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 Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807 bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

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.

Tolerance and adaptation mechanism of Solanaceous crops under salinity stress.

Solanaceous crops act as a source of food, nutrition and medicine for humans. Soil salinity is a damaging environmental stress, causing significant reductions in cultivated land area, crop productivity and quality, especially under climate change. Solanaceous crops are extremely vulnerable to salinity stress due to high water requirements during the reproductive stage and the succulent nature of fruits and tubers. Salinity stress impedes morphological and anatomical development, which ultimately affect the production and productivity of the economic part of these crops. The morpho-physiological parameters such as root-to-shoot ratio, leaf area, biomass production, photosynthesis, hormonal balance, leaf water content are disturbed under salinity stress in Solanaceous crops. Moreover, the synthesis and signalling of reactive oxygen species, reactive nitrogen species, accumulation of compatible solutes, and osmoprotectant are significant under salinity stress which might be responsible for providing tolerance in these crops. The regulation at the molecular level is mediated by different genes, transcription factors, and proteins, which are vital in the tolerance mechanism. The present review aims to redraw the attention of the researchers to explore the mechanistic understanding and potential mitigation strategies against salinity stress in Solanaceous crops, which is an often-neglected commodity.