Development of reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay for rapid detection of large cardamom chirke virus.

Large cardamom chirke virus (LCCV) causing chirke disease of large cardamom is a major production constraint of this crop. Rapid and accurate detection of LCCV is important for managing the disease. In the present study an isothermal assay namely, reverse transcriptase-recombinase polymerase amplification (RT-RPA) was developed for the detection of LCCV. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00861-2.

Complete genome sequence of a divergent strain of cardamom mosaic virus.

Cardamom mosaic virus (CdMV; genus Macluravirus), which causes mosaic (katte) disease in cardamom, is a highly variable member of the family Potyviridae. So far, the complete genome sequence of one isolate from Karnataka (KS) has been reported. In the present study, we determined the complete genome sequence of a CdMV isolate from Kerala (KI) and the complete CP gene sequences of nine isolates of CdMV from Kerala, Karnataka, and Tamil Nadu, India. The complete genome of CdMV (KI) consists of 8255 nucleotides (nt) with two open reading frames (ORFs). The large ORF, potentially coding for a polyprotein of 2638 amino acids (aa), is further processed into nine mature proteins at eight cleavage sites. The second ORF, PIPO (pretty interesting Potyviridae ORF) starting with a C(A)6 motif, encodes a small protein of 56 aa. The viral genome contains an additional 13 nt in the 5' untranslated region (UTR) and 6 nt in the CP gene, as well as a deletion of 13 nt at the 3' UTR in comparison to the KS isolate of CdMV. The complete viral genome and polyprotein share 76% and 85% sequence identity with the KS isolate of CdMV, indicating that the present isolate is highly divergent from the KS isolate. Sequencing and analysis of the CP sequences of 16 CdMV isolates from different regions revealed high heterogeneity among them, suggesting that they should be considered members of more than one species.

Rapid onsite detection of piper yellow mottle virus infecting black pepper by recombinase polymerase amplification-lateral flow assay (RPA-LFA).

Piper yellow mottle virus (PYMoV) is a pararetrovirus associated with stunt disease in black pepper. As the primary spread of the virus occurs through vegetative propagation, effective diagnostics are required for the production of virus-free plants. Currently available assays are time-consuming, require expensive equipment, and are not suitable for on-site detection. In the present study, two rapid assays based on the recombinase polymerase amplification (RPA) coupled with lateral flow assay (LFA) using (i) 6-carboxyfluorescein (FAM) labeled nfo probe and biotin-labeled reverse primer and (ii) FAM labeled forward and biotin-labeled reverse primer was developed for the detection of PYMoV. The assays were performed using TwistAmp DNA amplification reagents and crude extract from the infected plant and mealybug as templates. Both assays were optimized for parameters like concentration of magnesium acetate, temperature, and time. The RPA product was then diluted and applied to the sample pad of a lateral flow device for visualizing the results. The formation of a colored line at the test line was considered positive for PYMoV. The entire process from sample preparation to visualization of results could be completed in about 30 min. The developed assays were specific and 10 times more sensitive than PCR. The assays were validated using field samples of black pepper and mealybug vectors.

Piper DNA virus 1 and 2 are endogenous pararetroviruses integrated into chromosomes of black pepper (Piper nigrum L).

A previous study named 7178 and 892 bp contigs obtained through high-throughput sequencing (HTS) of black pepper as Piper DNA virus 1 (PDV-1) and PDV-2 respectively. In the present study, HTS results were confirmed through polymerase chain reaction and Sanger sequencing. The sequenced region of both PDV-1 and PDV-2 contained partial genomes with motifs characteristic of pararetroviruses. BLAST analysis of PDV-1 and PDV-2 against the whole genome sequence of the black pepper showed integration of the PDV-1 at 22 loci in chromosome number 14, and PDV-2 at two loci in chromosome number 12 of black pepper. The integration was confirmed through amplification and sequencing of the junction regions. The present study suggests that both PDV-1 and PDV-2 occur as endogenous viruses in black pepper. Further studies are needed to determine whether these endogenous viruses occur in episomal forms, their complete genome sequence and whether they are activable under abiotic stress conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-021-00752-w.

Symptoms of piper yellow mottle virus in black pepper as influenced by temperature and relative humidity.

Masking of symptoms in winter and their re-appearance in black pepper (Piper nigrum L.) infected with piper yellow mottle virus (PYMoV) in summer is common, especially on new flushes that appear after pre-monsoon showers. Plants of nineteen cultivars of black pepper infected with PYMoV but without any visible symptoms were grown in a polyhouse under natural conditions and in a greenhouse under controlled conditions from January 2019 to January 2020. The number of plants expressing symptoms in the polyhouse increased gradually from 1% during the 3rd standard meteorological week (SMW) (16 January) to 41% during the 21st SMW (22 May), when the afternoon temperature was 30-40 °C and relative humidity (RH) was 75-93%, but began declining thereafter until the 53rd SMW (1 January), when the afternoon temperature was 30-36 °C and RH was 65-86%. The proportion of plants expressing symptoms varied with the cultivar. However, in the greenhouse, in which temperature and RH were maintained at approximately 26 °C and 80%, respectively, not more than 2% of the plants expressed symptoms. The number of symptomatic plants was positively correlated to maximum temperature (T Max) and maximum relative humidity (RH Max) in the afternoon. Based on this observation, a model for predicting the percentage of symptomatic plants was developed using stepwise regression analysis. Plants at the two sites did not differ significantly in the concentration of virus (virus titre) but differed significantly in the content of total carbohydrates, lipid peroxidase, and phenols. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00686-3.

Reverse transcriptase loop-mediated isothermal amplification and reverse transcriptase recombinase amplification assays for rapid and sensitive detection of cardamom vein clearing virus.

In the present study, two isothermal molecular assays viz. reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and reverse transcriptase recombinase amplification (RT-RPA) were developed to detect the cardamom vein clearing virus (CdVCV) infecting cardamom. Assays were optimized for parameters like duration, temperature and concentration of magnesium sulfate, and betaine in the case of RT-LAMP and magnesium acetate in the case of RT-RPA. Detection limits of both assays were determined and compared with conventional RT-PCR and SYBR Green-based real-time RT-PCR. RT-LAMP was found 10,000 times additional sensitive than RT-PCR and one-tenth that of real-time RT-PCR. RT-RPA was found 1000 times additional sensitive than RT-PCR and one-hundredth that of real-time RT-PCR. Both assays were specific, rapid, and sensitive for detecting CdVCV. Compared to real-time RT-PCR, these assays are economical and can be employed in large scale screening of cardamom plants against CdVCV for the selection of virus-free plants.

Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays for the detection of two novel viruses infecting ginger.

Our recent studies have shown the association of two novel viruses namely, ginger chlorotic fleck-associated virus 1 (GCFaV-1) and ginger chlorotic fleck-associated virus 2 (GCFaV-2) with chlorotic fleck disease of ginger. As ginger is propagated through vegetative means, the development of diagnostics would aid in the identification of virus-free plants. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays were developed and validated for the quick detection of GCFaV-1 and GCFaV-2. The detection limits of viruses by these assays, when compared with conventional and real-time RT-PCR, showed that RT-LAMP was up to 1000 times more sensitive than conventional RT-PCR and one-hundredth that of real-time RT-PCR for both the viruses. The detection limit of RT-RPA for GCFaV-1 was up to 100 times more than that of RT-PCR and one-thousandth that of real-time RT-PCR. On the other hand, for detecting GCFaV-2, RT-RPA was found up to 1000 times more sensitive than conventional RT-PCR and one hundredth that of real-time RT-PCR. Based on the cost-effectiveness and duration, RT-LAMP and RT-RPA assays can be suggested for the rapid detection of both viruses.

Recombinase polymerase amplification assay for the detection of piper yellow mottle virus infecting black pepper.

Recombinase polymerase amplification (RPA) is a quick, specific, sensitive molecular tool carried out at a constant temperature for pathogen detection. In the present study, RPA and reverse transcription (RT) RPA assays were optimized for the detection of piper yellow mottle virus (PYMoV) infecting black pepper. Out of the eight primer pairs targeted to amplify open reading frames (ORFs) 2 and 3 of the virus, the primer pair targeted to ORF2 gave specific amplification only with DNA isolated from infected plant but not with healthy plant. A magnesium acetate concentration of 18 mM, 40 min of incubation time and a temperature of 37-42 °C was found optimum for detection of the virus in RPA assay. Comparison of sensitivity of detection revealed that RPA could detect the virus up to 10-5 dilution of the total DNA while PCR could detect the virus up to 10-4 dilution indicating that RPA is 10 times more sensitive than PCR. RPA was further simplified using crude extract as template which could detect the virus up to 10-3 dilution. RT-RPA was optimized for the detection of PYMoV using total RNA isolated from infected plants as the template. Both RT-RPA and RPA assays were validated using field samples of black pepper representing different varieties and geographical regions by using CTAB isolated DNA, crude DNA extract and cDNA. Our study showed that RPA and RT-RPA can be successfully adopted as a substitute to PCR for detection of PYMoV infecting black pepper.

Complete genome sequencing of banana bract mosaic virus isolate infecting cardamom revealed its closeness to banana infecting isolate from India.

The complete genome of banana bract mosaic virus (BBrMV), a Potyvirus belonging to the family Potyviridae causing chlorotic streak disease of cardamom (Elettaria cardamomum) in India was determined for the first time from a naturally infected cardamom var. Njallani Green Gold through reverse transcription PCR using nine sets of primers designed to different overlapping regions of the genome. The complete genome has 9708 nucleotides excluding poly (A) tail and has the genome organization similar to that of BBrMV isolates infecting banana and flowering ginger (Alpinia purpurata). The virus has a single open reading frame of 9372 nucleotides that encodes for a polypeptide of 3124 amino acids which is later cleaved into ten matured proteins. The length and arrangements of different proteins in BBrMV-Cardamom was similar to other BBrMV isolates except for the P1 protein that showed a single amino acid deletion. Comparison with three available complete genome sequences revealed that, BBrMV-Cardamom isolate is more closer to BBrMV-Banana isolate from India (BBrMV-TRY) (96.7% identity) than to BBrMV-Banana isolate from Philippines and flowering ginger isolates from USA (94.5%). Analysis of polyprotein and their individual proteins also showed close identity of BBrMV-Cardamom and BBrMV-TRY. The phylogenetic analysis also suggested that BBrMV-Cardamom isolate is closely related to other BBrMV isolates.

Occurrence of endogenous Piper yellow mottle virus in black pepper.

Some badnaviruses are known to occur as endogenous viruses integrated into their host genome. In the present study, Piper yellow mottle virus (PYMoV), a badnavirus infecting black pepper was shown to occur as endogenous virus based on the PCR, reverse transcription (RT)-PCR, ELISA and Southern hybridization tests. Black pepper plants that tested positive in PCR for PYMoV gave negative reaction in RT-PCR indicating that they harbour endogenous PYMoV (ePYMoV) sequences. The RT-PCR (-ve) plants tested negative in ELISA and also in PCR using outword primers to amplify the full circular genome. Further, the presence of ePYMoV sequences in the black pepper genome was confirmed by Southern hybridization analysis using cloned PYMoV genomic fragments as probes. Among different open reading frames (ORFs) of the virus, ORF 3 was more frequently integrated. This is the first report of occurrence of ePYMoV sequences in black pepper genome.

Complete genome sequencing of cucumber mosaic virus from black pepper revealed rare deletion in the methyltransferase domain of 1a gene.

The complete genome of cucumber mosaic virus (CMV) from black pepper was sequenced and compared with CMV isolates from subgroups I and II reported worldwide. Percent identity and phylogenetic analyses clearly indicated that the CMV isolate from black pepper (BP) belongs to subgroup IB. Sequence analyses also showed the presence of a rare deletion of nine nucleotides in the putative methyltransferase domain of 1a gene which was observed only in two more isolates of CMV among one hundred 1a gene sequences of CMV for which sequence information is available in the database. Interestingly this deletion is not present in the black pepper isolate of CMV from China (WN1) and from Indian long pepper that is closely related to black pepper. Percent identity analyses showed that the 3'untranslated region (UTR) of the three RNAs of the BP isolate were conserved with 91% identity whereas the 5'UTR of three RNAs showed 52-80% identity. The level of gene conservation among the subgroups was highest in coat protein and lowest in 2b. The values of nucleotide diversity studies were further consistent with the above data. The ratio of non-synonymous to the synonymous substitution of the five genes of three RNAs was in the order 1a > 2a > 2b > 3a > 3b and less than one for all the genes, indicating purifying selection. These clearly reflect that the protein encoded by RNA1 is highly tolerant to amino acid changes followed by that of RNA2 and, RNA3 is the least tolerant correlating to its functional importance.

Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies.

Complete genome sequencing of Piper yellow mottle virus infecting black pepper, betelvine, and Indian long pepper.

The complete genome of the Piper yellow mottle virus (PYMoV), a Badnavirus belonging to the family Caulimoviridae, was sequenced from three naturally infected hosts namely, black pepper, betelvine, and Indian long pepper. The genome length of the three virus strains (one from each of the three host species) varied from 7,559 to 7,584 nucleotides, and all the three strains possessed four open reading frames (ORFs) I to IV that potentially encode proteins of 15.67, 17.08, 218.6, and 17.22 kDa, respectively. ORF III encodes a polyprotein consisting of viral movement protein, trimeric dUTPase, zinc finger, aspartic protease, reverse transcriptase, and RNase H whereas ORF I, II, and IV encode proteins of unknown functions. The complete genome sequences at the nucleotide level were 89-99 % identical with one available sequence of PYMoV and 39-56 % identical with other badnaviruses, indicating that all three are strains of PYMoV. Nucleotide and amino acid sequences of ORF I-IV and of the intergenic region (IR) were 80-100 % identical among PYMoV strains. Phylogenetic analysis of ORF III amino acid sequences showed the PYMoV strains forming a distinct cluster well separated from other badnaviruses. Among other badnaviruses, Fig badnavirus 1 (FBV-1) was the one most closely related to PYMoV.

Detection of Cardamom mosaic virus and Banana bract mosaic virus in cardamom using SYBR Green based reverse transcription-quantitative PCR.

Cardamom being perennial, propagated vegetatively, detecting viruses in planting material is important to check the spread of viruses through infected material. Thus development of effective and sensitive assay for detection of viruses is need of the time. In this view, assay for the detection of Cardamom mosaic virus (CdMV) and Banana bract mosaic virus (BBrMV), infecting cardamom was developed using SYBR Green one step reverse transcription-quantitative PCR (RT-qPCR). The RT-qPCR assay amplified all isolates of CdMV and BBrMV tested but no amplification was obtained with RNA of healthy plants. Recombinant plasmids carrying target virus regions corresponding to both viruses were quantified, serially diluted and used as standards in qPCR to develop standard curve to enable quantification. When tenfold serial dilutions of the total RNAs from infected plants were tested through RT-qPCR, the detection limit of the assay was estimated to be 16 copies for CdMV and 10 copies for BBrMV, which was approximately 1,000-fold higher than the conventional RT-PCR. The RT-qPCR assay was validated by testing field samples collected from different cardamom growing regions of India. This is the first report of RT-qPCR assay for the detection of CdMV and BBrMV in cardamom.

The complete genome sequence of Piper yellow mottle virus (PYMoV).

This study reports the first complete genome sequence of Piper yellow mottle virus (PYMoV, KC808712) identified in black pepper. The genome is 7,622 nucleotides long, possessing four open reading frames (ORFs). ORF1, ORF2 and ORF4 of PYMoV are reported as hypothetical proteins of unknown function with a predicted molecular mass of 15.7, 17.1 and 17.9 kDa, respectively. ORF3 of PYMoV encodes a polyprotein of 218.6 kDa and consists of a viral movement protein (MP), trimeric dUTPase, zinc finger, retropepsin, RT-LTR, and RNAse H. Detailed PYMoV genome analysis confirmed that it is a member of the family Caulimoviridae, genus Badnavirus. Fragments of two additional novel sequences resembling those found in members of the family Caulimoviridae were also identified in the black pepper sample, and the viruses from which they were derived were tentatively named Piper DNA virus 1 and 2.

Sequence diversity among badnavirus isolates infecting black pepper and related species in India.

The badnavirus, piper yellow mottle virus (PYMoV) is known to infect black pepper (Piper nigrum), betelvine (P. betle) and Indian long pepper (P. longum) in India and other parts of the world. Occurrence of PYMoV or other badnaviruses in other species of Piper and its variability is not reported so far. We have analysed sequence variability in the conserved putative reverse transcriptase (RT)/ribonuclease H (RNase H) coding region of the virus using specific badnavirus primers from 13 virus isolates of black pepper collected from different cultivars and regions and one isolate each from 23 other species of Piper. Of these, four species failed to produce expected amplicon while amplicon from four other species showed more similarities to plant sequences than to badnaviruses. Of the remaining, isolates from black pepper, P. argyrophyllum, P. attenuatum, P. barberi, P. betle, P. colubrinum, P. galeatum, P. longum, P. ornatum, P. sarmentosum and P. trichostachyon showed an identity of >85 % at the nucleotide and >90 % at the amino acid level with PYMoV indicating that they are isolates of PYMoV. On the other hand high sequence variability of 21-43 % at nucleotide and 17-46 % at amino acid level compared to PYMoV was found among isolates infecting P. bababudani, P. chaba, P. peepuloides, P. mullesua and P. thomsonii suggesting the presence of new badnaviruses. Phylogenetic analyses showed close clustering of all PYMoV isolates that were well separated from other known badnaviruses. This is the first report of occurrence of PYMoV in eight Piper spp and likely occurrence of four new species in five Piper spp.

Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions.

Development, characterization and cross species amplification of polymorphic microsatellite markers from expressed sequence tags of turmeric (Curcuma longa L.).

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST-SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.

Survey and RT-PCR Based Detection of Cardamom mosaic virus Affecting Small Cardamom in India.

Mosaic or marble or katte disease caused by Cardamom mosaic virus (CdMV) is an important production constraint in all cardamom growing regions of the world. In the present study, 84 cardamom plantations in 44 locations of Karnataka and Kerala were surveyed. The incidence of the disease ranged from 0 to 85%. The incidence was highest in Madikeri (Karnataka) while no incidence was recorded in Peermade (Kerala). In general, incidence and severity of the disease was higher in cardamom plantations of Karnataka. A procedure for total RNA isolation from cardamom and detection of CdMV through reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the conserved region of coat protein was standardized and subsequently validated by testing more than 50 field cardamom samples originating from Karnataka and Kerala states. The method can be used for indexing the planting material and identifying resistant lines/cultivars before either they are further multiplied in large scale or incorporated in breeding.

Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper.

Isolation of intact high quality RNA suitable for RT-PCR from black pepper is greatly hindered by the presence of polyphenols and polysaccharides. These compounds adversely affect the sensitivity of virus detection by RT-PCR. The present study evaluated the effect of sodium sulphite in enhancing RNA yield and quality in a modified acid guanidium thiocyanate-phenol-chloroform (AGPC) protocol. The results were compared with the standard AGPC method and RNeasy Plant Mini Kit (Qiagen) for detection of Cucumber mosaic virus through RT-PCR. The addition of sodium sulphite in the extraction buffer increased the sensitivity of virus detection. Higher sensitivity of detection (than obtained from the kit) was seen when sodium sulphite was used at 0.5%. Similar levels of sensitivity were also observed for the detection of Cucumber mosaic virus from Piper longum.