Identification of a putative novel cholivirus in the transcriptome of Gymnema sylvestre R. Br.

Gymnema sylvestre is a tropical climber species that is widely used in traditional medicine since ages. In the present study, the transcriptome datasets of G. sylvestre available in public domain were screened for the presence of novel plant viral sequences and a putative novel virus tentatively named as Gymnema sylvestre virus 1 (GysV1) was identified. Coding-complete genome segments of GysV1 that are 6.35 kb (RNA1) and 3.98 kb (RNA2) long possessed a single large open reading frame coding for a polyprotein. BLASTp, sequence identity and phylogenetic analyses revealed the relatedness of GysV1 to the members of the subgenus Cholivirus (genus Sadwavirus; family Secoviridae; order Picornavirales). Based on the species demarcation criteria of the family Secoviridae, GysV1 can be regarded as a new cholivirus member.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Mining of the water hyssop (Bacopa monnieri) transcriptome reveals genome sequences of two putative novel rhabdoviruses and a solendovirus.

The genomes of three putative novel viruses, tentatively named "Bacopa monnieri virus 1" (BmV1), "Bacopa monnieri virus 2" (BmV2), and "Bacopa monnieri virus 3" (BmV3) were identified in the transcriptome dataset of a medicinally important herb - water hyssop (Bacopa monnieri (L.) Wettst.). The BmV1 and BmV2 genomes resemble those of plant rhabdoviruses. The 13.3-kb-long BmV1 genome contains eight antisense ORFs in the order 3' l-N-P2'-P-P3-M-G-P6-L-t 5', with P2' ORF overlapping with P, while the 13.2-kb BmV2 genome contains six interspersed ORFs in the antisense orientation (3' l-N-P-P3-M-G-L-t 5'). The 8-kb BmV3 genome possesses five overlapping ORFs, with ORFs 2 to 5 being similar to those of solendoviruses. Based on genome organization, sequence similarity, and phylogeny, BmV1, BmV2, and BmV3 can be regarded as new members of the genera Cytorhabdovirus, Betanucleorhabdovirus, and Solendovirus, respectively.

Complete genome sequence and genetic organization of a Garlic virus D infecting garlic (Allium sativum) from northern India.

The present paper describes first full genome sequence of the Garlic virus D (GarV-D) from northern India with a genome size of 8425 bp long ssRNA. The infected leaves and bulbs of garlic variety Yamuna Safed (G-282) plants suspected for GarV-D infection were collected with the aim to identify contagion virus during March, 2018. The total RNA was extracted from the pooled garlic plants using TRIzol reagent and sequenced using an Illumina HiSeq 2000 platform. BLASTn search in the NCBI database identified contagion as GarV-D (MK518067). It shared 83.63-85.83% nucleotide sequence identities with other (GarV-D) isolates from Argentina (KF550407, KF555653, KR819505) and 83.15% with isolates from China (MF795136, MF363012). Keywords: Allium sativum; Allexivirus; Garlic virus D; India.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India.

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

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.

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection.

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.