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Plant viruses are a global concern for sustainable crop production. Among the currently available antiviral approaches, nanotechnology has been overwhelmingly playing an effective role in circumventing plant viruses. Alfalfa mosaic virus (AMV) was isolated and identified from symptomatic pepper plants in Egypt using symptomatology, serological tests using the direct ELISA technique, differential hosts and electron microscopy. The virus was biologically purified from a single local lesion that developed on Chenopodium amaranticolor. The AMV infection was further confirmed using an AMV coat protein-specific primer RT-PCR. We further evaluated the antiviral potential of chitosan nanoparticles (CS-NPs) and chitosan silver nanocomposites (CS-Ag NC) in different concentrations against AMV infections in pepper plants. All tested concentrations of CS-NPs and CS-Ag NC induced the inhibition of AMV systemically infected pepper plants when applied 24 h after virus inoculation. The foliar application of 400 ppm CS-NPs or 200 ppm CS-Ag NC produced the highest AMV inhibitory effect (90 and 91%) when applied 24 h after virus inoculation. Treatment with CS-NPs and CS-Ag NC considerably increased the phenol, proline and capsaicin contents compared to the infected plants. Moreover, the agronomic metrics (plant height, fresh and dry pod weights and number of pods per plant) were also significantly improved. According to our results, the potential applications of CS-NPs and CS-Ag NC may provide an effective therapeutic measure for better AMV and other related plant virus management.

In 2020, the leaves and fruit of 50 pumpkin plants with suspected cucumber mosaic virus (CMV) symptoms of leaf mosaic, vein yellowing, and mild leaf curling were collected from Sharq El-Owainat (Al Wadi El-Gaded governorate), a new reclamation land in Egypt. This study was aimed at deciphering and characterizing the causal agent of the leaf yellowing disease associated with pumpkin plants in Egypt. The causal agent was identified by serological, cytological, and molecular means. The serological identification by DAS–ELISA confirmed the presence of CMV in 20% of the plants. The cytological identification by electron microscopy revealed typical cucumovirus isometric particles of 28–30 nm diameter in the cytoplasm of the leaf parenchyma, epidermal cells, the integument, and the nucleus. Molecular characterization by one-step reverse transcriptase-PCR yielded the required size of amplicon (678 bp) for CMV. Additionally, mechanical sap inoculation was used to determine the host range and symptomatology of the isolated CMV in seventeen different plant species belonging to six different plant families. CMV replicated, moved systemically, and induced a range of symptoms in sixteen plant species. The isolated CMV was transmitted to pumpkin plants at a 16.4% rate by seeds. CMV-infected pumpkin plant leaves were characterized by a substantially low concentration of photosynthetic pigments, a high level of reducing sugars, relatively low protein levels, and a significant increase in total phenol contents, implying their potential role as antiviral agents. Ultrathin sections of infected cells revealed histological changes and cytological abnormalities in comparison to healthy plants. This is the first identification of CMV on new reclamation land in Egypt, pinpointing its swift spread, which could pose a major constraint to pumpkin production in Egypt.

The current study is aimed to assess water use efficiency and evaluate economic viability of hydroponic and conventional production of barley green fodder by keeping in view the water scarcity challenges in Saudi Arabia. A hydroponic system and open field experimental plot was used to evaluate the water use efficiency for different irrigation regimes. Economic indicators for both production systems are estimated and compared to accomplish economic assessment. Estimated indicators include returns from inputs and net profit; benefit-cost ratio; break-even levels of prices, production, and yield; returns over variable cost; and returns on investment. Results indicated that the yield of barley green fodder produced under hydroponic conditions overtopped the yield under conventional cultivation. Under hydroponic and conventional conditions, WUE was decreased with increasing the harvesting date. However, WUE for the hydroponic technique was much higher than the conventional one. The returns and net profits supported the conventional cultivation methods, where lower dry matter content coupled with higher fixed and variable costs incurred by the hydroponic technique outweighed returns leading to economic loss. Cost-benefit ratios, returns over investment, and break-even prices and yield suggested that growing barley fodder under the hydroponic technique is economically not suitable for small-scale farming. However, regarding water conservation, hydroponic barley cultivation showed superiority over conventional field cultivation. Further research on the adoption of hydroponic fodder cultivation is highly recommended for water-scarce arid regions, such as the Kingdom of Saudi Arabia.

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, βC1, 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.

Pumpkin (Cucurbita maxima) plants showing begomovirus-like yellowing, curling and stunting symptoms were observed in the Barka Governate, Oman in December 2016. Cloning and sequencing of rolling circle amplified circular DNA molecules showed the plants to be infected with watermelon chlorotic stunt virus (WmCSV); a bipartite Old World begomovirus that is a serious pathogen of cucurbit crops across the Middle East, Arabian Peninsula and North Africa. Although not known to interact with satellites, the risk of WmCSV possibly interacting with betasatellites and alphasatellites to agriculture in Oman, and beyond, was assessed. Tomato leaf curl betasatellite (ToLCB) and Ageratum enation alphasatellite (AEA) are commonly associated with other begomoviruses in Oman. A cognate pair of WmCSV DNA-A and DNA-B clones was highly infectious to Nicotiana benthamiana by Agrobacterium-mediated inoculation, inducing severe leaf curling and crumpling symptoms. Inoculation of only the DNA-A component with ToLCB or AEA did not result in symptoms or significant levels of the DNA-A or satellites in plants. Inoculation of both DNA-A and DNA-B with ToLCB or AEA resulted in a slight change in symptom phenotype and reduced DNA-A and DNA-B titers. Although ToLCB DNA levels in co-infected plants were low, the levels of AEA were high. This is the first identification of WmCSV infecting pumpkin in Oman and the isolates identified were typical of the virus previously identified in Oman, being distinct from the isolates occurring in the western Arabian Peninsula. Additionally the infectivity analyses suggest that WmCSV interaction with satellites, in contrast to some other bipartite begomoviruses, is unlikely to result in more serious disease in the field.

Background Soil salinity causes huge economic losses to agriculture productivity in arid and semiarid areas worldwide. The affected plants face disturbances in osmotic adjustment, nutrient transport, ionic toxicity and reduced photosynthesis. Conventional breeding approaches produce little success in combating various stresses in plants. However, non-conventional approaches, such as in vitro tissue culturing, produce genetic variability in the development of salt-tolerant plants, particularly in woody trees. Results Embryogenic callus cultures of the date palm cultivar Khalas were subjected to various salt levels ranging from 0 to 300 mM in eight subcultures. The regenerants obtained from the salt-treated cultures were regenerated and evaluated using the same concentration of NaCl with which the calli were treated. All the salt-adapted (SA) regenerants showed improved growth characteristics, physiological performance, ion concentrations and K+/Na+ ratios than the salt non-adapted (SNA) regenerants and the control. Regression between the leaf Na+ concentration and net photosynthesis revealed an inverse nonlinear correlation in the SNA regenerants. Leaf K+ contents and stomatal conductance showed a strong linear relationship in SA regenerants compared with the inverse linear correlation, and a very poor coefficient of determination in SNA regenerants. The genetic fidelity of the selected SA regenerants was also tested using 36 random amplified polymorphic DNA (RAPD) primers, of which 26 produced scorable bands. The primers generated 1–10 bands, with an average of 5.4 bands per RAPD primer; there was no variation between SA regenerants and the negative control. Conclusion This is the first report of the variants generated from salt-stressed cultures and their potential adaptation to salinity in date palm cv. Khalas. The massive production of salt stress-adapted date palm plants may be much easier using the salt adaptation approach. Such plants can perform better during exposure to salt stress compared to the non-treated date palm plants.

Among major constraints to crop production, salt stress affects the morphological structure and yield in crop plants. Nano-fertilizers, have made their hierarchy to supplement the traditional ferti...

A complex of begomoviruses and their associated DNA-satellites is causing leaf curl diseases in Asia, Africa and the Mediterranean Region. Begomoviruses and DNA-satellites native to Asia and Africa are distinct from each other. Different combinations of geographically distinct monopartite begomoviruses, betasatellites and an alphasatellite were analyzed in infection experiments: cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMuV) from Pakistan along with their associated cotton leaf curl Multan betasatellite (CLCuMuB); okra yellow crinkle virus (OYCrV), cotton leaf curl Gezira betasatellite (CLCuGeB), ageratum leaf curl Cameroon betasatellite (ALCCMB) and ageratum leaf curl Cameroon alphasatellite (ALCCMA) from Cameroon; tomato yellow leaf curl virus (TYLCV) from the Mediterranean Region. The infectivity assays were carried out in the model host species Nicotiana benthamiana and agro-infectious clones containing partial tandem repeats of the begomovirus and satellite genomes. The tested begomoviruses from different regions could functionally interact with non-cognate betasatellites and alphasatellite. Plants co-inoculated with CLCuKoV/CLCuGeB or CLCuMuV/CLCuGeB developed, for both combinations, more severe symptoms than with the begomovirus alone and the infection was verified by Southern blot hybridization. Nonetheless, CLCuKoV and CLCuMuV were shown also to support infection by ALCCMB, whereas OYCrV and TYLCV were able to support infection with CLCuMuB, ALCCMB and CLCuGeB. Moreover, all begomoviruses from different geographical origins supported ALCCMA from Africa. The results confirm that geographically diverse begomoviruses can functionally interact with non-cognate DNA-satellites, which may cause the emergence of new begomovirus-satellite complexes. Possibly, exchange of DNA-satellites may occur across continents leading to the emergence of new viral diseases.

Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.

Potato (Solanum tuberosum L.), with approximately 388 million mt annual world production, is ranked fourth among food crops after rice, wheat and corn. However, substantial potato production is vulnerable due to the susceptibility of potato crop to different devastating pathogens especially RNA viruses. The present potato breeding approaches are laborious, intensive and require a protracted time-consuming procedure to develop new resistant and elite cultivars with desired traits. On the other hand, the rapid ability of potato pathogens to evolve has enabled them to cope with the host defense; therefore, a quick technique having a fine-tuned, broad-spectrum and durable mechanism of resistance is required. In addition, such techniques should meet GMO guidelines to win public acceptance. The disclosures of CRISPR-Cas variants, especially Cas13a as a programmable RNA-guided RNase ability, could be reprogramed to target the potato infecting pathogens. Successful execution of a CRISPR-Cas13a system against different pathogens and obtaining transgene-free plants have been described in recent ongoing investigations. The present chapter features the plausible implication of CRISPR-Cas systems to accelerate potato breeding programs. Besides, a multiplexed CRISPR-Cas13a based theoretical model is discussed to combat the menacing worldwide spread of the pathogen potato virus Y (PVY).

Date palm (Phoenix dactylifera L.) is an economically important crop in the oases agroecological zones. In vitro mutagenesis has been an effective strategy for genetic improvements in several traits of crop plants. However, studies related to the genetic improvement of this fruit tree are very limited. Several conventional approaches including physical and chemical mutagens, insertional and somaclonal mutations have been practiced creating the desired traits. However, contemporary site-directed mutation approaches, like TALENS, ZNFs and CRISPR-Cas, have not yet been put into practice for the date palm. The prospects and applications of currently accessible mutagenesis methods for date palm genetic improvement are discussed in this chapter. New breeding tools (NBTs) for targeted mutagenesis through CRISPR-Cas-based genome editing (GE) and its base editing (BE) versions can be very effective to engineer date palm genomes. However, with a large and complex genome, heterozygosity and outcrossing, somaclonal variation during in vitro regeneration, the presence of single-nucleotide polymorphism (SNP) and ultimate genetic instability caused by these SNPs pose challenges. Such challenges could be addressed effectively by the execution of site-specific CRISPR-Cas versions, like BEs, coupled with high-throughput screening techniques. Finally, the hierarchy of targeted mutagenesis over random mutagenesis is addressed as a potential approach for futuristic studies of date palm genetic improvement.

Both biotic and abiotic stresses impact date palm cultivation in harsh and dry desert environments around the world. There is a dire need to improve the adaptability, productivity, and robustness of date palms cultivated under stressful environments. The modern—omics tools coupled with next-generation sequencing (NGS) have progressed in date palm genomics during the current decade. However, date palm has long been an ignored crop for the application of modern approaches. Mutation breeding through physical and chemical mutagens has long been in use to bring about genetic modifications for improved yield and quality-related traits in crop plants. Nevertheless, these mutations are random in their impact and precise genetic modifications have long been a standing question for plant researchers. Modern genome editing (GE) tools have advanced from biomedical sciences to modern agriculture in this post-genomic era. Under these circumstances, CRISPR-Cas-based GE tool can pave the way towards innovations in date palm breeding. It has emerged itself as a state-of-the-art technology due to its robustness, sophistication, cost-effectiveness, versatility, precision, and efficiency. This technique is versatile in single base editing (BE), multiplexing to target gene families, gene knockout or knockin, gene transfer/replacement, epigenetic modifications, DNA-barcoding, genotyping, pathogen profiling and many other expanding applications beyond GE.

A genome represents an organelle’s complete DNA repertoire and it is the starting point of genetic studies and genome mapping. Since the advent of genome mapping techniques, researchers have devoted concerted efforts to link phenotype to genotype. Initially, efforts were made to accomplish genotyping by isozymes, followed by the execution of various kinds of molecular markers such as simple sequence repeats (SSR), single nucleotide polymorphism (SNP), and random amplified polymorphic DNA (RAPD). Nonetheless, genome mapping was dramatically revolutionized as next-generation sequencing (NGS) technologies emerged. NGS technologies have produced a deluge of genome sequencing. The analysis of such sequencing data is only possible through highly sophisticated computational tools. Presently, the low-cost sequencing technologies and highly sophisticated computational tools are of great significance in unraveling the complexity of plant genomes. Date palm (Phoenix dactylifera L.) is a socioeconomically important plant. Cultivar identification mostly relied on phenotypic traits rather than genotyping. Still, limited information is available on date palm genotyping and genome mapping. Nonetheless, advancements in whole-genome sequencing and genome assembly computational tools hold a substantial advantage to boost the identification of date palm cultivars, genome variants, and gene/genome annotation. In this chapter, advancements in date palm genome mapping are categorized into two main types: date palm genetic mapping in the pre-NGS and post-NGS eras. Various computational tools that hold an unparalleled advantage to conduct genome-wide association mapping, genome prediction, and selection, and with higher prediction accuracy of SNPs and other variations in date palm genomes are discussed. In addition, various databases related to date palm genomes are discussed, these databases encompass important information about the whole genome of certain date palm cultivars, molecular markers such as SNPs and SSR, and phylogenetic relationship of different date palm cultivars.

Symptomatic and non-symptomatic leaf samples of cucumber, zucchini, melon and watermelon plants were collected from three locations (Al-Ahsa in the East, Jizan in the South and Tabuk in the North) ...

The whitefly-transmitted begomoviruses (family Geminiviridae) infect dicotyledonous plants and occur in tropical and sub-tropical regions. Although most commonly found in herbaceous plants, recently begomoviruses have increasingly been identified in woody plants. Leaf samples from five mulberry (Morus alba L.) plants with leaf yellowing and curling symptoms were collected in Lahore (Pakistan) and shown by PCR to be associated with a begomovirus, an alphasatellite and a betasatellite. The complete sequences of two begomovirus clones, as well as an alphasatellite clone and a betasatellite clone, were determined. The begomovirus clones were shown to be isolates of ageratum enation virus (AEV), a virus most commonly identified in weeds but increasingly being identified in crops such as tomato, soybean and fenugreek. Analyses of the sequences of the alphasatellite and betasatellite clones showed them to be isolates of Guar leaf curl alphasatellite and Papaya leaf curl betasatellite, respectively. Both the virus and the alphasatellite sequences showed evidence of a recombination. This is the first report of the weed-infecting monopartite begomovirus AEV, and associated satellites, infecting the woody plant mulberry.

Complete begomovirus and betasatellite genomes were isolated from turnip (Brassica rapa) plants exhibiting leaf crumpling symptoms in Pakistan. In this study, two complete begomovirus genomes and one betasatellite molecule were cloned, sequenced and characterized. The identified begomovirus isolates were 100% identical to each other and shared their highest nucleotide (nt) sequence identity at 94.4 and 92.7% with pedilanthus leaf curl virus (PeLCV) and tomato leaf curl Kerala virus (ToLCKeV), respectively. However, in phylogenetic analysis both isolates grouped separately in the ToLCKeV clade. The detailed phylogenetic and recombination analysis showed that the isolates have unique and recombinant nature thus, representing new isolates of PeLCV from turnip in Pakistan. The cloned betasatellite isolate (1371 nt in length) was a non-recombinant molecule and shared highest nt sequence identity (89.8%) with tobacco leaf curl betasatellite (TbLCB). This is the first report of a unique isolate of PeLCV associated with TbLCB infecting B. rapa in Pakistan. The present study extends our knowledge about the current diversity of PeLCV in the important crops.

Date palm (Phoenix dactylifera L.) is the most important edible fruit crop in Saudi Arabia. Date palm cultivation and productivity are severely affected by various fungal diseases in date palm-producing countries. In recent years, black scorch disease has emerged as a devastating disease affecting date palm cultivation in the Arabian Peninsula. In the current survey, leaves and root samples were collected from deteriorated date palm trees showing variable symptoms of neck bending, leaf drying, tissue necrosis, wilting, and mortality of the entire tree in the Al-Ahsa region of Saudi Arabia. During microscopic examination, the fungus isolates growing on potato dextrose agar (PDA) media produced thick-walled chlamydospores and endoconidia. The morphological characterization confirmed the presence of Thielaviopsis punctulata in the date palm plant samples as the potential agent of black scorch disease. The results were further confirmed by polymerase chain reaction (PCR), sequencing, and phylogenetic dendrograms of partial regions of the ITS, TEF1-α, and β-tubulin genes. The nucleotide sequence comparison showed that the T. punctulata isolates were 99.9–100% identical to each other and to the T. punctulata isolate identified from Iraq-infecting date palm trees. The pathogenicity of the three selected T. punctulata isolates was also confirmed on date palm plants of Khalas cultivar. The morphological, molecular, and pathogenicity results confirmed that T. punctulata causes black scorch disease in symptomatic date palm plants in Saudi Arabia. Furthermore, seven commercially available fungicides were also tested for their potential efficacy to control black scorch disease. The in vitro application of the three fungicides Aliette, Score, and Tachigazole reduced the fungal growth zone by 86–100%, respectively, whereas the in vivo studies determined that the fungicides Aliette and Score significantly impeded the mycelial progression of T. punctulata with 40% and 73% efficiency, respectively. These fungicides can be used in integrated disease management (IDM) strategies to curb black scorch disease.

Leaf samples from Malva parviflora plants exhibiting vein yellowing and chlorosis were collected during 2013–14 from Lahore, Pakistan and analysed for the presence of begomovirus and associated DNA satellites through PCR and subsequent sequencing. The identified full-length begomovirus genome shared maximum nucleotide (nt) sequence identity at 92.5% with Hollyhock leaf curl virus (HoLCV), representing a new strain, which was tentatively designated as HoLCV-Mal. The betasatellite shared maximum nt sequence identity (92.8%) with Kenaf leaf curl betasatellite (KLCuB) and represents a new isolate of KLCuB. The two identified alphasatellites were 86% identical to each other and shared maximum nt sequence identities at 94.3 and 93.2% with Ageratum conyzoides symptomless alphasatellite (ACSLA) and Ageratum yellow vein India alphasatellite (AYVIA), respectively. Thus, these are the new isolates of ACSLA and AYVIA identified from M. parviflora in Pakistan. To the best of our knowledge, this is the first re...

There is a risk that Tomato yellow leaf curl virus (TYLCV) and its vector, whiteflies of the Bemisia tabaci species complex, will become established in greenhouses in temperate regions of the world ...

The white-fly borne begomoviruses (family Geminiviridae) have circular single-stranded (css) DNA genome, which is encapsidated as monopartite (DNA-A) or bipartite (DNA-A and DNA-B) in the twinned icosahedrons. During the course of their evolution and to escape host defense machinery, begomoviruses adopt small cssDNA satellites called alpha-, beta-, and deltasatellites. Alphasatellties are found to be associated with begomovirus–betasatellite complexes and encode their own replication-associated protein (Rep), thus capable of autonomous replication. These satellite-like molecules are not well known to serve any critical function for their helper begomovirus except for few reports about attenuation of helper-virus accumulation and/or occasionally suppression of the host defense. Most of the monopartite begomoviruses in the Old World (OW) are found to be associated with betasatellites; however, none of the New World (NW) begomoviruses are known to be associated with betasatellites. Begomoviruses replicate their genome through rolling circle replication (RCR), which requires the virus-encoded Rep to recognize and bind to the iterated sequences (iterons) in the origin of replication (ori) region. Betasatellites lack such iterated sequences; however, they can be transreplicated by a diverse range of begomoviruses, following a similar pattern for replication. Betasatellites play a significant role in viral pathogenesis by interacting with certain host factors, attenuation of disease symptoms, suppression of host defense, and sometimes inter- or intracellular shuttling of begomovirus genome. Likewise, the noncoding molecules deltasatellites depend upon their helper virus for their replication. However, their precise role in viral pathogenesis still needs to be explored.

Cotton has a tremendous economic value worldwide due to its high-quality fiber, edible oil and protein contents. However, the intensifying scenario of human population expansion and global environmental changes demand a proportionate increase in cotton production. In the past, several successful attempts have been made by introgression of many quality- and yield-related traits into elite cotton cultivars through conventional breeding. However, those measures are time consuming due to the reliance on introgression of naturally-existing genetic variation through extensive backcrossing. Nonetheless, plant breeding can be accelerated through modern genome editing (GE) tools. Various GE techniques including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced palindromic repeats and CRISPR-associated proteins systems (CRISPR/Cas)-based approaches have been successfully employed for various crop plants. Among them, CRISPR/Cas-based approaches hold great GE potential due to their simplicity, competency and versatility. In cotton, this system can regulate the gene expression associated with quality traits, to circumscribe phytopathogens and/or to stack molecular traits at a desired locus. In gene stacking through site-specific endonucleases, the desired genes can be introduced in close proximity to a specific locus in the cotton genome with a low risk of segregation. However, such executions are tedious to achieve through classical breeding techniques. Moreover, through the CRISPR/Cas-based approaches, transgene-free cotton plants can easily be produced by selfing or backcrossing to meet the current genetically modified organisms (GMO) guidelines. In this chapter, we address the potential application of CRISPR/Cas-based approaches in available whole cotton genomes to sustain cotton productivity, and achieve genetic improvement, pathogen resistance and agronomic traits. Future prospects of GE applications in cotton breeding are also addressed.

RNA viruses are the most diverse phytopathogens which cause severe epidemics in important agricultural crops and threaten the global food security. Being obligatory intracellular pathogens, these viruses have developed fine-tuned evading mechanisms and are non-responsive to most of the prophylactic treatments. Additionally, their sprint ability to overcome host defense demands a broad-spectrum and durable mechanism of resistance. In context of CRISPR-Cas discoveries, some variants of Cas effectors have been characterized as programmable RNA-guided RNases in the microbial genomes and could be reprogramed in mammalian and plant cells with guided RNase activity. Recently, the RNA variants of CRISPR-Cas systems have been successfully employed in plants to engineer resistance against RNA viruses. Some variants of CRISPR-Cas9 have been tamed either for directly targeting plant RNA viruses' genome or through targeting the host genes/factors assisting in viral proliferation. The new frontiers in CRISPR-Cas discoveries, and more importantly shifting towards RNA targeting will pyramid the opportunities in plant virus research. The current review highlights the probable implications of CRISPR-Cas system to confer the pathogen-derived or host-mediated resistance against phytopathogenic RNA viruses. Furthermore, a multiplexed CRISPR-Cas13a methodology is proposed here to combat Potato virus Y (PVY); a globally diverse phytopathogen infecting multiple crops.

Cotton crop has been severely affected by multiple begomoviruses in Pakistan and India. In our previous study, we found okra enation leaf curl virus (OELCuV), cotton leaf curl Multan betasatellite (CLCuMuB) and cotton leaf curl Multan alphasatellite (CLCuMuA) infecting cotton in Pakistan. The current study was designed to investigate the infectivity of OELCuV and its ability to trans-replicate non-cognate CLCuMuB. Agro-infectious clones containing the partial tandem repeats of OELCuV and CLCuMuB were constructed and the infectivity assays were carried out through Agrobacterium mediated transformation in the model host species Nicotiana benthamiana under controlled conditions. The results showed that in the inoculated plants OELCuV alone can cause downward curling and yellowing of leaves with thickened veins. However, when co-inoculated with the non-cognate CLCuMuB it could functionally trans-replicate CLCuMuB resulting in a more severe phenotype. The expression of Pre-coat/V2 protein in the N. benthamiana plants through the potato virus X (PVX) system caused localized cell death after severe leaf curling in the infiltrated leaves. The tissue tropism of the virus was associated with the systemic development of a hypersensitive response (HR), which ultimately lead to the plant death. The results indicated the involvement of V2 protein in the pathogenicity of OELCuV and its ability to trigger the host defense machinery. This study also demonstrated the ability of OELCuV to trans-replicate CLCuMuB resulting in typical leaf curl disease symptoms in N. benthamiana.

The genetic modifications through breeding of crop plants have long been used to improve the yield and quality. However, precise genome editing (GE) could be a very useful supplementary tool for improvement of crop plants by targeted genome modifications. Various GE techniques including ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), and most recently clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9)-based approaches have been successfully employed for various crop plants including fruit trees. CRISPR/Cas9-based approaches hold great potential in GE due to their simplicity, competency, and versatility over other GE techniques. However, to the best of our knowledge no such genetic improvement has ever been developed in date palm—an important fruit crop in Oasis agriculture. The applications of CRISPR/Cas9 can be a challenging task in date palm GE due to its large and complex genome, high rate of heterozygosity and outcrossing, in vitro regeneration and screening of mutants, high frequency of single-nucleotide polymorphism in the genome and ultimately genetic instability. In this review, we addressed the potential application of CRISPR/Cas9-based approaches in date palm GE to improve the sustainable date palm production. The availability of the date palm whole genome sequence has made it feasible to use CRISPR/Cas9 GE approach for genetic improvement in this species. Moreover, the future prospects of GE application in date palm are also addressed in this review.

Cotton leaf curl disease (CLCuD), the most complex disease of cotton, is a major limiting biotic factor to worldwide cotton productivity. Several whitefly-transmitted monopartite begomoviruses causing CLCuD have been characterized and designated as CLCuD-associated begomoviruses. Despite of being reported over 100 years ago in Africa, CLCuD became economically pandemic causing massive losses to cotton production in Pakistan and India during past couple of decades. In Asia, cotton has faced two major epidemics during this period viz. “Multan epidemic” and “Burewala epidemic.” The “Multan epidemic” era was 1988–1999 after which the virus remained calm until 2002 when “Burewala epidemic” broke into the cotton fields in Indo-Pak subcontinent, till 2013–2014. However, both the epidemics were caused by monopartite begomovirus complex. Similarly in Africa, Cotton leaf curl Gezira virus with associated DNA-satellites causes CLCuD. Quite recently, in the Old World (both Asia and Africa), bipartite begomoviruses have started appearing in the areas under cotton cultivation. Under such aggravated circumstances, it seems we are heading toward another epidemic of CLCuD in the Old World. Here we articulate the causes and potential emergence of the third epidemic of CLCuD in Asia. The current situation of CLCuD in Asia and Africa is also discussed.

Imported tomato fruits infected with Tomato yellow leaf curl virus (TYLCV) were identified on the market in northern Europe using paper-based FTA Classic Cards (Whatman), polymerase chain reaction (PCR) and partial DNA sequence analysis. Trade tomatoes originating from southern Europe, Africa and the Middle East were sampled in Estonia and Sweden, and tested for infection with begomoviruses. Out of 100 batches analysed with five fruits sampled in each batch (58 batches from Estonia and 42 from Sweden), 20 batches were positive (16 from Estonia and four from Sweden). Rolling circle amplification (RCA) and full-length genome sequence analysis of one isolate collected in Estonia and one isolate in Sweden, revealed highest nucleotide sequence identity at 99% to TYLCV-IL for the Estonian isolate and at 97% to TYLCV-Mld for the Swedish isolate. In this study, TYLCV was identified for the first time in imported tomato fruits on the market in northern Europe. FTA cards proved to be an effective means to collect, extract and store begomovirus DNA from tomato fruits and the subsequent molecular analysis.

Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

Okra leaf curl disease (OLCD) is the most important viral disease of okra in West Africa. In this study, a complex of begomoviruses and associated DNA satellites were identified in symptomatic okra plants from southwestern Cameroon. Sequence analyses showed that two of the plants (Lik1 and Njo5) were infected with a begomovirus being a recombinant of cotton leaf curl Gezira virus (CLCuGeV) and okra yellow crinkle virus (OYCrV). The recombinant genome shared highest nucleotide identity with isolates of CLCuGeV at 87.8% and is therefore considered to be member of a new begomovirus species, Okra leaf curl Cameroon virus (OLCuCMV). One plant (Mue5) was infected by a begomovirus with 95.8% nucleotide identy to CLCuGeV, while in the plants Lik1, Mue1 and Njo5, a begomovirus was identified showing highest nucleotide identity at 93.7% with OYCrV. The nucleotide comparisons and phylogenetic analyses suggest that these isolates represent new Cameroonian strains of CLCuGeV and OYCrV (CLCuGeV-CM and OYCrV-CM). Mixed infection of OLCuCMV and OYCrV-CM was found in two of the plants. A betasatellite and two divergent alphasatellites were also associated with the begomoviruses. The betasatellite was identified as cotton leaf curl Gezira betasatellite (CLCuGeB) with the highest nucleotide identity at 93.3% to other African isolates of CLCuGeB. The alphasatellites, herein named Alpha-1 and Alpha-2, shared 97.3% and 95.2% identity, respectively, with cotton leaf curl Gezira alphasatellite (CLCuGeA) and okra leaf curl Burkina Faso alphasatellite (OLCuBFA). These collective results emphasize the extent of diversity among okra-infecting begomovirus–satellite complexes in western Africa.