Tobacco Streak Virus Infection Research Articles

First Report of Tobacco Streak Virus in Cannabis sativa in New York.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

Thrips diversity of cotton ecosystem and the role of parthenium pollen grains in the transmission of tobacco streak virus (TSV) infection in cotton.

The online version contains supplementary material available at 10.1007/s13205-021-02967-6.

Rapid detection of Tobacco streak virus (TSV) in cotton (Gossypium hirsutum) based on Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP)

Tobacco Streak Virus (TSV) belongs to the genus Ilarvirus of the family Bromoviridae an emerging pathogen posing threat to the crop species worldwide. Identification of symptoms due to TSV infection by visual observation of plants often results in misdiagnosis as symptoms produced by this virus can match with those reflecting physiological and nutritional disorders affecting cotton. Development of diagnostic tools with rapidity will have immense role to play in detection and management of the emerging virus. The protocol for rapid diagnosis of TSV infected samples by using Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) was optimised and this is the first report of its use for diagnosis of TSV on cotton and Soybean. The colorimetric detection for diagnostic simplicity of amplified RT-LAMP product by using different dyes lead to enhanced applicability of this technique. The RT-LAMP diagnostic tool can be utilized not only for laboratory research but also for quarantine and field diagnosis of this important emerging pathogen affecting cotton.

Detection, discrimination and discovery of a new Tobacco streak virus strain

Soybean plants that exhibited symptoms of virus infection were sampled from different counties of Oklahoma. These plants were tested serologically for 15 viruses known to infect soybean plants. Fifty-seven samples that exhibited typical virus-like symptoms did not test positive for any of the 15 viruses used in a dot-immunobinding assay (DIBA). Four samples were pooled and used for next generation sequencing using the 454-Roche protocol. Sequence and phylogenetic analysis of the sequences obtained revealed infection with a distinct strain of Tobacco streak virus (TSV). TSV was one of the 15 viruses initially tested for using DIBA and had tested negative. TSV belongs to the genus Ilarvirus and has been reported as a causal agent of bud blight in soybean crops in Brazil and the United States. Out of 10 reported primer pairs for TSV reverse transcription-polymerase chain reaction (RT-PCR), only two had the potential, based on sequence similarity, to amplify part of the genome of the distinct strain of TSV found in Oklahoma and only one was actually able to amplify the region. In this study, a new primer pair, specific to all known TSV and capable of amplifying the Oklahoma strain (TSV-OK), was designed from a highly conserved region of coat protein (CP) sequences and end-point PCR and quantitative RT-PCR detection methods were developed and their sensitivity assayed. This is the first report of specific primers designed from this highly conserved region in the CP of TSV for detection of TSV. Twenty-three of the 57 DIBA soybean samples that initially tested negative were retested with the new specific end-point PCR method and found positive for TSV infection.

Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation.

The absence of resistance genes against biotic stresses like Tobacco streak virus (TSV) within compatible peanut germplasm necessitates the deployment of genetic engineering strategy to develop transgenic resistance. Transgenic resistance in peanut (Arachis hypogaea L.) to peanut stem necrosis disease caused by TSV was obtained by transferring coat protein (CP) gene of TSV through Agrobacterium-mediated transformation of de-embryonated cotyledons and immature leaves of peanut cultivars Kadiri 6 (K6) and Kadiri 134 (K134). Integration of the transgene in T1, T2 and T3 generations were confirmed by PCR with gene-specific primers. On the basis of segregation analysis of the PCR amplicons, homozygosity was confirmed in progeny from five transgenic lines. Six transgenic plants from three different single copy transgenic lines homozygous for the transgene were selected for challenge inoculation in T3 generations. The transgenic lines remained symptomless throughout and showed traces or no systemic accumulation of virus indicating the tolerance/resistance to the TSV infection. CP gene expression was observed in transgenic lines by RT-PCR, real-time PCR and ELISA. The findings provide an effective strategy for developing peanut with resistance to peanut stem necrosis disease.

Engineering resistance against Tobacco streak virus (TSV) in sunflower and tobacco using RNA interference

The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.

Dahlia mosaic virus and Tobacco streak virus in Dahlia (Dahlia variabilis) in New Zealand.

Dahlia (Dahlia variabilis Hort.) is a significant ornamental plant in New Zealand. Symptoms such as mosaic, ring spots, mottling, and veinal chlorosis, suggestive of a viral infection, are often seen in various dahlia collections. To better understand the incidence of viruses in dahlia in New Zealand, several popularly grown cultivars were evaluated for viruses that are known to infect dahlia. Viruses that were tested included Cucumber mosaic virus (CMV), Dahlia mosaic virus (DMV), Impatiens necrotic spot virus (INSV), Tobacco streak virus (TSV), and Tomato spotted wilt virus (TSWV). At least one symptomatic plant was tested from each of the following cultivars: Akito Dawn, Cincinnati Dancer, Hamari Accord, Hamari Rose, LeBatts Prime, LeVonne Splinter, Riverlea Tropicana, Spartacus, Tartan, Tui Connie, and Wandas Antartica. Except for DMV, initial testing was done by ELISA with commercially available kits for the above viruses. In the case of dahlia mosaic, samples were tested for DMV that was described previously (4) and two additional and distinct caulimoviruses (DMV-D10 and DMV-Holland) that were found to be associated with dahlia (1,2). Primer pairs, ORF6st: ATG GAA GAA ATT AAG GCG T and ORF6end: TTG TCT TCA TCC ATA AAG CAG; DenF1: CAG CAA GAA ACA GGA ATT GA and DenR: TTA CAG TCG AAG CTG CTA AA; and Kapht-F: ATG AGT AAT GCT TCA GCA A and Kapht-R: TGA CCA TGG CTT CTA ACT GT were used for the specific detection of DMV-D10, DMV-Holland, and DMV, respectively (1). None of the samples tested were ELISA positive for CMV, INSV, or TSWV. To verify the TSV infection, TSV-specific primers (5'-GTC CAG ACC ATC CAT CCA AC-3' and 5'-TTG ATT CAC CAG GAA ATC TT-3'), designed based on sequences available in GenBank, were used in reverse transcription (RT)-PCR. For DMV, the diagnostic tests used were electron microscopy and PCR followed by amplicon cloning and sequencing. Electron microscopic observation of leaf-dip preparations showed near isometric virions, approximately 50 to 60 nm in all samples tested. PCR showed that all samples tested were positive for DMV-Holland and DMV-D10. While DMV-Holland is a typical caulimovirus, DMV-D10 was found to exist as an endogenous plant pararetroviral sequence in dahlia (3). One sample each from two cultivars, Spartacus and Tui Connie, were positive for TSV by ELISA, RT-PCR, followed by the sequence analysis of the cloned amplicon. The impact of TSV-infected dahlias as a potential source of inoculum remains to be seen. Our results suggested the prevalence of dahlia mosaic-associated caulimoviruses in several dahlia cultivars and the presence of TSV in New Zealand dahlias. Dahlia mosaic continues to be prevalent in several parts of the world (1), and with the current findings in New Zealand, testing for these viruses should be conducted to ensure virus-free status of the propagating material.

First Report of Tobacco streak virus on Dahlia in the Czech Republic.

Dahlia is an important ornamental crop in the Czech Republic where they have been grown for more than 150 years. New dahlia cultivars have been selected by Czech plant breeders. Virus diseases, including mosaic and stunt caused mostly by Dahlia mosaic virus, have been a problem. From 2003 to 2005, color breaking was observed in several dahlia cultivars of foreign and Czech origin. White stripes in blossoms were most frequently expressed in the second half of the flowering season. No symptoms are visible in flowers of white and yellow cultivars. It was difficult to characterize symptoms on leaves because most cultivars were infected simultaneously by Dahlia mosaic virus. Sap inoculations of Chenopodium quinoa produced local lesions after 5 to 7 days, followed by systemic chlorosis, necrosis of younger leaves, and death of the shoot apex, indicating possible Tobacco streak virus (TSV) infection (2). Spherical particles (25 to 30 nm) were observed in leaf-dip preparations of samples from experimentally infected C. quinoa plants and analyzed by using transmission electron microscopy. These particles became decorated when using immunoelectron microscopy with TSV IgG (Bioreba, Reinach, Switzerland and Neogen, Ayrshire, Scotland). Samples of 80 dahlia cultivars were tested for TSV infection by ELISA using commercially available kits (Bioreba and Neogen). Most of the samples were grown in a collection of dahlia cultivars of Czech and foreign origin and some were obtained from growers in the Czech Republic. Fifty six dahlia cultivars were shown to be TSV infected. ELISA also indicated a higher concentration of the virus in flowers. The identity of the virus isolated from symptomatic plants was confirmed by reverse transcription (RT)-PCR using total RNA extraction from symptomatic plants. RT-PCR (4), using a primer pair (1) derived from the coat protein gene sequence of TSV (3), was followed by electrophoresis on 1.0% agarose gels. Products of the predicted size (approximately 700 bp) were found in naturally infected dahlia plants (n = 10), systemically infected host plants C. quinoa (n = 10), and symptomatic Nicotina megalosiphon (n = 10) that scored as TSV positive by ELISA. No bands of this size were seen in negative controls. To our knowledge, this is the first detection of TSV in the Czech Republic.

Sources of Resistance to Tobacco streak virus in Wild Arachis (Fabaceae: Papilionoidae) Germplasm.

Stem necrosis disease caused by Tobacco streak virus (TSV), first recognized in 2000, has emerged as a potential threat to peanut (Arachis hypogaea) in southern states of India. The virus induces severe necrosis of shoots leading to death of the plant, and plants that survive are malformed, with severe reduction in pod yield. All the currently grown peanut cultivars in India are highly susceptible to the virus. Therefore, wild relatives of peanut were evaluated to identify potential sources of resistance to TSV infection. In all, 56 germplasm accessions from 20 wild Arachis spp. in four sections (Arachis, Erectoides, Procumbente, and Rhizomatosae), along with susceptible peanut cultivars (JL 24 and K 1375), were evaluated for resistance to TSV under greenhouse conditions using mechanical sap inoculations. Systemic virus infection, determined by enzyme-linked immunosorbent assay (ELISA), in the test accessions ranged between 0 and 100%. Twenty-four accessions in section Arachis that had 0 to 35% systemically infected plants were retested, and systemic infection was not detected in eight of these accessions in repeated trials in the greenhouse. These are International Crops Research Institute for the Semi-Arid Tropics groundnut (ICG) accession nos. 8139, 8195, 8200, 8203, 8205, and 11550 belonging to A. duranensis; ICG 8144 belonging to A. villosa; and ICG 13210 belonging to A. stenosperma. Even though the resistant accessions had 0 to 100% TSV infection in inoculated leaves, TSV was not detected in the subsequently emerged leaves. This is the first report of TSV resistance in Arachis spp. The eight TSV resistant accessions are cross compatible with A. hypogaea for utilization in breeding for stem necrosis disease resistance.

First report of the natural occurrence of Tobacco streak virus on blackgram (Vigna mungo)

; Fabaceae) is an important pulse crop in India,Nepal, Bangaladesh and Myanmar. Between June and September 2002, anew disease was observed in the majority of the blackgram-growing areasof India. Symptoms of the disease consisted of brown necrotic lesions onyoung leaves, with brown streaks on petioles and stems, with death ofplant in severe cases. Mechanical inoculation of plant sap from infectedplants onto various plant species – Vigna unguiculata cv. 152; Nicotianatabacum; Chenopodium amaranticolor and Gomphrena globosaproduced necrotic lesions on leaves and streaks on stems.Diseased leaf samples of blackgram tested positive for Tobacco streakvirus (TSV) by DAC-ELISA (Bhat et al., 2001) using a specific polyclonalantibody (a gift from G. Cook, Plant Protection Research Institute, SouthAfrica). Electron microscopic observations of negatively stained prepara-tions of purified virus from infected blackgram leaves revealed sphereswith a diameter of 27 nm, consistent with TSV infection. RT-PCR tests oftissue from diseased blackgram plants using primers specific for the coatprotein gene of TSV (Bhat et al., 2002) resulted in an amplicon of theexpected size: approximately 700 bp. TSV infects a wide range of hosts inIndia (Prasada Rao et al., 2003). Alternative hosts may also play a rolein disease epidemiology, since TSV infects many widely distributedweeds. For example, Parthenium hysterophorus probably plays a majorrole in the spread of TSV in peanut ( Arachis hypogaea ) (Prasada Rao et al.,2003).This is the first report of the natural occurrence of TSV on Vigna mungo.ReferencesBhat AI, Anil K, Jain RK, Chander Rao S, Ramiah M, 2001. Development of serological based assays for the diagnosis of sunflower necrosis disease.

High Incidence of Tobacco Streak Virus in Tobacco and Its Transmission byMicrocephalothrips abdominalisand Pollen fromAgeratum houstonianum

Incidence of tobacco streak virus (TSV) in an Australian tobacco crop at flowering was shown by enzyme-linked immunosorbent assay (ELISA) to be 58−59% in outside rows near weeds infected with TSV, decreasing to 10−18% in the 12th row inside the crop. The most prevalent dicotyledonous weed, Ageratum houstonianum, had a 50% incidence of infection and its flower heads were commonly infested with Microcephalotrips abdominalis. In transmission tests with either five or 10 adult thrips and pollen taken from the nearby A. houstonianum, TSV infection occurred in 32 of 45 tobacco, 11 of 12 cucumber, two of 12 Nicotiana clevelandii, and 18 of 22 Chenopodium amaranticolor test seedlings (.)

Transgenic tobacco expressing tobacco streak virus or mutated alfalfa mosaic virus coat protein does not cross-protect against alfalfa mosaic virus infection

Transgenic tobacco plants expressing the coat protein (CP) genes of tobacco streak virus (TSV) and alfalfa mosaic virus (AIMV) were used in studies on cross-protection and genome activation. Plants expressing the TSV CP gene were highly resistant to infection with TSV nucleoproteins but were susceptible to infection with AIMV nucleoproteins. Moreover, these plants could be infected with a mixture of AIMV RNAs 1, 2, and 3 in contrast to the nontransformed control plants. This demonstrates that the endogenously produced TSV CP is able to activate the AIMV genome but does not cross-protect against this virus. Conversely, it was shown that plants expressing the AIMV CP gene did not resist TSV infection. Transgenic tobacco plants transformed with an AIMV CP gene with a frame-shift mutation in the reading frame were found to accumulate viral transcripts to a level similar to that obtained in plants expressing a wild-type AIMV CP gene. However, these plants did not produce detectable amounts of viral protein and showed no resistance to infection with AIMV nucleoproteins in contrast to transgenic plants accumulating wild-type AIMV CP. This demonstrates that it is the CP that is responsible for cross-protection in transgenic plants and not the chimeric CP mRNA.

Leaf-Curl of Impatiens Caused by Tobacco Streak Virus Infection

Leaf-Curl of Impatiens Caused by Tobacco Streak Virus Infection