornamental Plant Biotechnol. 3:531, 2006. (3) R. P. Singh et al. J. Virol. Methods 132:204, 2006.

Identification of sugarcane grassy shoot-associated phytoplasma and one of its putative vectors in India

During a survey of sugarcane fields at the Sugarcane Research Institute, Shahjahanpur, Uttar Pradesh, India, in August–September 2012, 6% to 28% incidence of sugarcane grassy shoot disease was observed in different fields of sugarcane variety CoS 7250. The association of phytoplasma with symptomatic sugarcane was confirmed by direct and nested PCR amplification of phytoplasma ribosomal gene. Four different delphacid leafhopper species, viz. Cofana unimaculata Signoret, Exitianus indicus (Distant), Sogatella kolophon Kirkaldy and Hishimonus phycitis (Dist.) were the prevalent feeding species of the Auchenorrhyncha fauna in the symptomatic sugarcane fields. Out of these four leafhopper species, only E. indicus tested positive for phytoplasma presence. Phylogenetic analysis suggested that the phytoplasmas from sugarcane and E. indicus in the present study were members of 16Sr XI. The confirmation of association of sugarcane grassy shoot phytoplasma in E. indicus population is important to understand the secondary spread of this phytoplasma in sugarcane plants.

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.

Letter to the editor: Characterization of a Grapevine leafroll-associated virus 3 from India showing incongruence in its phylogeny.

Survey conducted during 2010–2011 in the vineyards of Nashik and Pune regions of India revealed the association of an Ampelovirus antigenically related to Grapevine leafroll-associated virus 3 (GLRaV-3) with seven cultivars of grapevine. Upon sequencing of the coat protein (CP) and partial heat shock protein 70 homologue (HSP70h) genes of GLRaV-3 present in the cultivar Cabernet Souvignon showed distinct grouping behaviour. It clustered in group 2 and group 1 on the basis of CP and HSP70h sequences, respectively. Incongruent clustering pattern observed on the basis of two genomic regions suggest that GLRaV-3 is a distinct isolate from India.

Transmission and detection of toria [Brassica rapa L. subsp. dichotoma (Roxb.)] phyllody phytoplasma and identification of a potential vector

Phyllody disease associated with 16SrIX phytoplasma was observed in the range of 4.1–11% in 10 different lines of toria [Brassica rapa L. subsp. dichotoma (Roxb.)] in experimental fields of the Indian Agricultural Research Institute, New Delhi, India during 2008 and 2009. The toria phyllody (TP) phytoplasma was detected in all the symptomatic and 13.3% of asymptomatic toria plants by nested PCR. The phytoplasma was detected in midrib, flower part, siliquae, stem, and root of infected plants as well as seeds. TP was transmitted by grafting and by dodder to toria and nine other rapeseed/mustard species as confirmed by nested PCR. However, symptoms of phytoplasma infection were induced only in toria, yellow sarson [Brassicarapa L. subsp. trilocularis (Roxb.)], brown sarson [Brassicarapa L. subsp. sarson (Prain)], rapeseed (B. napus subsp. oleifera), and rocket or taramira (Eruca sativa) but not in mustard (B. juncea), black mustard (B. nigra), Ethiopian mustard (B. carinata), B. tournefortii and white mustard (Sinapis alba). Transmission of TP phytoplasma to periwinkle (Catharanthus roseus) was successful only through dodder, but no transmission to tomato (Lycopersicon esculentum) or brinjal (Solanum melongena) was found. TP phytoplasma was detected in Laodelpax striatellus, an abundant planthopper in toria fields, which indicates that this planthopper may be a potential vector for TP phytoplasma.

Simultaneous Detection of Mixed Infection of Onion yellow dwarf virus and an Allexivirus in RT-PCR for Ensuring Virus Free Onion Bulbs

Reduced seed production in onion is associated with Onion yellow dwarf virus (OYDV), a filamentous Potyvirus. Onion is also infected with other filamentous virus particles suspected to be Allexivirus. RT-PCR was used to detect mixed infection of both the viruses in leaves and bulbs. A duplex RT-PCR was developed, which simultaneously detected the presence of these two viruses in winter (Rabi) onion bulb. In summer (Kharif) onion bulbs only Allexivirus was detected. The absence of OYDV in summer crop is discussed. The sequencing of RT-PCR amplified products confirmed the identity of OYDV and Allexivirus, the latter showing closer identity to Garlic virusC (GVC)/Garlic mite-borne mosaic virus. This makes the first detection of an Allexivirus in onion crop in India. The duplex RT-PCR to detect these viruses (OYDV and Allexivirus) would be an improvement for indexing of viruses in onion bulbs for seed production.

Development of an immunocapture–reverse transcription–polymerase chain reaction (IC-RT-PCR) using modified viral RNA release protocol for the detection of Grapevine leafroll-associated virus 3 (GLRaV-3)

Grapevine leafroll-associated virus 3(GLRaV-3) is the most destructive virus causing leaf roll disease in grapevine. ELISA has been widely used to screen the propagating materials for indexing of this virus at nursery stage. But the uneven distribution of GLRaV-3 in vines, its confinement to phloem tissues and impact of seasonal influences on its concentration limit the scope of ELISA. RT-PCR (reverse transcription-polymerase chain reaction), is a more sensitive technique, but not feasible for large scale screening purpose because of the tedious process of RNA isolation. Furthermore, location of virus particles and the presence of inhibitory compounds in the woody tissues of grapevine make RNA isolation problematic. Immunocapture-RT-PCR (IC-RT-PCR), more sensitive than ELISA and RT-PCR alone, is a technique where the virus can be detected without isolating the RNA. In this study, IC-RT-PCR was performed using different combinations of three virus extraction buffers and two virus nucleic acid releasing buffers along with one virus RNA releasing condition for the detection of GLRaV-3. The modified extraction and RNA release protocol developed in this study was validated for specific detection of the virus in the vines of five infected grapevine cultivars. This protocol can help in complementing the GLRaV-3 specific certification program of the country.

Simultaneous detection of four garlic viruses by multiplex reverse transcription PCR and their distribution in Indian garlic accessions

Indian garlic is infected with Onion yellow dwarf virus (OYDV), Shallot latent virus (SLV), Garlic common latent virus (GarCLV) and allexiviruses. Identity and distribution of garlic viruses in various garlic accessions from different geographical regions of India were investigated. OYDV and allexiviruses were observed in all the garlic accessions, while SLV and GarCLV were observed only in a few accessions. A multiplex reverse transcription (RT)-PCR method was developed for the simultaneous detection and identification of OYDV, SLV, GarCLV and Allexivirus infecting garlic accessions in India. This multiplex protocol standardized in this study will be useful in indexing of garlic viruses and production of virus free seed material.