Genetic Diversity, Evolutionary Dynamics, and Ongoing Spread of Pedilanthus Leaf Curl Virus.

Pedilanthus leaf curl virus (PeLCV) is a monopartite begomovirus (family Geminiviridae) discovered just a few decades ago. Since then, it has become a widely encountered virus, with reports from ca. 25 plant species across Pakistan and India, indicative of its notable evolutionary success. Viruses mutate at such a swift rate that their ecological and evolutionary behaviors are inextricably linked, and all of these behaviors are imprinted on their genomes as genetic diversity. So, all these imprints can be mapped by computational methods. This study was designed to map the sequence variation dynamics, genetic heterogeneity, regional diversity, phylogeny, and recombination events imprinted on the PeLCV genome. Phylogenetic and network analysis grouped the full-length genome sequences of 52 PeLCV isolates into 7 major clades, displaying some regional delineation but lacking host-specific demarcation. The progenitor of PeLCV was found to have originated in Multan, Pakistan, in 1977, from where it spread concurrently to India and various regions of Pakistan. A high proportion of recombination events, distributed unevenly throughout the genome and involving both inter- and intraspecies recombinants, were inferred. The findings of this study highlight that the PeLCV population is expanding under a high degree of genetic diversity (π = 0.073%), a high rate of mean nucleotide substitution (1.54 × 10-3), demographic selection, and a high rate of recombination. This sets PeLCV apart as a distinctive begomovirus among other begomoviruses. These factors could further exacerbate the PeLCV divergence and adaptation to new hosts. The insights of this study that pinpoint the emergence of PeLCV are outlined.

Efficacy of Biogenic Zinc Oxide Nanoparticles in Treating Wastewater for Sustainable Wheat Cultivation.

Water scarcity due to overuse and growing water pollution has led to the need for upgrading of conventional methods of wastewater treatment. The biological synthesis of zinc oxide nanoparticles (ZnO-NPs) and their photocatalytic capacity to degrade contaminants offer a promising and environment-friendly approach to municipal wastewater treatment. This technique is advantageous due to its cost-effectiveness, sustainability, and reduction in toxic residual substances. In this study, microbial-synthesized ZnO-NPs were used for the treatment of municipal wastewater. The objective of this study was to evaluate the potential of treated wastewater for wheat crop cultivation. Zinc oxide nanoparticles were synthesized from a pre-isolated bacterial strain, namely Shewanela sp., and characterized using UV-VIS, X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR) analyses. The results showed that after the treatment of wastewater, the concentration of total dissolve solids (TDS), the chemical oxygen demand (COD), and sulfate and phosphate levels decreased by 76.5%, 57.1%, 81.1%, and 67.4%, respectively. However, the application of treated wastewater increased chlorophyll, carotenoids, and antioxidants by 45%, 40.8%, and 10.5 to 30.6%, respectively. Further, the application of treated wastewater also significantly decreased oxidative stress induced by hydrogen peroxide (H2O2) and malondialdehyde (MDA) by 8.1% and 30.1%, respectively. In conclusion, biosynthesized ZnO-NPs could be an important choice to treat municipal wastewater and to improve wheat productivity.

Antiviral Activity of Chitosan Nanoparticles and Chitosan Silver Nanocomposites against Alfalfa Mosaic Virus.

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.

Population Dynamics of Phytophthora infestans in Egypt Reveals Clonal Dominance of 23_A1 and Displacement of 13_A2 Clonal Lineage.

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt.

Genotypic and Phenotypic Structure of the Population of Phytophthora infestans in Egypt Revealed the Presence of European Genotypes.

Late blight disease of potato and tomato, caused by Phytophthora infestans, results in serious losses to Egyptian and global potato and tomato production. To understand the structure and dynamics of the Egyptian population of P. infestans, 205 isolates were collected from potato and tomato plants during three growing seasons in 2010-2012. The characterization was achieved by mating-type assay, metalaxyl sensitivity assay, and virulence pattern. Additionally, genotyping of 85 Egyptian isolates and 15 reference UK isolates was performed using 12 highly informative microsatellite (SSR) markers David E. L. Cooke and five effector (RxLR) genes. Mating-type testing showed that 58% (118 of 205) of the isolates belonged to mating type A1, 35% (71 isolates) to mating type A2, and the rest 8% (16 isolates) were self-fertile. The phenotype of metalaxyl response was represented as 45% resistant, 43% sensitive, and 12% as intermediate. Structure analysis grouped the 85 identified genotypes into two main clonal lineages. The first clonal lineage comprised 21 isolates belonging to A2 mating type and 8 self-fertile isolates. This clonal lineage was identified as Blue_13 or EU_13_A2. The second main clonal lineage comprised 55 isolates and was identified as EU_23_A1. A single isolate with a novel SSR genotype that formed a distinct genetic grouping was also identified. The effector sequencing showed good correspondence with the virulence data and highlighted differences in the presence and absence of loci as well as nucleotide polymorphism that affect gene function. This study indicated a changing population of P. infestans in Egypt and discusses the findings in the context of late blight management.

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience.

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.

Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective.

In recent years, next-generation sequencing (NGS) and contemporary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have revolutionized the life sciences and the field of plant virology. Both these technologies offer an unparalleled platform for sequencing and deciphering viral metagenomes promptly. Over the past two decades, NGS technologies have improved enormously and have impacted plant virology. NGS has enabled the detection of plant viruses that were previously undetectable by conventional approaches, such as quarantine and archeological plant samples, and has helped to track the evolutionary footprints of viral pathogens. The CRISPR-Cas-based genome editing (GE) and detection techniques have enabled the development of effective approaches to virus resistance. Different versions of CRISPR-Cas have been employed to successfully confer resistance against diverse plant viruses by directly targeting the virus genome or indirectly editing certain host susceptibility factors. Applications of CRISPR-Cas systems include targeted insertion and/or deletion, site-directed mutagenesis, induction/expression/repression of the gene(s), epigenome re-modeling, and SNPs detection. The CRISPR-Cas toolbox has been equipped with precision GE tools to engineer the target genome with and without double-stranded (ds) breaks or donor templates. This technique has also enabled the generation of transgene-free genetically engineered plants, DNA repair, base substitution, prime editing, detection of small molecules, and biosensing in plant virology. This review discusses the utilities, advantages, applications, bottlenecks of NGS, and CRISPR-Cas in plant virology.

CRISPR technology to combat plant RNA viruses: A theoretical model for Potato virus Y (PVY) resistance.

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.

Infectivity of okra enation leaf curl virus and the role of its V2 protein in pathogenicity.

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.

Detection of Verticillium species in Swedish soils using real-time PCR.

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.