Pyramiding Ty-1/Ty-3 and Ty-2 in tomato hybrids dramatically inhibits symptom expression and accumulation of tomato yellow leaf curl disease inducing viruses

Tomato yellow leaf curl disease is a major constraint for tomato production worldwide and availability of new resistant materials is of great importance for breeding programmes. A phenotypic survey was undertaken to evaluate the level of resistance to the main tomato yellow leaf curl disease-inducing viruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, in several commercial tomato cultivars, never characterised before. Seven weeks post inoculation, two cultivars resulted in high resistant phenotypes to both begomoviruses, and four were tolerant to at least one of them. In the two highly resistant hybrids (SJ12, RFT112), symptoms were completely absent and viral DNA was from 10² to 10⁵ fold lower than in susceptible plants. Molecular marker analysis revealed that these genotypes harbour the resistant genes Ty-1/Ty-3 and Ty-2. Given their high resistance, they can be considered good candidates for cultivation and breeding in areas where incidence of TYLCD is very elevated.     

Development of PCR-based diagnostic probe to detect begomoviruses infecting chilli in the hot arid region of Rajasthan

A survey was conducted in major chilli-growing hot arid regions of Rajasthan, namely, Bikaner, Nagur, Jodhpur and Jalore districts, during November 2009. Among the four districts surveyed for chilli leaf curl disease (ChLCD), maximum disease incidence was recorded in Jodhpur (98%) followed by Jalore district (88%). The number of whiteflies was also counted in top, middle and bottom leaf of chilli plants grown in these areas. The average number of whiteflies per plant ranged from 0.0 to 4.0. Higher number of whiteflies (4.0) was recorded in Jodhpur and lowest (1.8) in Jalore district. On the basis of conserved region in the genome of begomoviruses infecting chilli, a set of primers was designed to amplify all begomoviruses infecting chilli by PCR; ChCPF 5'-ATTAGGGCTAAGAATTATGTC-3' and ChCPR 5'-AAATTCCAATCTTTATTAATT-3'. These primers were validated by cloning and sequencing (HM004433) of PCR-amplified products and detection from infected chilli leaf samples. These primers were utilised while screening chilli cultivars against begomovirus infection in the asymptomatic plants. Our investigation suggests that the leaf curl disease of chilli is widespread in the hot arid regions of Rajasthan and is caused by a begomovirus associated with a satellite DNA β. The PCR primers designed in this study could be highly useful in chilli breeding programmes.     

Molecular evidence of occurrence of Tomato Leaf Curl New Delhi Virus infecting cucurbits in several states in India

Abstract

Tomato Leaf Curl New Delhi Virus (ToLCNDV) is an important begomovirus constraint to the production of cucurbitaceous and other crops in India and its subcontinent. In this study first time, symptomatic samples of different cucurbits were collected from Telangana, Uttar Pradesh (Varanasi) and Madhya Pradesh location in India. The symptomatic samples were associated with begomovirus infection was confirmed by PCR amplification using specific universal primers Deng 540/541. Thirteen out of fifteen samples were amplified with newly designed bipartite specific primers JKR 58?F and JKR 59?R. In eleven out of thirteen bipartite samples contain betasatellite, confirmed by PCR amplification using specific primer CLB36F and CLB37R. The amplified PCR product with JKR58F and 59R of bitter gourd sample collected from Telangana was sequenced. The sequence was share 97.53% similarity with ToLCNDV infecting bottle gourd in Haryana in India (FN645905). Phylogenic analysis revealed that ToLCNDV infecting cucurbits are different, from ToLCNDV infecting tomato.     

Molecular in silico structure and recombination analysis of betasatellite in Calotropis procera associated with begomovirus

The uncharacterised betasatellite of begomovirus associated with Calotropis procera was characterised by using molecular and in silico tools and techniques. Attempts to identify the presence of a DNA-β in the infected C. procera samples, using rolling circular amplification (RCA) followed by restriction digestion, produced a ca. 1.4 kb product, corresponding to that expected for a full-length amplicon from a betasatellite, which was sequenced (accession number HQ631430). During BLASTp, analysis of second reading frame of HQ631430 (HQ631430/2-f) against Protein Databank revealed 35% identity with Tryptophanyl–tRNA synthetase of Giardia lamblia (3FOC). Ramachandran plot of HQ631430/2-f.pdb had only 57.1% residues in the most favoured region while 3FOC.pdb had 94.2% residues in the most favoured region; therefore, only template 3FOC.pdb model could be placed in good quality category. The protein binding function was predicted for HQ631430/2-f as an important functional site of the model with 0.29 confidence level through 3d2GO. The Croton yellow vein mosaic betasatellite (GU111995 CroYVMB) serve as major parent and Croton yellow vein mosaic betasatellite-Panipat 8 (HM143908 PaLCuVM) as minor parent for HQ631430. Perhaps this is the first report of recombination in Croton yellow vein mosaic betasatellite (HQ631430).     

Complete Nucleotide Sequence of Ageratum enation virus and an Alphasatellite Infecting a New Host Glycine max in India

During 2011, leaf crumpling, yellowing and stunting were observed on soya bean (Glycine max) in Himachal Pradesh, India. PCR‐based detection confirmed the presence of a begomovirus. The viral genome was amplified by rolling circle amplification, cloned and sequenced. The complete nucleotide sequence of DNA‐A showed highest nucleotide identity to an isolate of Ageratum enation virus infecting a weed Ageratum conyzoides. In addition, a DNA molecule was found which shared 95% nucleotide identity with an alphasatellite infecting ageratum. Neither beta satellite nor DNA‐B was detected in the infected samples.     

Characterisation of full genome of Dolichos yellow mosaic virus based on sequence comparison,genetic recombination and phylogenetic relationship

Yellow mosaic disease (YMD) is one of the most important diseases affecting different leguminous crops and causes significant yield losses in Indian sub‐continent. Eight different bipartite begomovirus species are known to cause YMD in more than 10 leguminous crops. These species are collectively known as legume yellow mosaic viruses (LYMVs), and their full genomes have been characterised except for Dolichos yellow mosaic virus (DoYMV). In this study, full genome of DoYMV isolate (KJ481204 and KJ481205) infecting dolichos has been characterised. The DNA‐A of DoYMV consists of 2761 nucleotides and DNA‐B of 2733 nucleotides with a genome organisation typical of Old World bipartite begomoviruses. Nucleotide identity of DNA‐B (KJ481205) of DoYMV with DNA‐B of other legumoviruses was 57.5–61.0%. Both components contain a nonanucleotide and conserved inverted repeat sequences with the potential to form a stem‐loop. Nucleotide identity of common region of DoYMV was 90.3%, above the threshold nucleotide identity (>85%) for considering a DNA‐B molecule as cognate of DNA‐A of a begomovirus. Four recombination events in DNA‐A and two in DNA‐B of DoYMV isolate were detected. Mungbean yellow mosaic virus, Rhynchosia yellow mosaic virus and Horsegram yellow mosaic virus were identified as probable parents.     

RNA silencing suppressor activity of Velvet bean severe mosaic begomovirus DNA-A encoded proteins

Velvet bean severe mosaic virus (VbSMV) is a bipartite DNA virus infecting Mucuna pruriens (Velvet bean), belongs to the genus Begomovirus in the family Geminiviridae. Velvet bean is a medicinal plant of enormous medicinal value. In the present study, it was delineated that proteins encoded by VbSMV viz. AV2 (pre-coat protein), AC2 (TrAP), AV1 (coat protein) are suppressors of RNA silencing as identified through Agrobacterium co-infiltration assays using Nicotiana benthamiana as a host plant. AV2 showed strong suppressor activity whereas AC1 and AV1 were found to be weak suppressors. To the best of our knowledge, this is the first report on identification of suppressor of RNA silencing encoded by VbSMV infecting a medicinal plant.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Molecular identification of three isolates of Jatropha mosaic India virus associated with mosaic disease of Withania somnifera in India

The association of begomovirus with mosaic disease of Withania somnifera was detected at three locations in India by PCR using begomovirus-specific degenerate primers. The resulting amplicons of ～1.2 kb from three locations (Aligarh, Lucknow and Hindaun City) were sequenced. The begomovirus isolates of three locations shared 96–97% identities among them and 91% identities with Jatropha mosaic India virus (JMIV). Based on highest sequence identities and close phylogenetic relationships with JMIV, these begomovirus isolates associated with mosaic disease of W. somnifera were identified as isolates of JMIV.     

Interplay between abiotic (drought) and biotic (virus) stresses in tomato plants

With climate warming, drought becomes a vital challenge for agriculture. Extended drought periods affect plant–pathogen interactions. We demonstrate an interplay in tomato between drought and infection with tomato yellow leaf curl virus (TYLCV). Infected plants became more tolerant to drought, showing plant readiness to water scarcity by reducing metabolic activity in leaves and increasing it in roots. Reallocation of osmolytes, such as carbohydrates and amino acids, from shoots to roots suggested a role of roots in protecting infected tomatoes against drought. To avoid an acute response possibly lethal for the host organism, TYLCV down-regulated the drought-induced activation of stress response proteins and metabolites. Simultaneously, TYLCV promoted the stabilization of osmoprotectants' patterns and water balance parameters, resulting in the development of buffering conditions in infected plants subjected to prolonged stress. Drought-dependent decline of TYLCV amounts was correlated with HSFA1-controlled activation of autophagy, mostly in the roots. The tomato response to combined drought and TYLCV infection points to a mutual interaction between the plant host and its viral pathogen.     

Detection of begomovirus associated with okra leaf curl disease

Leaf curl and yellow vein mosaic viral disease is the major constraint on okra (Abelmoschus esculentus L.) production in India. Amplified fragment sequence of DNA-β showed highest similarity of 91.7% with Bhendi yellow vein mosaic virus-Tamil Nadu (AJ308425, NC_003405) and lowest similarity of 48.5% with OKLCV (NC_004093), whereas coat protein specific amplified sequence showed highest homology with isolate of Madurai, Haryana, Ludhiana and lowest homology of 92% with Mesta yellow vein mosaic Bahraich virus (MYVMBV) (EU360303). The results obtained in the present study confirm that both the viral diseases of okra reported in southern India are caused by a begomovirus associated with DNA-β in which the plants show leaf curl symptoms and never develops yellow vein mosaic and those plants which show yellow vein mosaic, never develops leaf curl symptoms even in the same rows and field. The okra leaf curl is an emerging virus disease in India.     

Characterization and Experimental Host Range of a Brazilian Tomato Isolate of Tomato severe rugose virus

We report the complete molecular characterization of the DNA‐A and DNA‐B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using whitefly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.     

Mixed infections of begomoviruses in pumpkins with yellow vein mosaic disease in north India

Yellow vein mosaic disease of pumpkin (Cucurbita moschata) is a serious problem for the cultivation of pumpkin throughout India. Symptomatic samples collected from Varanasi region of North India showed mixed infection of the three begomoviruses which could represent up to three species namely Tomato leaf curl New Delhi virus, Squash leaf curl China virus and Tomato leaf curl Palampur virus.     

番茄黄化曲叶病毒对温室白粉虱适合度的影响

【目的】温室白粉虱Trialeurodes vaporariorum(Westwood)是为害北方地区花卉蔬菜的主要粉虱种类,烟粉虱Bemisia tabaci(Gennadius)则在近些年逐渐频繁的花卉贸易活动中扩散至黑龙江省部分地区并取代了白粉虱成为当地温室害虫的优势物种,番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)是烟粉虱传播的一种重要双生病毒,对作物的危害十分严重,然而该病毒对本地白粉虱的影响及对烟粉虱与白粉虱种间竞争关系的影响尚待研究。【方法】本研究观察记录感染番茄黄化曲叶病毒的番茄植株上温室白粉虱和烟粉虱的种群动态及番茄形态和部分生理指标的变化。【结果】结果表明:1)有烟粉虱滋生的番茄植株矮小,根系发达;2)有白粉虱滋生的番茄略微矮粗,影响较小;3)感染病毒的番茄矮粗或矮小,根部无明显变化;4)染毒带虫相对于带虫处理而言,在白粉虱试验中,番茄植株矮小,根系生物量也骤减;与此相反,在烟粉虱试验中,番茄的反应相对缓和;5)此外,不同酶类对植株染毒的响应不同:Ach E酶活高不利于植株,而GST酶活高则有利于植株。【结论】总体而言:烟粉虱单独作用很不利于苗期番茄,白粉虱对苗期番茄没有明显的直接影响;而感染病毒会缓解烟粉虱对番茄的强烈刺激,而加重白粉虱对番茄的作用,即染毒使带白粉虱的苗期番茄生长发育受到明显抑制。与番茄变化情况相对应的是,染毒番茄上烟粉虱产卵较少,但在发育前期(从卵到伪蛹)存活率较高;染毒番茄上白粉虱产卵较多,但在前期存活率较低。本研究可为高纬度地区粉虱综合防控提供参考。     

The DNA-1 like alphasatellites in association with the bipartite Mungbean yellow mosaic virus in natural infections

Yellow mosaic disease is the major limitation in the production of grain legumes in India. This disease is caused by bipartite begomovirus, Mungbean yellow mosaic virus. In addition to the bipartite genomic components, the yellow mosaic disease affected urdbean plants which contain satellite like DNA-1 component called as alphasatellites. The present study has been attempted to characterise the alphasatellites associated with Mungbean yellow mosaic virus. Nucleotide sequence analysis of alphasatellites showed 98% identity with Vernonia yellow vein Fijian alphasatellite, VYVFA (JF733780). Since the sequence identity is more than 98%, the threshold value for demarcation of alphasatellites species, the alphasatellites of the present study are named as Vernonia yellow vein Fijian alphasatellite. Comparison with other, alphasatellites shared 51–55% identity with alphasatellites associated with monopartite begomovirus and it shared only 41–42% identity with an unusual alphasatellites, DNA-2. This is the first report on characterisation of alphasatellites associated with Mungbean yellow mosaic virus.     

Universal primers for the PCR-mediated amplification of DNA β

DNA β is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA β sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA β component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.     

Emerging geminivirus problems: A serious threat to crop production

Geminiviruses form the second largest family of plant viruses, the Geminiviridae, represented by four genera: Mastrevirus, Curtovirus, Topocuvirus and Begomovirus. During the last two decades these viruses have emerged as devastating pathogens, particularly in the tropics and subtropics, causing huge economic losses and threatening crop production. Epidemics caused by re‐emerging and newly emerging geminiviruses are becoming frequent even in regions that were earlier free from these viruses. Compared to mastreviruses and curtoviruses, begomoviruses have emerged as more serious problems in a variety of crops, for example, cassava, cotton, grain legumes and vegetables. Major contributory factors for the emergence and spread of new geminivirus diseases are the evolution of variants of the viruses, the appearance of the whitefly ‘B’ biotype and the increase in the vector population. Variability in geminiviruses has arisen through mutations, recombination and pseudorecombination. Genomic recombination in geminiviruses, not only between the variants of the same virus but also between species and even between genera, has resulted in rapid diversification. From the disease point of view, most virulent variants have developed through recombination of viral genomes such as those associated with cassava mosaic, cotton leaf curl, and tomato leaf curl diseases. Heterologous recombinants containing parts of the host genome and/or sequences from satellite‐like molecules associated with monopartite begomoviruses provide unlimited evolutionary opportunities. Human activity has also played an important role in the emergence of serious geminivirus diseases across the globe, like the changes in cropping systems, the introduction of new crops, the movement of infected planting materials and the introduction of host susceptibility genes through the exchange of germplasm.