Genome sequencing of cucumber mosaic virus (CMV) isolates infecting chilli and its interaction with host ferredoxin protein of different host for causing mosaic symptoms.

Chilli (Capsicum annuum L.) is an important vegetable crop grown in the Indian sub-continent and is prone to viral infections under field conditions. During the field survey, leaf samples from chilli plants showing typical symptoms of disease caused by cucumber mosaic virus (CMV) such as mild mosaic, mottling and leaf distortion were collected. DAC-ELISA analysis confirmed the presence of CMV in 71 out of 100 samples, indicating its widespread prevalence in the region. Five CMV isolates, named Gu1, Gu2, BA, Ho, and Sal were mechanically inoculated onto cucumber and Nicotiana glutinosa plants to study their virulence. Inoculated plants expressed the characteristic symptoms of CMV such as chlorotic spots followed by mild mosaic and leaf distortion. Complete genomes of the five CMV isolates were amplified, cloned, and sequenced, revealing RNA1, RNA2, and RNA3 sequences with 3358, 3045, and 2220 nucleotides, respectively. Phylogenetic analysis classified the isolates as belonging to the CMV-IB subgroup, distinguishing them from subgroup IA and II CMV isolates. Recombination analysis showed intra and interspecific recombination in all the three RNA segments of these isolates. In silico protein-protein docking approach was used to decipher the mechanism behind the production of mosaic symptoms during the CMV-host interaction in 13 host plants. Analysis revealed that the production of mosaic symptoms could be due to the interaction between the coat protein (CP) of CMV and chloroplast ferredoxin proteins. Further, in silico prediction was validated in 13 host plants of CMV by mechanical sap inoculation. Twelve host plants produced systemic symptoms viz., chlorotic spot, chlorotic ringspot, chlorotic local lesion, mosaic and mild mosaic and one host plant, Solanum lycopersicum produced mosaic followed by shoestring symptoms. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03777-8.

Transcriptome analysis during ToLCBaV disease development in contrasting tomato genotypes.

Tomato leaf curl Bangalore virus (ToLCBaV) is one of the most important plant viruses. The infection causes substantial yield losses in tomato crop. The current viral disease management is based mainly on introgression of Ty locus into new tomato cultivars. Unfortunately, strains of the leaf curl virus have been evolving and are breaking Ty based tolerance in tomato. In this study, the defence response to ToLCBaV infection has been compared between contrasting tomato genotypes, resistant line (IIHR 2611; without any known Ty markers) and the susceptible line (IIHR 2843). We carried out comparative transcriptome profiling, and gene expression analysis in an effort to identify gene networks that are associated with a novel ToLCBaV resistance. A total of 22,320 genes were examined to identify differentially expressed genes (DEGs). We found that 329 genes of them were expressed significantly and differentially between ToLBaV-infected samples of both IIHR 2611 and IIHR 2843. A good number of DEGs were related to defence response, photosynthesis, response to wounding, toxin catabolic process, glutathione metabolic process, regulation of transcription DNA-template, transcription factor activity, and sequence-specific DNA binding. A few selected genes such as, nudix hydrolase 8, MIK 2-like, RING-H2 finger protein ATL2-like, MAPKKK 18-like, EDR-2, SAG 21 wound-induced basic protein, GRXC6 and P4 were validated using qPCR. The pattern of gene expression was significantly different in resistant and susceptible plants during disease progression. Both positive and negative regulators of virus resistance were found in the present study. These findings will facilitate breeding and genetic engineering efforts to incorporate novel sources of ToLCBaV resistance in tomatoes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03629-5.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant.

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Unraveling different begomoviruses, DNA satellites and cryptic species of Bemisia tabaci and their endosymbionts in vegetable ecosystem.

Bemisia tabaci species complex contains more than 46 cryptic species. It has emerged as an important pest causing significant yield loss in many cultivated crops. This pest is also a vector for more than 100 species of begomoviruses, that are a major threat for the cultivation of many crops in different regions of the world. The relation between cryptic species of the B. tabaci species complex and associated begomoviruses that infect different crops remains unclear. In the present study, four cryptic species (Asia I, China 3, Asia II 5 and Asia II-1) of B. tabaci and four associated endosymbionts (Arsenophonus, Cardinium, Rickettsia and Wolbachia) were identified in different vegetable crops. The vector-based PCR detection revealed five different begomoviruses such as okra enation leaf curl virus (OELCuV), tomato leaf curl Palampur virus (ToLCPalV), squash leaf curl China virus (SLCCNV), chilli leaf curl virus (ChiLCuV), and tomato leaf curl New Delhi virus (ToLCNDV). Of these begomoviruses, the maximum infection rate was observed (9.1%) for OELCuV, followed by 7.3% for ToLCNDV. The infection rate of the other three viruses (SLCCNV, ChiLCuV, ToLCPalV) ranged from 0.9 to 2.7% in cryptic species of B. tabaci. Further, each cryptic species was infected with multiple virus species and the virus infection rate of Asia I, Asia II-5, China 3 and Asia II-1 was 21.2%, 15.1%, 15.1% and 0.6% respectively. Similarly, in case of betasatellites the highest infection rate was 12% for ToLCBDB, followed by 6% for OLCuB and PaLCB. With regard to alphasatellites, the highest infection rate was 18.2% for AEV and 3% for CLCuMuA. This study demonstrates the distribution of cryptic species of whitefly and their endosymbionts, and associated begomoviruses and DNA satellites in vegetable ecosystem. We believe that the information generated here is useful for evolving an effective pest management strategies for vegetable production.

Random distribution of nucleotide polymorphism throughout the genome of tomato-infecting begomovirus species occurring in India: implication in PCR based diagnosis.

Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00785-9.

Molecular characterization of novel bipartite begomovirus associated with enation leaf disease of Garden croton (Codiaeum variegatum L.).

Garden croton (Codiaeum variegatum L.) plants showing typical begomovirus symptoms of vein twisting, enation and curling were collected from different gardens at Varanasi, Uttar Pradesh state of India ranged from 20 to 30%. All the 10 ten (CR1-CR10) infected samples of garden croton resulted in expected amplicon of 1.2 Kb in PCR specific to begomoviruses. No amplification was obtained for betasatellite and alphasatellite specific primers. The complete genome sequence of DNA-A and DNA-B for two isolates (CR1 and CR2) was obtained through rolling cycle amplification (RCA) and comparisons were made with other begomoviruses using Sequence Demarcation Tool (SDT) which revealed that, DNA-A of two isolates, CR1 (Acc.No.: MW816855) and CR2 (Acc.No.: MW816856) showed maximum nucleotide (nt) identity of 85.7-85.9% with Tomato leaf curl Karnataka virus, which is below the threshold percentage of begomovirus species demarcation, hence considered as novel begomovirus and proposed the name Garden croton enation leaf curl virus (CroELCuV) [IN: Varanasi: Croton: 18]. Further, DNA-B these isolates shared maximum nt identity of 91.0-92.2% (DNA-B) with Tomato leaf curl New Delhi virus. Recombination and GC plot analysis showed that the recombination occured at in low GC content regions of DNA-A and DNA-B of the CroELCuV and are derived from the previously reported several begomoviruses. This is the first record of novel bipartite begomovirus associated with vein twisting, enation and leaf curling of disease of garden garden croton in India and world. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00772-0.

Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta.

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus (CTV) of genus Closterovirus and family Closteroviridae. The disease has caused tremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595 fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 × 107-1.4 × 108/100 mg) and the lowest in partial dead twigs (1 × 103-1.7 × 104/100 mg), and shoot tip (2.3 × 103-4.5 × 103/100 mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 × 107-1.1 × 108/100 mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.

Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus.

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to 'Kinnow mandarin' hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 109) to 0.000948 amol (5.709 × 102), with detection limit of 5.709 × 102 copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

Transmission, characterization and occurrence of recombination in Indian strain of squash leaf curl China virus associated with yellow mosaic and leaf curl disease of Summer squash.

Summer squash is one of the important vegetable crops and its production is hampered by various abiotic and biotic stresses. Of the different biotic stresses, viral infections are responsible for causing great losses to this crop. Diseases caused by begomoviruses are becoming a major constraint in the cultivation of summer squash. Samples from summer squash plants exhibiting severe yellow mosaic and leaf curl symptoms were collected from the Varanasi district of Uttar Pradesh (India) and begomovirus associated with these plants was transmitted through whiteflies (Bemisia tabaci) to healthy squash plants. The relationship between causal virus and whitefly vector was determined. The minimum acquisition access period (AAP) and inoculation feeding period (IFP) required by B. tabaci to transmit the virus was determined to be 10 min and female insects have greater efficiency in transmitting virus than male insects. The partial genome of the virus was amplified by PCR (1.2 kb), cloned and sequenced from the ten infected plant samples collected from field. Partial genome sequence analysis (1.2 kb) obtained from the ten samples revealed that they are associated with begomovirus species closely related to the Indian strain of Squash leaf curl China virus (SLCCNV). Therefore, one representative sample (Sq-1) was selected and complete genome of the virus was amplified by rolling circle amplification (RCA) method. Sequence analysis by Sequence Demarcation Tool (SDT) showed that the current isolate has maximum nucleotide (nt) identity of 93.7-98.4% and 89-98.1% with respect to DNA A DNA B, respectively with Indian strains of SLCCNV infecting cucurbits in India. Recombination analysis of genomes (DNA A and DNA B components) showed that a major part of genomes likely to be originated from already known begomoviruses (ToLCNDV, SLCCNV-CN and SLCCNV-IN) are infecting cucurbitaceous crops. Serological assays such as triple antibody sandwich-enzyme-linked immune-sorbent (TAS-ELISA) assay, dot blot immunobinding assay (DIBA), immuno-capture polymerase chain reaction (IC-PCR) were developed for the detection of SLCCNV. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02821-9.

A Rapid and Sensitive Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus.

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the 'Kinnow mandarin', a commercial citrus crop cultivated in the northwest of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcription polymerase chain reaction (RT-PCR) that is time consuming. Here, we describe a novel, one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. To standardize the RT-LAMP assay, four different primers were designed and tested to target the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. The ICRSV RT-LAMP assay developed in the present study is a simple, rapid, sensitive, specific technique. Moreover, the assay consists of only a single step and is more cost effective than existing methods. This is the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large-scale indexing of field samples in diagnostic laboratories, in nurseries, and for quarantine applications.

Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection.

Diseases caused by begomoviruses are becoming the major limiting factors for the production of watermelon in India. Survey for the incidence of plants showing symptoms typical to begomovirus infection was conducted in watermelon fields. The study revealed that 40% of the watermelon plants were showing the yellowing and downward curling symptoms. Twenty infected samples were collected from the different farmer's fields to know the association of begomoviruses. The PCR amplification using begomovirus-specific primers resulted in an expected 1.2 kb PCR product indicating the begomovirus association with the watermelon samples. The sequence comparison results of 1.2 kb representing partial genome revealed that all sequences obtained from watermelon samples have a nucleotide (nt) identity of more than 98% among them and are maximum homology with Tomato leaf curl New Delhi virus (ToLCNDV). One watermelon sample (WM1) was selected for complete genome amplification using RCA method (rolling-circle amplification). Amplification of DNA B and no amplification of betasatellites and alphasatellite indicated this virus as bipartite. Sequence Demarcation Tool (SDT) analysis of the DNA A component of the WM1 isolate showed the maximum nt identity of 94.6-97.9% and 85.2-95.8% with ToLCNDV infecting cucurbits. The recombinant analysis showed that the genome was likely to be derived from the recombination of already reported begomoviruses (ToLCNDV, ToLCPalV, and MYMIV) infecting diverse crops. The whitefly cryptic species predominant in the begomovirus-infected watermelon fields were identified as Asia-II-5 group. The LAMP assay developed based on coat protein gene sequence was able to detect the ToLCNDV in the infected samples. Visual detection of the LAMP-amplified products was observed with the hydroxy naphthol blue. LAMP assay was also validated with ToLCNDV infected sponge gourd, spine gourd, ivy gourd, ridge gourd, and cucumber. This is the first report of ToLCNDV association with leaf curl and yellowing disease of watermelon from India and World based on complete genome sequencing.

Association of Tomato Leaf Curl New Delhi Virus, Betasatellite, and Alphasatellite with Mosaic Disease of Spine Gourd (Momordica dioica Roxb. Willd) in India.

BACKGROUND: Spine gourd (Momordica dioica Roxb. Willd) is one of the important cucurbitaceous crops grown across the world for vegetable and medicinal purposes. Diseases caused by the DNA viruses are becoming the limiting factors for the production of spine gourd reducing its potential yield. For the commercial cultivation of the spine gourd, propagation material used by most of the growers is tuberous roots and stem cuttings, which in turn results in an increased occurrence of the mosaic disease. There is a need for understanding the causal agent; through characterization of which will lead to the designing management strategies for the spine gourd mosaic disease control. OBJECTIVES: Characterization of a begomovirus and its satellites associated with mosaic disease on spine gourd. MATERIALS AND METHODS: Total DNA was extracted from spine gourd samples exhibiting symptoms typical to the begomoviruses infection (mosaic mottling, leaf curl) and was tested by PCR using begomovirus specific primers. Furthermore, the complete genome of begomo viruses (DNA A, DNA B, alpha satellite, and beta satellite) was amplified by rolling circle amplification (RCA) method. RESULTS: The full-length sequences of DNA A, DNA B, alpha satellite, and beta satellite isolated from symptomatic spine gourd were determined. The full length genomes (DNA A and DNA B) of the Tomato leaf curl New Delhi Virus (ToLCNDV) infecting spine gourd were compared with the other begomovirus genomes available in the data base. The sequence analysis has revealed that DNA A and DNA B components of the begomovirus infecting spine gourd share 95.4-96.2 and 86.7-91.2% identical sequence (i.e., nucleotide (nt) identity) with that of ToLCNDV infecting potato and cucurbits in the Indian subcontinent isolates reported earlier (available in GenBank), respectively. Further, alpha satellite and beta satellite were also detected in the begomovirus infected spine gourd samples. The recombination analysis of the DNA A, DNA B, beta satellite, and alpha satellite of the begomovirus infecting spine gourd showed the associated begomovirus and satellite DNAs were driven from the different begomoviruses, leading to emergence as a new variant of the begomovirus infecting spine gourd. CONCLUSIONS: The commercial cultivation of the spine gourd by most growers depends on the tuberous roots and stem cutting. The occurrence of begomovirus in spine gourd gives an alarming signal against utilization of such infected plant materials in the crop breeding and improvement programs. Using the clean virus-free vegetative propagation material is considered as one of the most important methods for controlling viral diseases. The study is highly useful for detection of the begomovirus infecting spine gourd in the detection of the virus infection in the clonally propagated planting material.

ICTV Virus Taxonomy Profile: Bromoviridae.

Bromoviridae is a family of plant viruses with tri-segmented, positive-sense, single-stranded RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain subgenomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and are transmitted between hosts mechanically, in/on the pollen and non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomato, cucurbits, bananas, fruit trees and alfalfa. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bromoviridae, which is available at www.ictv.global/report/bromoviridae.

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Molecular detection and characterization of phytoplasma in association with begomovirus in eggplant.

The samples from eggplants showing mixed symptoms of little leaf and mosaic were collected from two districts (Mirzapur and Varanasi) of Uttar Pradesh, India. The total nucleic acid extracted from these samples was amplified by PCR using universal 16S rRNA primers specific to phytoplasma and primers specific to DNA-A-like sequence of begomovirus. A total of eighteen eggplant samples showing the symptoms of little leaf and mosaic tested positive for the presence of both begomovirus and phytoplasma. The phytoplasma associated with the mixed symptoms of mosaic and little leaf in the eggplant samples was identified as a member belonging to Clover proliferation group (16SrVI) (nucleotide sequence identity of 97.5-97.8%). The characterized begomovirus from the eggplant samples was identified as a strain of previously described bipartite begomovirus tomato leaf curl Palampur virus (ToLCPalV) (92.5-94.1% nucleotide sequence identity), which is known to infect cucurbits and solanaceous crops in India and Ireland. Further, putative recombination events were detected within the 16S rRNA gene F2n/R2 fragment of phytoplasma and DNA-A of strain of ToLCPalV. Most of the sequence variations observed within the phytoplasma were due to intra and interspecific recombination events between eggplant little leaf-16SrVI-D, Ca. P. asteris-16SrI and Ca. P. pruni-16SrIII. Similarly, most of the DNA fragments of newly characterized strain of ToLCPalV appear to have been derived from tomato leaf curl New Delhi virus (ToLCNDV), squash leaf curl China virus (SLCCNV) and ToLCPalV like ancestors. This perhaps is the first evidence of mixed infection of both phytoplasma-begomovirus in eggplant in India.

Quantitative distribution of Citrus yellow mosaic badnavirus in sweet orange (Citrus sinensis) and its implication in developing disease diagnostics.

Citrus yellow mosaic badnavirus (CMBV) is the etiologic agent of citrus yellow mosaic disease, which has caused serious economic losses to Indian citrus industry. CMBV is a quarantined pathogen that is geographically restricted to India. To prevent unintentional movement of the virus to other major citrus-growing countries in fruits, root stocks or grafted citrus plants and facilitate trade, a sensitive, validated diagnostic tool is needed. In the present study, we developed a SYBR Green real-time PCR-based method to detect and quantify CMBV in different tissues of infected Mosambi sweet orange (Citrus sinensis) and compared its sensitivity to conventional PCR protocols. Primers were designed to recognize a portion of the CMBV capsid protein gene. Conventional and real-time PCR were performed on several different tissues: shoot tips, leaves displaying typical CMBV symptoms, asymptomatic leaves, senescent leaves, thorns, green stems and feeder roots. The detection limit of CMBV by conventional PCR was 2.5 × 104 copies per 5 ng of total genomic DNA, while the detection limit of real-time PCR was found to be 4.6 × 102 virus copies per 5 ng of viral DNA. The viral load varied between different tissues. The highest concentration occurred in feeder roots (3.5 × 108 copies per 5 ng of total genomic DNA) and the lowest in thorns (1 × 106 copies per 5 ng of total genomic DNA). The variation in viral load within different tissues suggests movement of the virus within an infected plant that follows the path of photo-assimilates via the phloem. In symptomatic leaves, the CMBV concentration was highest in the lamella followed by midrib and petiole, suggesting that virus resides inside these sections of a leaf and side by side symptoms develop. On the other hand, in asymptomatic leaves, the petiole contained higher virus load than the lamella and midrib suggesting that the pathogen gets established from the stem through the phloem into petiole then infects the lamella and midrib. In addition to information on virus movement, the distribution of CMBV in different tissues helps with the selection of tissues with relatively higher viral load to sample for early and sensitive diagnosis of the disease, which will be useful for better management of the disease in endemic areas.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

Diversity and phylogeography of Begomovirus-associated beta satellites of Okra in India.

BACKGROUND: Okra (Abelmoschus esculentus; family Malvaceae) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus Begomovirus. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India. RESULTS: The full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect ßC1 gene. ßC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi. CONCLUSION: The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the ßC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.

HIV sero-prevalance in general population of Warangal, A.P., South India.

HIV/AIDS pandemic has devastated many countries reversing national development; HIV was not seen in Asia and India till 1980. Now India has become epicenter of AIDS pandemic. During April 2002 to March 2003 the HIV+ ve pregnant women and their husbands were encouraged to enroll in the prospective study with informed consent. The study results consist of most of the females who are in the age group between 16-25 years who were affected by HIV High infection is observed in people with lower socio-economic and education background. High infection rate is observed in house wives (26.7%), laborers (23%) and agricultural workers (12.1%) followed by toddy tapers (5%), drivers (5.96%) and others (6 47%). HIV +ve subjects at Mother To Child Transmission (MTCT) centers are surprisingly clinically very healthy. No disease manifestation was noticed.