Exogenous delivery of dsRNA for management of mungbean yellow mosaic virus on blackgram.

MAIN CONCLUSION: Exogenous application of dsRNA molecules targeting MYMV genes offers a promising approach to effectively mitigate yellow mosaic disease in blackgram, demonstrating potential for sustainable plant viral disease management. The exogenous application of double-stranded RNA (dsRNA) molecules to control plant viral diseases is gaining traction due to its advantages over conventional methods, such as target specificity, non-polluting nature, and absence of residue formation. Furthermore, this approach does not involve genome modification. In this study, dsRNA molecules targeting the coat protein gene (dsCP) and replication initiator protein gene (dsRep) of mungbean yellow mosaic virus (MYMV) were synthesised using an in vitro transcription method. To evaluate the effectiveness of dsRNA treatment, blackgram plants exhibiting MYMV symptoms at the first trifoliate stage were subjected to exogenous application of dsRNA. Second, third, and fourth trifoliate leaves, which emerged at 7, 15, and 21 days after dsRNA application, respectively, were monitored for MYMV symptoms. Remarkably, a significant reduction in yellow mosaic disease (YMD) symptoms was observed in the newly emerged trifoliate leaves of MYMV-infected blackgram plants after treatment with dsRNA targeting both gene regions. This reduction was evident as a decrease in the intensity of yellow mosaic coverage on the leaf lamina compared to control. dsCP effectively reduced the MYMV titre in the treated plants for up to 15 days. However, dsRep demonstrated greater efficiency in conferring resistance to MYMV at 15 days post-application. These findings were supported by quantitative real-time PCR analysis, where the observed Ct values for DNA extracted from dsRep-treated plants were significantly higher compared to the Ct values of DNA from dsCP-treated plants at 15 days post-application. Similarly, higher viral copy numbers were observed in dsCP-treated plants 15 days after dsRNA treatment, in contrast to plants treated with dsRep.

Molecular studies on tobacco streak virus (TSV) infecting cotton in Tamil Nadu, India.

Tobacco streak virus (TSV), the causal agent of cotton necrosis, is of emerging importance in the recent years. Unfortunately, all the cotton varieties and hybrids are susceptible to this virus. Cotton plants cultivated in different districts of Tamil Nadu were surveyed during 2014-2016. Samples collected from different locations confirmed the presence of TSV in cotton. TSV infection was confirmed by direct antigen coating-enzyme linked immuno sorbent assay (DAC-ELISA), dot immuno binding assay (DIBA), and reverse transcriptase polymerase chain reaction (RT-PCR). The virus was morphologically confirmed by transmission electron microscope (TEM). TSV isolate collected from Coimbatore was sequenced to obtain the full-length genome. Full length analysis was done for RNA 1 and RNA 3, whereas there was problem in obtaining few nucleotides in 5' and 3' end in spite of using different primers. Critical domain search in the nucleotide sequences revealed the presence of characteristic viral methyl transferase domain and helicase domain (RNA 1), 'GDD' motif and 'DFSKFD' of viral replicase in RNA 2 and Zinc finger motif in RNA 3. Phylogenetic analysis reveals high nucleotide similarity with TSV isolates of India and USA. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03437-3.

Molecular epidemiology on seasonal variation of yellow mosaic disease incidence in blackgram (Vigna mungo L. Hepper) with its vector Bemisia tabaci.

The yellow mosaic disease (YMD) of blackgram caused by Mungbean yellow mosaic virus has emerged as a serious threat to grain legume production, especially in Southeastern Asia. Seasonal incidence of YMD with its vector population was assessed in three different agroclimatic zones of Tamil Nadu in India for three consecutive cropping seasons namely, Rabi 2018 (October-December), Summer 2019 (March-May), and Kharif 2019 (June-August) at three different time intervals viz., 20, 40, and 60 days after sowing (DAS). For all three seasons, disease incidence and whitefly count were recorded for a resistant and susceptible variety of blackgram in fields without any vector control intervention. The highest disease incidence (87%) was observed in the Panpozhi location during the summer season followed by Vamban and Coimbatore locations. The whitefly count was made through both visual count and yellow sticky traps. The whitefly population was highest at 20 DAS and decreased with the increasing age of crop for all the three locations assessed. Molecular epidemiology was analyzed by determining latent infection of mungbean yellow mosaic virus (MYMV) using molecular diagnosis. Latent infection was found to be well pronounced in the Coimbatore location during the Kharif season, where the crop was asymptomatic in both the resistant and susceptible varieties for all the three time periods assessed. The latent infection of MYMV observed in Coimbatore and Vamban ranged from 16.6 to 83.3% in both resistant and susceptible varieties for all three seasons. In Panpozhi, the latent infection of MYMV ranged from 16.6 to 66.6% for the susceptible variety (CO-5) for all three seasons observed. However, in the Panpozhi location, the resistant variety (VBN-8) failed to record any latent infection.

Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity.

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

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

Survey in the cotton fields of Coimbatore and Erode districts confirmed the presence of three different thrips species including, Scirtothrips dorsalis, Scirtothrips oligochaetus, and Frankliniella occidentalis. Their identity was confirmed through morphometric analysis and molecular characterization. Tobacco streak virus (TSV) was detected in the leaves and pollen grains of both parthenium and cotton plants collected from infected cotton fields. The presence of TSV was confirmed through immuno-detection by direct antigen coating enzyme-linked immuno sorbent assay (DAC-ELISA). Further confirmation was accomplished by reverse transcriptase polymerase chain reaction (RT-PCR) using TSV coat protein-specific primers. Other than parthenium, weed plants in the cotton field such as Trianthema portulacastrum, Boerhavia diffusa, and Amaranthus sp. were also confirmed for TSV infection through RT-PCR. Parthenium hysterophorus plants acts as a silent carrier of TSV and they occasionally produced symptoms. Among all the randomly collected thrips samples, two pooled samples were detected positive for TSV through RT-PCR. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02967-6.

Molecular modelling of coat protein of the Groundnut bud necrosis tospovirus and its binding with Squalene as an antiviral agent: In vitro and in silico docking investigations.

Bud blight disease caused by groundnut bud necrosis virus (GBNV) is a serious constraint in the cultivation of agricultural crops such as legumes, tomato, chilies, potato, cotton etc. Owing to the significant damage caused by GBNV, an attempt was made to identify suitable organic antiviral agents through molecular modelling of the nucleocapsid Coat Protein of GBNV; molecular docking and molecular dynamics that disclosed the interaction of the ligands viz., Squalene and Ganoderic acid-A with coat protein of GBNV. Invitro inhibitory effect of Squalene and Ganoderic acid-A was examined in comparison with different concentrations, against GBNV in cowpea plants under glasshouse condition. The different concentrations of Squalene (50, 100, 150, 250 and 500 ppm) tested in vitro resulted in reduction of lesion numbers (1.69 cm2) as well as reduced virus titre in co-inoculation spray. The present study suggests the antiviral activity of Squalene by effectively fitting into binding site of coat protein of GBNV with favourable hydrophilic as well as strong hydrophobic interactions thereby challenging and blocking the binding of viral replication RNA with coat protein and propagation. The present organic antiviral molecules will be helpful in development of suitable eco-friendly formulations to mitigate GBNV infection disease in plants.

Antiviral activity of basidiomycetous fungi against Groundnut bud necrosis virus in tomato.

Tomato is an important vegetable crop which is severely affected by Groundnut bud necrosis virus (GBNV). Until now effective antiviral agents have not been reported for the management of necrosis disease caused by GBNV. Therefore, a study was undertaken to manage the necrosis disease caused by GBNV using culture filtrate of basidiomycetous fungi viz., Coprinopsiscinerea, Ganoderma lucidum and Lentinula edodes. In vitro studies were conducted in the indicator host cowpea and primary host tomato in glasshouse under insect proof condition; co-inoculation spraying of culture filtrate of Ganoderma lucidum at 0.1% concentration reduced the lesion numbers and inhibited the virus population build-up when compared to inoculated control in the indicator host cowpea upto 77.83%. DAC-ELISA test was performed to quantify the virus titre, indicated reduced virus titre in co- inoculation spray of culture filtrate of G. lucidum treated cowpea with OD value 0.17 ± 0.01 at 405 nm and in tomato plants 0.14 ± 0.01 respectively. The viral copy numbers were quantified by qPCR. About 2.0 × 101 viral copy numbers were observed in tomato plants treated with G. lucidum (co-inoculation) which was lesser than untreated inoculated control plants (2.4 × 108). In order to identify the antiviral properties of G. lucidum, GCMS analysis was carried out and we found the triterpenoid compound Squalene. This is the first study to analyse and confirm the antiviral activity of G. lucidum against a plant virus.

Induction of in planta resistance by flagellin (Flg) and elongation factor-TU (EF-Tu) of Bacillus amyloliquefaciens (VB7) against groundnut bud necrosis virus in tomato.

Role of plant growth promoting rhizobacteria (PGPR) in growth promotion and induction of resistance against various plant pathogens have been extensively studied. However, MAMP (Microbe Associated Molecular Pattern) triggered immunity (MTI) against plant viruses are not well exploited. The present study enlightens the role of two MAMP genes including, flagellin (Flg) and elongation factor (EF-Tu) in the induction of plant defense against GBNV infecting tomato. Secondary metabolites of Bacillus amyloliquefaciens (VB7), effectively suppressed GBNV symptom expression up to 84% compared to untreated control in cowpea, the indicator host plant. Agrobacterium tumefaciens EHA105 clones expressing the MAMP genes were drenched in the root zone to assess the induction of resistance against GBNV in tomato. Treatment with A. tumefaciens EHA105 clones containing flagellin (Ag- Ba.Flg) and elongation factor-TU (Ag-Ba.EF-Tu) genes as soil drench and foliar spray, reduced virus titre,0.369 OD and 0.379 OD respectively as compared to control 1.249 OD. The disease severity was reduced up to 15% in Ag-Ba.Flg treated plants compared to 88.25% in inoculated control. Further there was an increased expression of defense associated genes including, MAPKK1, WRKY33BB, NPR1 and PR1.The present investigation clearly indicated the efficiency of MAMP genes in triggering defense mechanism in tomato against GBNV.

ssDNA viruses: key players in global virome.

Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. High throughput genome sequencing and improved bioinformatics tools have yielded vast information on presence of ssDNA viruses in diverse habitats. The simple genome of ssDNA viruses have high propensity to undergo mutation and recombination often emerging as threat to human civilization. Interestingly their genome is found embedded in fossils dating back to million years. The unusual evolutionary history of ssDNA viruses reveal evidences of horizontal gene transfer, sometimes between different species and genera.

Evidence of seed transmission of dolichos yellow mosaic virus, a begomovirus infecting lablab-bean in India.

Yellow mosaic disease in field bean caused by begomoviruses belonging to the family Geminiviridae is a major threat to the cultivation of crop in South India. Appearance of bright yellow mosaic symptom in emerging seedlings in farmers field was suggestive of seed transmission of the begomovirus associated with the disease which was investigated in the present study. The begomovirus causing yellow mosaic disease was identified as dolichos yellow mosaic virus (DoYMV) and the presence of DoYMV in matured seeds was confirmed by DAS-ELISA with OD value up to 3.268. In PCR with DoYMV specific primer (DoYMV-CP) the virus was detected in different parts of the seeds viz., seed coat, endosperm and embryo. In embryo the virus was detectable up to 100 per cent followed by endosperm (69.23%). When the non symptomatic leaves of 30 days old grow-out test plants were subjected to DAS-ELISA, the virus was detected up to 46.6%. This is the first evidence of seed transmission of DoYMV.

A distinct seed-transmissible strain of tomato leaf curl New Delhi virus infecting Chayote in India.

Chayote (Sechium edule (Jacq. Sw.) is a single seeded cucurbitaceous vegetable crop mainly grown for its fruit. During 2015-2016, mosaic and leaf distortion type of symptoms were observed in chayote plants in hilly regions of Tamil Nadu. The disease incidence was 50. 3-100% and yield loss was about 69.9% in Dindigul district. The infected chayote plants showed yellow spots, yellow mosaic, leaf curling, puckering, and enations. The fruits of infected plants were malformed and were not marketable. The begomovirus causing the disease was identified as a variant of tomato leaf curl New Delhi virus. The chayote isolates of ToLCNDV share only 91 to 92% identity with other ToLCNDV isolates, deserving to be designated as distinct strain. The phylogenetic analysis on the basis of DNA A component nucleotides clearly indicated common origin of chayote, ridge gourd, ash gourd isolates of India along with Spanish isolates of ToLCNDV. This was contrasting to diverse origin of ToLCNDV isolates from other countries. The virus was sap transmissible to selected cucurbitaceous hosts. The whitefly population (Asia-I) reared in the glass house transmitted the virus to bottle gourd with 4 h of acquisition access period (AAP) and 24 h of inoculation feeding period (IFP). Heavy infestation of greenhouse whiteflies, Trialeurodes vaporariorum on infected chayote plant in Kodaikanal paved way to investigations on its role in vector transmission of ToLCNDV. The field population of T. vaporariorum was found to be viruliferous in PCR using virus specific primers. The greenhouse whitefly efficiently transmitted the virus with 4 h of AAP and 24 h IFP. Seed-borne nature of ToLCNDV was confirmed in PCR by using Roja's and ToLCNDV specific primers in different parts of the fruit viz., pericarp, mesocarp, seed coat, endosperm and embryo. This is the first report of seed transmissible nature of ToLCNDV, its implication in transboundary movement of the virus across several countries is discussed.

Biocontrol Potentials of Antimicrobial Peptide Producing Bacillus Species: Multifaceted Antagonists for the Management of Stem Rot of Carnation Caused by Sclerotinia sclerotiorum.

Bacillus species are widely exploited as biocontrol agents because of their efficiency in impeding various plant pathogens with multifaceted approach. In this study, Bacillus species were isolated from rhizosphere of various plants viz., carnations, cotton, turmeric, and bananas in Tamil Nadu state of India. Their potential to control the mycelial growth of Sclerotinia sclerotiorum was assessed in vitro by dual plate and partition plate techniques. B. amyloliquefaciens strain VB7 was much effective in inhibiting mycelial growth (45% inhibition of over control) and sclerotial production (100%). PCR detection of AMP genes revealed that B. amyloliquefaciens (VB7) had a maximum of 10 diverse antibiotic biosynthesis genes, namely, ituD, ipa14, bacA, bacD, bamC, sfP, spaC, spaS, alba, and albF, that resulted in production of the antibiotics iturin, bacilysin, bacillomycin, surfactin, subtilin, and subtilosin. Further, metabolites from B. amyloliquefaciens strains VB2 and VB7, associated with inhibition of S. sclerotiorum, were identified as phenols and fatty acids by gas chromatography mass spectrometry (GC-MS). Delivery of bacterial suspension of the effective strains of Bacillus spp. as root dip was found promising for the management of stem rot of cultivated carnations. Minimal percent disease incidence (4.6%) and maximum plant growth promotion was observed in the plants treated with B. amyloliquefaciens (VB7).

Revision of Begomovirus taxonomy based on pairwise sequence comparisons.

Viruses of the genus Begomovirus (family Geminiviridae) are emergent pathogens of crops throughout the tropical and subtropical regions of the world. By virtue of having a small DNA genome that is easily cloned, and due to the recent innovations in cloning and low-cost sequencing, there has been a dramatic increase in the number of available begomovirus genome sequences. Even so, most of the available sequences have been obtained from cultivated plants and are likely a small and phylogenetically unrepresentative sample of begomovirus diversity, a factor constraining taxonomic decisions such as the establishment of operationally useful species demarcation criteria. In addition, problems in assigning new viruses to established species have highlighted shortcomings in the previously recommended mechanism of species demarcation. Based on the analysis of 3,123 full-length begomovirus genome (or DNA-A component) sequences available in public databases as of December 2012, a set of revised guidelines for the classification and nomenclature of begomoviruses are proposed. The guidelines primarily consider a) genus-level biological characteristics and b) results obtained using a standardized classification tool, Sequence Demarcation Tool, which performs pairwise sequence alignments and identity calculations. These guidelines are consistent with the recently published recommendations for the genera Mastrevirus and Curtovirus of the family Geminiviridae. Genome-wide pairwise identities of 91 % and 94 % are proposed as the demarcation threshold for begomoviruses belonging to different species and strains, respectively. Procedures and guidelines are outlined for resolving conflicts that may arise when assigning species and strains to categories wherever the pairwise identity falls on or very near the demarcation threshold value.

Complete genome sequence of a new begomovirus associated with yellow mosaic disease of Hemidesmus indicus in India.

The complete DNA A genome of a virus isolate associated with yellow mosaic disease of a medicinal plant, Hemidesmus indicus, from India was cloned and sequenced. The length of DNA A was 2825 nucleotides, 35 nucleotides longer than the unit genome of monopartite begomoviruses. Comparison of the nucleotide sequence of DNA A of the virus isolate with those of other begomoviruses showed maximum sequence identity of 69 % to DNA A of ageratum yellow vein China virus (AYVCNV; AJ558120) and 68 % with tomato yellow leaf curl virus- LBa4 (TYLCV; EF185318), and it formed a distinct clade in phylogenetic analysis. The genome organization of the present virus isolate was found to be similar to that of Old World monopartite begomoviruses. The genome was considered to be monopartite, because association of DNA B and ß satellite DNA components was not detected. Based on its sequence identity (<70 %) to all other begomoviruses known to date and ICTV (International Committee on Taxonomy of Viruses) species demarcating criteria (<89 % identity), it is considered a member of a novel begomovirus species, and the tentative name "Hemidesmus yellow mosaic virus" (HeYMV) is proposed.

Characterization of betasatellite associated with the yellow mosaic disease of grain legumes in Southern India.

Yellow mosaic disease caused by mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus (the family Geminiviridae) is a major constraint in cultivation of grain legumes in India. The urdbean (Vigna mungo (L.) Hepper) and mungbean (Vigna radiata (L.) R. Wilczek) samples affected with yellow mosaic disease exhibits yellow mosaic symptoms along with leaf puckering and leaf distortion in Tamil Nadu. Hence the study was performed to find out if there was any association and influence of betasatellite DNA on the symptom expression of MYMV. Full length viral clones of DNA A and DNA B were obtained through rolling circle amplification from YMD infected samples and identified as mungbean yellow mosaic virus. Interestingly, betasatellite was found to associate with MYMV, and its nucleotide sequence analysis showed its 95% identity with papaya leaf curl betasatellite (DQ118862) from cowpea. The present study represents the first report about the association of papaya leaf curl betasatellite with MYMV and represents a new member of the emerging group of bipartite begomovirus associated with betasatellite DNA.

Response of chickpea genotypes to Agrobacterium-mediated delivery of Chickpea chlorotic dwarf virus (CpCDV) genome and identification of resistance source.

Chickpea stunt disease caused by Chickpea chlorotic dwarf virus (CpCDV) (genus Mastrevirus, family Geminiviridae) is the most important biotic stress affecting chickpea crops worldwide. A survey conducted on the incidence of stunt disease clearly revealed high incidence of the disease with severe symptom expression in both indigenous and imported genotypes. To manage the disease in a sustainable way, resistant genotypes need to be bred by adopting objective and precise assessment of the disease response of chickpea genotypes. At present, evaluation of CpCDV resistance is conducted on the basis of natural infection in the field, which is bound to be erroneous due to vagaries in vector population. To circumvent the above problems, we devised an agroinoculation technique that involves the delivery of viral genomic DNA through Agrobacterium tumefaciens. An objective scoring system assigning quantitative value to different symptoms has been evolved to assess the response of chickpea genotypes to CpCDV inoculation. Using the inoculation and scoring techniques, we screened 70 genotypes, which helped in differentiating field resistance that is more due to resistance to vector feeding than resistance to the virus.

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites.

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF beta C1 in satellite DNA beta which are predicted to function as silencing suppressors. In the present study suppressor function of ORF beta C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB-[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing betaC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with betaC1 gene from ToLCBB. However, in the case of 35S-beta C1 CLCuMB and 35S- beta C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which beta C1 interfere in RNA-silencing pathway is different.