Cotton Leaf Curl Multan Betasatellite Research Articles

Molecular characterization of cotton leaf curl Multan beta-satellite occurance on chili plants in Multan, Pakistan

The prevalence of cotton leaf curl disease (CLCuD) has significantly hampered chili (Capsicum spp.) production, presenting a formidable challenge in Pakistan. During a chili field survey in 2018, distinct symptoms, including stunted growth, yellowing, and severe leaf curling, were observed on several plants. Subsequently, a comprehensive sampling effort was undertaken, collecting a total of 39 symptomatic samples from diverse locations across Multan, Punjab. The DNA extraction from these samples was conducted at the plant virology laboratory at Bahauddin Zakariya University, Multan, marking a crucial step in the investigation of this debilitating disease and its impact on chili production in the region. Molecular analysis with PCR using Av/Ac Core, Beta 01/02, and CLCuMuBF11/R33 primers confirmed begomovirus infection in chili plants. Positive amplification demonstrated a 71.79% infection rate, with 579 bp, 1.4 kb, and 481 bp amplicons for Av/Ac Core, Beta 01/02, and CLCuMuBF11/R33, respectively. Sequencing identified cotton leaf curl Multan beta-satellite (MT668934) infecting the chili plant. Effectively managing these begomoviruses is crucial to curbing their multiplication and protecting vital crops like chili. Addressing the distributions of beta-satellites in agricultural fields, particularly chili, is imperative to prevent further viral spread.

Cotton leaf curl Multan betasatellite impaired ToLCNDV ability to maintain cotton leaf curl Multan alphasatellite.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded (ss) DNA molecules of ~1350 nucleotide in size that have been characterized in both the Old and New Worlds. Alphasatellites have inherent ability to self-replicate, which is accomplished by a single protein, replication-associated protein (Rep). Although the precise function of alphasatellite is yet unknown, and these consider dispensable for infectivity, however, their Rep protein functions as a suppressor of host defence. While alphasatellites are most frequently associated with begomoviruses, particularly with monopartite than bipartite begomoviruses, they have recently been found associated with mastreviruses. The in planta maintenance of alphasatellites by helper geminivirus is still an enigma, with no available study on the topic. This study aimed to investigate whether a widely distributed bipartite begomovirus, tomato leaf curl New Delhi virus (ToLCNDV), can maintain cotton leaf curl Multan alphasatellite (CLCuMuA) in the presence or absence of cotton leaf curl Multan betasatellite (CLCuMuB). The findings of this study demonstrated that ToLCNDV or its DNA A could maintain CLCuMuA in Nicotiana benthamiana plants. However, the presence of CLCuMuB interferes with the maintenance of CLCuMuA, and mutations in the CP of ToLCNDV further reduces it. Our study highlighted that the maintenance of alphasatellites is impaired in the presence of a betasatellite by ToLCNDV. Further investigation is needed to unravel all the interactions between a helper virus and an alphasatellites.

Comparative taxonomical, biological and pharmacological potential of healthy and geminivirus infected leaves of Hibiscus rosa-sinensis L.: First report

In the present research project, the first report on comparative analysis of the taxonomical, biological and pharmacological potential of healthy and geminivirus infected Hibiscus rosa sinensis (L.) leaves of the family Malvaceae was done by using different micro and macroscopic techniques. First of all, leaves were characterized for Cotton leaf curl Multan virus (CLCuMuV) and its associated betasatellite (Cotton leaf curl Multan Betasatellite; CLCuMB). Different morphological parameters like shape and size of stem, leaves, seeds and roots, presence and absence of ligule, distance between nodes and internodes and type of inflorescence etc. were analyzed. CLCuMuV infected H. rosa-sinensis revealed systematic symptoms of infection like chlorosis of leaves, stunted growth, decrease in size of roots, shoots and distortion etc. Anatomical investigation was performed under light ad scanning electron microscope. Different anatomical features like length and shape of guard cells, subsidiary cells, presence or absence of stomata, secretory ducts and trichomes were examined. In both plant samples anomocytic types of stomata and elongated, non-glandular and pointed tip trichomes were present, but the size (especially length and width) of trichomes and other cells like epidermal, subsidiary, and guard cells were highest in virus infected plants likened to healthy one. In the antibacterial activity, the maximum antibacterial potentail was seen in methanolic extract of K. pneumonea while antifungal activity was shown by methanolic extract of A. solani. Plants interact with different biological entities according to environmental conditions continuously and evolved. These types of interactions induce changes positively and negatively on plant metabolism and metabolites production. Many plant viruses also attacked various host plants consequently alter their secondary metabolism. To overcome such virus infected plants produces many important and different types of secondary plant metabolites as a defense response. Subsequent analysis of this n-hexane plant extract using Gas chromatography mass spectroscopy technique revealed that Hibiscus eluted contained 10 main compounds in Healthy sample and 13 compounds in infected one. Presence of essential secondary metabolites were also analyzed by FTIR analysis. The present study provides a comprehensive and novel review on taxonomy (morphology, anatomy) and antimicrobial potential of both healthy and geminivirus infected H. rosa-sinensis.

Detection of Cotton Leaf Curl Disease using Betasatellite-based Molecular Marker

Cotton leaf curl disease (CLCuD) caused by whitefly-transmitted begomoviruses has hampered cotton production across the Punjab and Sindh provinces of Pakistan and northeastern India. Eight species of begomoviruses in association with a single betasatellite “Cotton leaf curl Multan betasatellite (CLCuMB)” have been reported to cause CLCuD. Objective: To detect early and efficiently Cotton leaf curl disease (CLCuD) using betasatellite-based molecular marker. Methods: 3-7 samples leaves were collected from symptomatic cotton fields in selected five areas of cotton production in Pakistan. Total DNA was extracted from collected leaves using the Cetyl trimethylammonium bromide (CTAB) method. Primers were designed by MUSCLE alignment tool and target region was amplified by PCR and amplification confirmed by performing gel electrophoresis. After DNA sequencing Phylogenetic analysis of the was carried out using software MEGA-X. Results: Amplified target region of483bp was observed by running 1% agarose gel. Comparison of DNA sequences revealed two nucleotide substitutions in DNA sequence from samples collected from Multan, Sakrand, Rahim Yar Khan and, while four nucleotide substations in sample collected from Vehari. High nucleotide substation in DNA sequence from Vehari as compared to other regions. Conclusions: In conclusion all of eight distinct begomoviruses causing CLCuD with CLCuMB is indicative of the fact that CLCuMB based molecular marker can be developed for detection of the disease. Early detection of disease will help the breeders and farmers to manage the disease.

Sentinel plot surveillance of cotton leaf curl disease in Pakistan- a case study at the cultivated cotton-wild host plant interface

A sentinel plot case study was carried out to identify and map the distribution of begomovirus-betasatellite complexes in sentinel plots and commercial cotton fields over a four-year period using molecular and high-throughput DNA ‘discovery’ sequencing approaches. Samples were collected from 15 study sites in the two major cotton-producing areas of Pakistan. Whitefly- and leafhopper-transmitted geminiviruses were detected in previously unreported host plant species and locations. The most prevalent begomovirus was cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu). Unexpectedly, a recently recognized recombinant, cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra) was prevalent in five of 15 sites. cotton leaf curl Alabad virus (CLCuAlV) and cotton leaf curl Kokhran virus-Kokhran, ‘core’ members of CLCuD-begomoviruses that co-occurred with CLCuMuV in the ‘Multan’ epidemic were detected in one of 15 sentinel plots. Also identified were chickpea chlorotic dwarf virus and ‘non-core’ CLCuD-begomoviruses, okra enation leaf curl virus, squash leaf curl virus, and tomato leaf curl New Delhi virus. Cotton leaf curl Multan betasatellite (CLCuMuB) was the most prevalent CLCuD-betasatellite, and less commonly, two ‘non-core’ betasatellites. Recombination analysis revealed previously uncharacterized recombinants among helper virus-betasatellite complexes consisting of CLCuKoV, CLCuMuV, CLCuAlV and CLCuMuB. Population analyses provided early evidence for CLCuMuV-Ra expansion and displacement of CLCuKoV-Bu in India and Pakistan from 2012-2017. Identification of ‘core’ and non-core CLCuD-species/strains in cotton and other potential reservoirs, and presence of the now predominant CLCuMuV-Ra strain are indicative of ongoing diversification. Investigating the phylodynamics of geminivirus emergence in cotton-vegetable cropping systems offers an opportunity to understand the driving forces underlying disease outbreaks and reconcile viral evolution with epidemiological relationships that also capture pathogen population shifts.

Transmission efficiency of Cotton leaf curl Khokhran virus /Cotton leaf curl Multan betasatellite complex by two whitefly cryptic species in Pakistan

Transmission efficiency of Cotton leaf curl Khokhran virus /Cotton leaf curl Multan betasatellite complex by two whitefly cryptic species in Pakistan

Interaction estimation of pathogenicity determinant protein βC1 encoded by Cotton leaf curl Multan Betasatellite with Nicotiana benthamiana Nuclear Transport Factor 2.

Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant βC1, located on the betasatellite associated with monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of βC1 in disease pathogenicity and to unveil potential interacting partners of βC1 protein in the model plant Nicotiana benthamiana. The βC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of N. benthamiana and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and in-silico protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families. In-silico analyses were performed to validate the binding affinity of βC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of βC1 protein with NTF2 were identified by using PyMOL and Chimera. The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the βC1. The NTF2 in Solanaceae and Nicotiana was found to be evolved from the Brassica and Gossypium species. The in-silico interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of βC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.

Identification and estimation of sequence variation dynamics of Tomato Leaf curl Palampur virus and betasatellite complex infecting a new weed host

Leaf samples from the symptomatic Solanum nigrum plants showing curling on leaf margins, leaf chlorosis, leaf crumpling and stunting in growth were collected during spring 2018. Subsequently, the standard procedures of total genomic DNA isolation, enrichment via rolling circle amplification, polymerase chain reaction, cloning and complete genome sequencing were performed. Pairwise nucleotide (nt) sequence comparison and phylogenetic studies of the inferred genome sequences confirmed the identity of a bipartite begomovirus Tomato leaf curl Palampur virus (ToLCPalV) in the symptomatic plant samples sharing 97.6 and 95.5% nt identity with ToLCPalV isolates identified from Pakistan and India. In the phylogenetic analysis, ToLCPalV grouped with ToLCPalV isolates previously described from Pakistan and India. Additional attempts to identify any putative DNA-satellites in S. nigrum plant samples revealed an associated betasatellite component, cotton leaf curl Multan betasatellite (CLCuMuB). This is the first identification of cotton leaf curl disease-associated betasatellite with ToLCPalV in this region. The nt diversity analysis showed that ToLCPalV populations have a high mutation rate and evolve independently under strong purifying selection, but not via recombination.

First report of Cotton leaf curl Multan virus infecting Millettia pinnata in Pakistan

Millettia pinnata (Pongame oiltree, Sukh chain; family Fabaceae) is an important leguminous tree widely grown and utilised as a herb in Southeast Asia and the Indian subcontinent. In October 2020, symptoms of stunted growth, leaf curling (upward and downward) and vein thickening (Figures 1 and 2), which are associated with infection by Begomovirus species in other hosts, were observed on M. pinnata, growing near cotton fields (30.3°N, 73.0°E), in Faisalabad, Punjab Province, Pakistan. DNA was isolated from ten leaves (S1 to S10), one each from ten diseased plants, and one leaf from two asymptomatic plants (AS11 and AS12) using a cetyl trimethyl ammonium bromide protocol (Doyle & Doyle, 1990). Rolling circle amplification in combination with PCR using begomovirus universal primers (BegomoF/BegomoR) (Briddon & Markham, 1994) amplified a c. 2.8 kb product from all the diseased samples. PCR assays for the Begomovirus alphasatellite (primer pair DNA101F/DNA101R) (Bull et al., 2003) and betasatellite (primer pair Beta01F/Beta02F) (Briddon et al., 2002) each amplified a c. 1.4 kb product from all the diseased samples. Neither begomovirus genome nor satellite DNA was detected in the asymptomatic samples. Amplicons from the begomovirus genome (AV-2 and AV-3 from S1 and S2; MZ268122-MZ268123), alphasatellite (AV-36 from S1; MZ057253) and betasatellite (AV-8 and AV-10 from S1, AV-11 and AV-12 from S2, and AV-13 from S3; MZ057231- MZ057235), were cloned in a general purpose PTZ57R/T cloning vector (Thermo Fisher Scientific, USA), Sanger sequenced, and the sequences deposited in GenBank. The genome sequences shared >99% nucleotide identity with Cotton leaf curl Multan virus (CLCuMuV) and were most closely related to CLCuMuV strain NAS-14 (MK357257), which was found in cotton in the same region of Faisalabad (Ahmed et al., 2021). They also had 94% and 87% nt similarity with the CLCuMuV-Raj (AF363011) and CLCuMuV-Dar strains (EU365613) respectively. Moreover, the alphasatellite and betasatellite sequences shared 98% similarity with Cotton leaf curl Multan alphasatellite (CLCuMuA; MK357286.1) and betasatellite (CLCuMuB; MT920327.1), respectively. The five CLCuMuB betasatellite sequences (MZ057231-MZ057235) shared 99% identity and the two Begomovirus genomic sequences (MZ268122-MZ268123) from the M. pinnata samples had 99% identity with each other. Southern hybridization (Zubair et al., 2017) confirmed the detection of Begomovirus genomic DNA and quantitative real-time PCR (Shafiq et al., 2017) assays confirmed the detection of CLCuMuV in three samples, S1, S2 and S3, selected for further analysis. To the best of our knowledge, this is the first report of CLCuMuV in Millettia pinnata which may serve as an alternative host of the virus in the off season when cotton is not grown. Routine monitoring and detection of CLCuMuV is essential to prevent the devastating leaf curl disease it causes from spreading worldwide. The authors acknowledge support in Sanger sequencing from Dr. Brian Scheffler and his group from USDA-ARS.

Prevalence, transmission and molecular characterization of Cotton leaf curl Multan virus infecting hollyhock plants in Pakistan.

Hollyhock (Alcea rosea) is an ornamental plant belonging to the Malvaceae family and has a remarkable aesthetic and medicinal value. A number of distinct infectious entity including fungi, nematode, bacteria and most importantly both single and double stranded DNA and RNA viruses are reported from infected hollyhock plant. Begomoviruses, the well reputed member of the family Geminiviridae infected the hollyhock recently with a new hollyhock vein yellowing virus and in the present study it infected the hollyhock plant with Cotton leaf curl Multan virus (CLCuMV) which cause the disease of leaf curling. The symptomatic leaves of the hollyhock plants were collected based on the characteristic symptoms of leaf curling, puckering as well as vein thickening. DNA was extracted by using the recommended 2× CTAB protocol and PCR technique was optimized for the detection of begomovirus followed by sequencing. The data of disease incidence of infection location wise was collected based on the positive results of PCR amplification. Virus free whitefly collected from cotton field and feed on infected hollyhock plant in cage for few days then used for the transmission study of begomovirus on healthy hollyhock plants. Results of PCR amplification indicated that the primers Av/Ac core, Begomo 01/02, and CLCV 01/02 showed the bands of 579bp, 2.8kb and 1.1kb respectively. The betasatellite was amplified by using beta01/02 and CLCuMuBF11/R33, which showed the band of 1400bp and 481bp. Disease incidence and Transmission study confirmed the begomovirus in hollyhock plants at molecular level. The sequence obtained with Av/Ac core primers showed the 99% identity with Cotton leaf curl Multan virus-Rajasthan strain and betasatellite primers showed 98% identity with Cotton leaf curl Multan betasatellite. Hollyhock plants infected by CLCuMV and associated betasatellite has been reported as a possible source of virus inoculum from Pakistan. These findings extend the range of Begomoviruses and betasatellites known to infect A. rosea and highlight this hollyhock species as an important reservoir of agriculturally important Begomoviruses and betasatellites.

A multiplex polymerase chain reaction for the simultaneous detection of the virus and satellite components associated with cotton leaf curl begomovirus disease complex

Cotton leaf curl disease (CLCuD) is caused by a complex of several whiteflies (Bemisia tabaci Genn.)-transmitted begomovirus species, Cotton leaf curl Multan virus (CLCuMuV), Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Alabad virus (CLCuAlV) by individual of mixed infection, associated with Cotton leaf curl Multan betasatellite (CLCuMB) and several alphasatellites. The disease causes major economic losses in cotton in the Indian subcontinent. For monitoring of epidemiology and development of management strategies of CLCuD, a quick, sensitive and effective method capable of detecting all the begomovirus, betasatellite and alphasatellite components associated with CLCuD is required. With this objective, a multiplex polymerase chain reaction (mPCR) assay was developed for the simultaneous detection of these three viral components associated with CLCuD of cotton. Primers for each component were designed based on the retrieved reference sequences from the GenBank. Each pair of primers, designed for each of the respective component, was evaluated for its sensitivity and specificity in both the component-specific simplex polymerase chain reaction (sPCR) and mPCR assay. This report identified three viral component-specific pairs of primers which, in all combinations, amplified simultaneously the CP gene (780 nts) of the begomovirus, the βC1gene (375 nts) of the betasatellite and the Rep gene (452 nts) of the alphasatellite associated with CLCuD in the mPCR assays. The amplified products specific to each component produced by these assays were identified based on their amplicon sizes, and the identities of the viral components amplified were confirmed by cloning and sequencing the amplicons obtained in the mPCR. The mPCR assay was validated using naturally CLCuD-affected cotton plants of the fields. This assay will be useful for rapid detection of CLCuD-associated begomovirus, betasatellite and alphasatellite DNA in field samples, extensive resistance screening in resistance breeding programme, and also monitoring epidemiology for detection of virus and its components when symptoms are mild or absent in the plant.

Transgenic Expression of Synthetic Coat Protein and Synthetic Replication Associated Protein Produces Mild Symptoms and Reduce Begomovirus-Betasatellite Accumulation in Nicotiana benthamiana

Cotton (Gossypium hirsutum L.) is the most essential fiber crop and is widely cultivated within the tropical regions of the world. It has a major impact on socio-economical get-together in Pakistan. Unfortunately, it has been tormented by cotton leaf curl disease (CLCuD) that is produced by distinct species of begomoviruses (Family; Geminiviridae) like Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bu) associated with specific betasatellite—Cotton leaf curl Multan betasatellite (CLCuMuB). Host resistance against CLCuD has not been achieved yet. The present study employed the use of synthetic genes considering the phenomenon of pathogen derived resistance (PDR). The coat protein (CP) and replication associated protein (Rep) genes sequence from CLCuKoV-Bu were codon optimized, synthesized and used to control CLCuKoV-Bu and associated betasatellite related to CLCuD. The Nicotiana benthamiana plants were Agrobacterium mediated transformed with synthetic CP (CPsyn) and synthetic Rep (Repsyn) under the control of Cauliflower mosaic virus 35S promoter. The transgenic plants harboring CPsyn and Repsyn genes were infiltrated with CLCuKoV-Bu alone and CLCuKoV-Bu- CLCuMuB inoculums showed relatively milder symptoms as compared with wild sorts and low virus concentration as checked by southern hybridization. The results confirmed that CPsyn, Repsyn genes may be utilized for the resistance of CLCuKoV-Bu and associated betasatellites related to CLCuD and PDR mechanism may also be developed for the control of CLCuD.

First report of Cotton leaf curl Kokhran virus associated with Cotton leaf curl Multan betasatellite infecting soybean in Pakistan

First report of Cotton leaf curl Kokhran virus associated with Cotton leaf curl Multan betasatellite infecting soybean in Pakistan

Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

Molecular phylogenetics and evolutionary analysis of a highly recombinant begomovirus, Cotton leaf curl Multan virus, and associated satellites.

Cotton leaf curl Multan virus (CLCuMuV) and its associated satellites are a major part of the cotton leaf curl disease (CLCuD) caused by the begomovirus species complex. Despite the implementation of potential disease management strategies, the incessant resurgence of resistance-breaking variants of CLCuMuV imposes a continuous threat to cotton production. Here, we present a focused effort to map the geographical prevalence, genomic diversity, and molecular evolutionary endpoints that enhance disease complexity by facilitating the successful adaptation of CLCuMuV populations to the diversified ecosystems. Our results demonstrate that CLCuMuV populations are predominantly distributed in China, while the majority of alphasatellites and betasatellites exist in Pakistan. We demonstrate that together with frequent recombination, an uneven genetic variation mainly drives CLCuMuV and its satellite’s virulence and evolvability. However, the pattern and distribution of recombination breakpoints greatly vary among viral and satellite sequences. The CLCuMuV, Cotton leaf curl Multan alphasatellite, and Cotton leaf curl Multan betasatellite populations arising from distinct regions exhibit high mutation rates. Although evolutionarily linked, these populations are independently evolving under strong purifying selection. These findings will facilitate to comprehensively understand the standing genetic variability and evolutionary patterns existing among CLCuMuV populations across major cotton-producing regions of the world.

Amplicon-based RNAi construct targeting beta-C1 gene gives enhanced resistance against cotton leaf curl disease.

The online version contains supplementary material available at 10.1007/s13205-021-02816-6.

Biologically prepared copper-graphene nanohybrid as the interface of microchips for sensitive detection of crop viruses

The development of portable and sensitive biosensors for the label-free detection of DNA has influenced fundamental biological research as well as advanced applications. Here, we report the novel microchip-based electronic devices for the efficient and sensitive detection of a crop virus i.e. Cotton leaf curl Khokran virus-Burewala strain (CLCuKoV-Bur), predominant cotton infecting virus worldwide. Three-dimensional copper nanostructures reinforced graphene nanohybrid (Cu Ns@GO) is developed via a biological synthesis approach. The hybrid consists of the typical graphene sheets, embellished with copper nanoparticles of 10–15 nm, to impart conducting and metallic character. This nanohybrid is applied as the active interface of microchips, and the surface charge of +38 mV enables successful anchoring of virus DNA as a probe to the interface. The hybridization events are manipulated as the change in the electron transport between sheets of graphene, leading to a corresponding decrease in conductance of the devices. Termed as Cu Ns@GO-microchip, the devices can detect the presence of viral DNA down to the detection limit of 200 pM. We further investigate specificity patterns and the non-complementary DNAs i.e. Cotton leaf curl Multan betasatellite, Cotton leaf curl Multan alphasatellite, and Maize insect resistance1-cystein protease genes show a negligible response generating only 10–20% of the signal. We apply this strategy to the virus-infected cotton field samples and using the devices, the level of virus infectivity can be discerned. This study suggests the potential of nanotechnology-based microchips for designing benchmark recognition interfaces, for the direct and facile monitoring of agricultural pathogens and other bio-threats.

Development of a LAMP assay using a portable device for the real-time detection of cotton leaf curl disease in field conditions.

Cotton production is seriously affected by the prevalent cotton leaf curl disease (CLCuD) that originated from Nigeria (Africa) to various parts of Asia including Pakistan, India, China and Philippines. Due to CLCuD, Pakistan suffers heavy losses approximately 2 billion USD per annum. Numerous reports showed that CLCuD is associated with multiple species of begomoviruses, alphasatellites and a single species of betasatellite, that is ‘Cotton leaf curl Multan betasatellite’ (CLCuMuB). The most prevalent form of CLCuD is the combination of ‘Cotton leaf curl Kokhran virus’-Burewala strain (CLCuKoV-Bur) and CLCuMuB. Thus, the availability of an in-field assay for the timely detection of CLCuD is important for the control and management of the disease. In this study, a robust method using the loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CLCuD. Multiple sets of six primers were designed based on the conserved regions of CLCuKoV-Bur and CLCuMuB-βC1 genes. The results showed that the primer set targeting the CLCuMuB-βC1 gene performed best when the LAMP assay was performed at 58°C using 100 ng of total plant tissue DNA as a template in a 25 µl reaction volume. The limit of detection for the assay was as low as 22 copies of total purified DNA template per reaction. This assay was further adapted to perform as a colorimetric and real-time LAMP assay which proved to be advantageously applied for the rapid and early point-of-care detection of CLCuD in the field. Application of the assay could help to prevent the huge economic losses caused by the disease and contribute to the socio-economic development of underdeveloped countries.

Circular DNA enrichment sequencing reveals the viral/satellites genetic diversity associated with the third epidemic of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is the most important limiting factor for cotton production in Pakistan. The CLCuD passed through two major epidemics in this region with distinct begomoviruses/satellites complexes. Since 2015 the disease has again started to appear in epidemic form, causing heavy losses to cotton crop, which we termed as the “third epidemic”. We applied CIDER-seq (Circular DNA Enrichment Sequencing), a recently developed sequencing method for PCR-free virus enrichment to produce a full length read of a single circular viral genome coupled with Sanger sequencing to explore the genetic diversity of the disease complex. We identified a highly recombinant strain of Cotton leaf curl Multan virus and a recently evolved strain of Cotton leaf curl Multan betasatellite that are dominant in all major cotton growing regions in the country. Moreover, we also identified multiple species of alphasatellites with one distinct species, Mesta yellow vein mosaic alphasatellite (MeYVMA) for the first time in cotton. Relative abundance of virus and associated satellites was also determined by real-time quantitative PCR. To the best of our knowledge, this is the first study that determined the CLCuD complex associated with its third epidemic.

Cotton Leaf Curl Multan Betasatellite as a Tool to Study the Localization of Geminiviruses in Plants

Cotton leaf curl Multan betasatellite (CLCuMB) is a ubiquitous betasatellite commonly found along with cotton leaf curl disease (CLCuD) associated begomoviruses in the Old World. It has a promiscuous replicative nature and trans-replicated by a diverse range of geminiviruses. CLCuMB encodes a single ORF, βCl, in the complementary direction and has pathogenicity, symptoms determinant, suppressor of post-transcription and transcription gene silencing functions. After substituting the βC1 gene with the target gene, it has been used successfully as a gene delivery vector. In the present study, the βC1 gene of CLCuMB was substituted with the green fluorescent protein (GFP) gene, and the resulting construct utilized as a reporter vector to decipher in planta localization of geminiviruses. The recombinant CLCuMB expressing GFP (CLCuMB-GFP) was co-inoculated to Nicotiana benthamiana plants either with Cotton leaf curl Kokharan virus (CLCuKoV) alone or in a combination with the wild type CLCuMB to investigate the objectives of the study. Results showed that CLCuKoV successfully supported the replication and systemic movement of CLCuMB-GFP either alone or in the presence of wild type CLCuMB. The presence of CLCuMB-GFP was readily detected with PCR and Southern blot hybridization. The modified CLCuMB may serve as a tool useful for in planta localization of geminiviruses.