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Plant Virus Metagenomics: Advances in Virus Discovery
Abstract
In recent years plant viruses have been detected from many environments, including domestic and wild plants and interfaces between these systems-aquatic sources, feces of various animals, and insects. A variety of methods have been employed to study plant virus biodiversity, including enrichment for virus-like particles or virus-specific RNA or DNA, or the extraction of total nucleic acids, followed by next-generation deep sequencing and bioinformatic analyses. All of the methods have some shortcomings, but taken together these studies reveal our surprising lack of knowledge about plant viruses and point to the need for more comprehensive studies. In addition, many new viruses have been discovered, with most virus infections in wild plants appearing asymptomatic, suggesting that virus disease may be a byproduct of domestication. For plant pathologists these studies are providing useful tools to detect viruses, and perhaps to predict future problems that could threaten cultivated plants.
... Recent proposals for a global surveillance system to mitigate pathogen introductions are being discussed (Carvajal Yepes et al., 2019). The increasing scalability and affordability of new sequencing technologies, combined with robotics and progress in nucleic acid extraction and enrichment, allow rapid virus identification (Ho and Tzanetakis, 2014;Roossinck et al., 2015;Massart et al., 2019). ...
... One of the caveats of these detection assays is that they are usually designed to target specific known viruses, meaning that a limited number of pathogens is tested in certified clean seed programs. Traditional diagnostics assays often fail at detecting novel viruses and genetic variants; however, with the implementation of HTS, all viruses can be recovered, as discussed above (Roossinck et al., 2015;Simmonds et al., 2017). Both DNA and RNA viruses can be detected using virions, small RNAs, dsRNAs, or total RNA, and these methods can be implemented in bulk or individual samples (Kreuze et al., 2009;Alcala-Briseño et al., 2020;Moubset et al., 2022). ...
... Complex interactions can sometimes protect crops, such as the tripartite interaction in papaya, which generated tolerance to papaya ringspot disease through an antagonistic effect of PapMV that elevated the expression of a pathogenesis-related protein (PR-1), reducing PRSV RNA accumulation (Chavez-Calvillo et al., 2016). Single or bulked samples have also been successfully used to identify resistance genes, and viruses (Roossinck et al., 2015;Shi et al., 2019;Kavil et al., 2021). Phenotypic and genotypic data can provide information for the development of disease-resistant cultivars, but the genetic structure underlying disease resistance regulatory networks is generally not simple. ...
Virome analysis via high-throughput sequencing (HTS) allows rapid and massive virus identification and diagnoses, expanding our focus from individual samples to the ecological distribution of viruses in agroecological landscapes. Decreases in sequencing costs combined with technological advances, such as automation and robotics, allow for efficient processing and analysis of numerous samples in plant disease clinics, tissue culture laboratories, and breeding programs. There are many opportunities for translating virome analysis to support plant health. For example, virome analysis can be employed in the development of biosecurity strategies and policies, including the implementation of virome risk assessments to support regulation and reduce the movement of infected plant material. A challenge is to identify which new viruses discovered through HTS require regulation and which can be allowed to move in germplasm and trade. On-farm management strategies can incorporate information from high-throughput surveillance, monitoring for new and known viruses across scales, to rapidly identify important agricultural viruses and understand their abundance and spread. Virome indexing programs can be used to generate clean germplasm and seed, crucial for the maintenance of seed system production and health, particularly in vegetatively propagated crops such as roots, tubers, and bananas. Virome analysis in breeding programs can provide insight into virus expression levels by generating relative abundance data, aiding in breeding cultivars resistant, or at least tolerant, to viruses. The integration of network analysis and machine learning techniques can facilitate designing and implementing management strategies, using novel forms of information to provide a scalable, replicable, and practical approach to developing management strategies for viromes. In the long run, these management strategies will be designed by generating sequence databases and building on the foundation of pre-existing knowledge about virus taxonomy, distribution, and host range. In conclusion, virome analysis will support the early adoption and implementation of integrated control strategies, impacting global markets, reducing the risk of introducing novel viruses, and limiting virus spread. The effective translation of virome analysis depends on capacity building to make benefits available globally.
... Recent advances in high-throughput sequencing technologies have enabled the accurate identification and comprehensive analysis of plant viromes, including those of crops of agricultural and horticultural importance 21,22 . In this context, various tools, encompassing reference-based mapping, de novo assembly, or k-mer analysis, have been created, contributing to the swift expansion of our understanding of the diverse virus species that infect crops and other plants [23][24][25][26] . In addition, metatranscriptomic data mining has proven to be a useful technique for finding hidden viral species in public databases generated for different purposes [27][28][29][30] . ...
... In this study, we integrated the analysis of sample pools derived from cultivars in La Mancha geographical region with the analysis of saffron transcriptomes sourced from the NCBI Sequence Read Archive (SRA). In recent years, the use of High-Throughput Sequencing (HTS) has enabled the identification and characterization of new viruses and some of which were uncovered through data mining in the NCBI database 25,26,28 . Our pipeline has combined hidden Markov probabilistic models (profile HMMs) and local similarity between protein or nucleotide sequences against several databases. ...
Saffron (Crocus sativus L.), a historically significant crop valued for its nutraceutical properties, has been poorly explored from a phytosanitary perspective. This study conducted a thorough examination of viruses affecting saffron samples from Spanish cultivars, using high-throughput sequencing alongside a systematic survey of transcriptomic datasets from Crocus sativus at the Sequence Read Archive. Our analysis unveiled a broad diversity and abundance, identifying 17 viruses across the 45 analyzed libraries, some of which were highly prevalent. This includes known saffron-infecting viruses and previously unreported ones. In addition, we discovered novel viruses from the Alphaflexiviridae, Betaflexiviridae, Potyviridae, Solemoviridae, and Geminiviridae families, with some present in libraries from various locations. These findings indicate that the saffron-associated virome is more complex than previously reported, emphasizing the potential of phytosanitary analysis to enhance saffron productivity.
... 18 However, the significant limitation of using transcriptome libraries for viral sequence detection is the low detection ability for low-abundance viruses. 19 2.2 Small RNA sequencing Small RNA (sRNA) sequencing is another profound technique for identifying viruses. [20][21][22] Most eukaryotes have an antiviral defense mechanism known as RNA interference (RNAi). ...
... 96 Baculovirus infection of the tracheal system servicing the central nervous system of caterpillars ensures that a supply of synthesized, secreted toxin is available in close proximity to axonal membranes. 19 Recent research has revealed the capability of positive-sense RNA viruses to encode exogenous genes and achieve expression within insect hosts. 64,65 However, the stability of exogenous genes tends to diminish with increasing passages. ...
Insect viruses have long been recognized for their potential as biological control agents in managing agricultural pests. Despite their promise, challenges such as host specificity, virulence, and limited viral resources have hindered their widespread application. The need for efficient discovery and utilization of insect viruses is paramount for sustainable agricultural practices. With the introduction of high‐throughput sequencing (HTS) technologies, the discovery and identification of new insect viruses have been significantly accelerated, enriching the available resources for pest management. Furthermore, advancements in reverse genetics tools have enabled the development of multifunctional viral expression vectors, enhancing the specificity and efficacy of insect viruses against targeted pests. This review provides a comprehensive overview of the methodologies for insect virus identification using HTS and the potential of genetically modified insect viruses in pest control. Genetically engineered insect viruses offer a promising avenue for targeted and efficient pest management. While HTS technologies have revolutionized the discovery of insect viruses, challenges remain. Addressing these challenges and harnessing the potential of these technologies will be pivotal in shaping sustainable agricultural management strategies in the future.
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... In plant virology in particular, advances in the development of viral metagenome analyses have been of great importance in terms of early detection of known viruses and discovery of novel plant viruses (4,7,13,14), as more than half of emerging diseases in plants are thought to be caused by viruses (15). HTS has a huge potential in plant virus diagnostics because it allows to picture the complete phytosanitary status of a plant and to differentiate between virus variants that may contribute differentially to disease etiology (14). ...
... For single plant samples or low-complexity samples, the use of total RNA or small interfering RNA (siRNA) sequencing are considered the most universal and straightforward options (24,25), but when the viromes of entire plant communities are analyzed from complex plant pools, VANA or dsRNA enrichment methods are generally preferred (4,7,19,21,26). A huge number of bioinformatic tools are available for HTS data analysis and have been, together with nucleic acid preparation strategies, extensively reviewed (13,27,28). The choice of a specific viral enrichment method or bioinformatic pipeline depends on the experimental objectives. ...
In contrast to microbial metagenomics, there has still been only limited efforts to benchmark performance of virome analysis approaches in terms of faithfulness to community structure and of completeness of virome description. While natural communities are more readily accessible, synthetic communities assembled using well-characterized isolates allow more accurate performance evaluation. Starting from authenticated, quality-controlled reference isolates from the DSMZ Plant Virus Collection, we have assembled synthetic communities of varying complexity up to a highly complex community of 72 viral agents (115 viral molecules) comprising isolates from 21 viral families and 61 genera. These communities were then analyzed using two approaches frequently used in ecology-oriented plant virus metagenomics: a virion-associated nucleic acids (VANA)-based strategy and a highly purified double-stranded RNAs (dsRNAs)-based one. The results obtained allowed to compare diagnostic sensitivity of these two approaches for groups of viruses and satellites with different genome types and confirmed that the dsRNA-based approach provides a more complete representation of the RNA virome. However, for viromes of low to medium complexity, VANA appears a reasonable alternative and would be the preferred choiceif analysis of DNA viruses is of importance. They also allowed to identify several important parameters and to propose hypotheses to explain differences in performance, in particular, differences in the imbalance in the representation of individual viruses using each approach. Remarkably, these analyses highlight a strong direct relationship between the completeness of virome description and sample sequencing depth which should prove useful in further virome analysis efforts.
IMPORTANCE
We report here efforts to benchmark performance of two widespread approaches for virome analysis, which target either virion-associated nucleic acids (VANA) or highly purified double-stranded RNAs (dsRNAs). This was achieved using synthetic communities of varying complexity levels, up to a highly complex community of 72 viral agents (115 viral molecules) comprising isolates from 21 families and 61 genera of plant viruses. The results obtained confirm that the dsRNA-based approach provides a more complete representation of the RNA virome, in particular, for high complexity ones. However, for viromes of low to medium complexity, VANA appears a reasonable alternative and would be the preferred choice if analysis of DNA viruses is of importance. Several parameters impacting performance were identified as well as a direct relationship between the completeness of virome description and sample sequencing depth. The strategy, results, and tools used here should prove useful in a range of virome analysis efforts.
... NGS could be thus helpful in controlling the introduction of foreign pathogens into a new country during the import of plant materials. Many novel and known viruses were detected from crop plants as well from wild hosts by using NGS in metagenomic methods (Roossinck et al., 2015;Roossinck, 2015;Stobbe & Roossinck, 2014). By using high-throughput sequencing techniques, complete genome sequencing of many known viruses were obtained which could be utilized for the identification and characterization of viral isolates in different novel and known host plants during infection. ...
... NGS could be thus helpful in controlling the introduction of foreign pathogens into a new country during the import of plant materials. Many novel and known viruses were detected from crop plants as well from wild hosts by using NGS in metagenomic methods (Roossinck et al., 2015;Roossinck, 2015;Stobbe & Roossinck, 2014). By using high-throughput sequencing techniques, complete genome sequencing of many known viruses were obtained which could be utilized for the identification and characterization of viral isolates in different novel and known host plants during infection. ...
... After applications of NGS technologies in plant virology during 2009 (Hadidi et al. 2016), their applications have been continuing in crop research across the world. It revolutionized the viral genomics mainly on the identification and discovery of novel viruses and redefining the characterization of previously identified viruses (Barba et al. 2014;Hadidi et al. 2016;Blawid et al. 2017;Pecman et al. 2017) from which the viral metagenomics or viromics arrived for the detection of all viruses present in a given sample (Roossinck et al. 2015). Since plant viruses are obligate/ biotrophic pathogens, the NGS based omics technologies have led to deciphering the blueprint of the genome, the molecular mechanism of host-virus interaction networks, and functional analysis of the gene pathways/genes, etc. Prior to the arrival of NGS technologies, suppression-subtractive hybridization (SSH), expressed sequence tag (EST), and cDNA-AFLP-sequencing were highly used to revel the genome details (Muthamilarasan et al. 2019). ...
... In plant virology, the viability of metagenomics was taken up during 2009 with the usage of the virus-derived small-interfering RNA (siRNA) based approach (Donaire et al. 2009;Kreuze et al. 2009). Subsequently, it quickly became popular; many studies of plant viruses and viroids have been published using this technique (Wu et al. 2015), along with identification of 86 unknown plant viruses (Roossinck et al. 2015). Plantviral metagenomics data also helps in analysing virus evolution and diversity studies, viral surveillance, developing new diagnostics and molecular viral epidemiology (Bernardo et al. 2013). ...
... The percentage of reads mapped to the expected target viruses and viroids ranged from 73 to 99.8%, with an average of 95.6% per sample (Supplementary Table S2). As previously reported in Costa et al. (2022a), this is one of the main advantages of the HiPlex amplicon sequencing approach when compared with regular high-throughput sequencing from total RNA for virus/viroid detection, in which only a small fraction of the reads, usually less than 1%, is derived from the viral or viroid genomes (Mehetre et al. 2021;Roossinck et al. 2015). ...
The implementation of reliable methods for the simultaneous detection of multiple viruses is challenging in plant viral diagnosis. In the present study, we report the development and effectiveness of an optimized version of a robust multiplexed PCR (HiPlex) followed by simultaneous targeted amplicon sequencing to detect 14 viruses and 5 viroids of pome fruit trees. We used amplicon sequencing data from 1,400 pome fruit tree samples previously diagnosed with the HiPlex v.1.0, which included 290 primer pairs for pome viruses. The analysis enabled the identification of highly efficient and specific primer pairs to be assembled in a new HiPlex (dubbed HiPlex v.2.0). Additionally, newly designed primers were implemented for 30% of targeted viruses and viroids. Overall, 116 primer pairs were assembled into the HiPlex v.2.0, resulting in a reduction of the number of primers and multiplex PCR reactions needed by 60 and 50%, respectively. The new HiPlex also includes two nepoviruses, tomato ringspot virus and tobacco ringspot virus, showing flexibility at augmenting and expanding the number of targeted diagnosed viruses using this approach. This optimized set of primers increased the cost-effectiveness and sensitivity while reducing background amplification. Comparable results for virus and viroid detection were obtained using the HiPlex v.2.0 and individual RT-qPCRs, even at a 10,000-fold dilution, demonstrating similar sensitivity to standard PCR-based diagnostics. This new HiPlex-based amplicon sequencing represents a powerful, sensitive, and low-cost tool for large-scale screening of pome fruit trees for all reported viruses and viroids infecting these crops.
[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
... The identification of many previously unknown viruses in symptomatic and asymptomatic wild plants improved the knowledge of viromes of specific environments/hosts. This allowed for predicting the potential risk for the emergence and spread of new plant diseases in cultivated plants in the future (Roossinck et al., 2015;Stobbe and Roossinck, 2016). ...
The European Virus Archive - Global (EVA-Global; https://www.european-virus-archive.com) is a non-profit organization involving a global network with expertise in animal, human, and plant virology, consisting of 38 partner laboratories both based in EU member states and abroad. EVA-Global, as part of the European Union’s Horizon 2020 research and innovation programme’s funding, is conceived to be an open-access entity aiming at developing synergies and complementary capabilities to offer improved access to researchers and industry. The web-based catalogue (https://www.european-virus-archive.com/evag-portal) provides easy access for the end user to the collections, including viruses, derived materials, services, and engineered cells. The consortium ensures that the available materials meet the highest scientific standards regarding safety, quality, and characterization. While the property of the provided materials remains with the originators, specific EU funding provides the option for free-of-charge access to viral resources for public research institutions. Besides virus material, antibodies, molecular detection assays, cutting-edge methodology for virus cultivation, preservation, and analyses are available. All partners agreed on implementing a quality management system. The large number of distinct viruses and isolates enables the concept of preparedness that has been beneficial to researchers worldwide during the current SARS-CoV-2 pandemic. Moreover, EVA-Global contributes to the preservation of virus biodiversity. Here, we summarize EVA-Global activities in building a network to actively share plant viruses and plant virus-derived products.
... Unlike Sanger sequencing, NGS is not limited by the length of a nucleotide sequence that can be read at a time and it doesn't require any prior information about nucleotide sequences. This provides NGS with a significant edge in the primary detection of plant viruses (Kesanakurti et al., 2016;Pecman et al., 2017;Roossinck et al., 2015). ...
Citrus yellow vein clearing virus (CYVCV) is a member of the Alphaflexiviridae family that causes yellow vein clearing symptoms on citrus leaves. A total of 118 leaf samples from nine regions of six provinces in Korea were collected from various citrus species in 2020 and 2021. Viral diagnosis using next-generation sequencing and reverse transcription polymerase chain reaction (RT-PCR) identified four viruses: citrus tristeza virus, citrus leaf blotch virus, citrus vein enation virus, and CYVCV. A CYVCV incidence of 9.3% was observed in six host plants, including calamansi, kumquat, Persian lime, and Eureka lemon. Among the citrus infected by CYVCV, only three samples showed a single infection; the other showed a mixed infection with other viruses. Eureka lemon and Persian lime exhibited yellow vein clearing, leaf distortion, and water-soak symptom underside of the leaves, while the other hosts showed only yellowing symptoms on the leaves. The complete genome sequences were obtained from five CYVCV isolates. Comparison of the isolates reported from the different geographical regions and hosts revealed the high sequence identity (95.2% to 98.8%). Phylogenetic analysis indicated that all the five isolates from Korea were clustered into same clade but were not distinctly apart from isolates from China, Pakistan, India, and Türkiye. To develop an efficient diagnosis system for the four viruses, a simultaneous detection method was constructed using multiplex RT-PCR. Sensitivity evaluation, simplex RT-PCR, and stability testing were conducted to verify the multiplex RT-PCR system developed in this study. This information will be useful for developing effective disease management strategies for citrus growers in Korea.
... After disinfection and surface washing of the plant samples, total RNA was extracted with the Pure Link RNA Mini kit (Invitrogen) according to the manufacturer's instructions. RNA samples were pooled (Roossinck et al. 2015), precipitated with 2 volumes of 100% ethanol and 0.1 volumes of 3M sodium acetate (pH 5.5) and resuspended in RNase-free water. Approximately 500 ng of precipitated RNA were sent to Novogene (Sacramento, USA) for HTS on the Illumina NovaSeq 6000 platform. ...
Tobacco (Nicotiana tabacum L.), an endemic species of South America that is grown worldwide, is a member of the Solanaceae family. Although it is not a common crop in Ecuador, tobacco is important to the nation's economy and a significant source of employment. Viral infections are particularly devastating to tobacco as they can reduce both its yield and quality. In July 2021, symptoms of chlorosis, mosaic and leaf distortion were observed in four commercial fields in the provinces of Guayas and Los Ríos in Ecuador. This study aims to comprehensively characterize a viral isolate obtained from symptomatic plants. Using high throughput sequencing (HTS), phylogenetic analysis of the different open reading frames, and transmission electron microscopy (TEM) of symptomatic plants, we report for the first time the molecular characterization and genome sequence of Cucumber mosaic virus (CMV) isolated from commercial tobacco plants in Ecuador. The ability to detect and identify CMV in tobacco commercial plant fields enables the development of efficient control strategies, thereby mitigating constraints on the production of high-quality tobacco leaves.
... The most severe illnesses that infect large cashew growers are fruit rot, anthracnose foliar blight, and gummosis of twigs and trunks [7]. Powdery mildew can cause crop losses ranging from 70 to 100 per cent [8], and anthracnose disease can harm up to 40 per cent of crops [9,10]. ...
Farming supplies nourishment for all humans, especially in times of rapid population growth. The cashew (Anacardium occidentale L.) tree provides nutrition, income, and health benefits through its agricultural products. However, diseases often affect cashew nuts and fruits, resulting in significant production losses. As a result, it is crucial to predict cashew diseases to ensure food supply for the entire population. This study created a database of healthy and diseased cashew nuts and fruits and divided it into two subsets: Training and Testing (Test). Data augmentation was applied to the Training dataset to enhance the images. The CAS-CNN model was developed, and the MobileNetV2 was modified. The performance of the developed CAS-CNN and CAS-MODMOBNET models was evaluated and compared with existing TL models. The CAS-MODMOBNET reached an average accuracy of 99.8% and an Area under the ROC Curve (AUC) of 1.0 on the Test subset. Furthermore, the presented system was analysed with the size, number of layers, and parameters of existing TL models.
... As expected, the growth of the different isolates varied between isolate and species. The strategy of sequencing purified dsRNA allowed us to identify RNA viruses, including dsRNA and single-stranded (ss) RNA viruses, as dsRNA molecules are replicative forms of viruses with ss RNA genomes [60,61]. This approach does not allow for the identification of DNA viruses. ...
Botryosphaeriaceae are fungi involved in the decay of various woody species, including the grapevine, leading to significant production losses. This fungal family is largely ubiquitous, and seven species of Botryosphaeriaceae have been identified in French vineyards, with variable levels of aggressiveness, both in vitro and in planta. Mycoviruses can impact the life traits of their fungal hosts, including aggressiveness, and are one of the factors influencing fungal pathogenicity. In this study, the RNA mycovirome of fifteen Botryosphaeriaceae isolates was characterized through the high-throughput sequencing of double-stranded RNA preparations from the respective samples. Eight mycoviruses were detected, including three potential novel species in the Narnaviridae family, as well as in the proposed Mycobunyaviridae and Fusagraviridae families. A large collection of Botryosphaeriaceae isolates was screened using RT-PCR assays specific for 20 Botryosphaeriaceae-infecting mycoviruses. Among the mycoviruses detected, some appeared to be specialists within a single host species, while others infected isolates belonging to multiple Botryosphaeriaceae species. This screening allowed us to conclude that one-third of the Botryosphaeriaceae isolates were infected by at least one mycovirus, and a significant proportion of isolates (43.5%) were found to be coinfected by several viruses, with very complex RNA mycoviromes for some N. parvum isolates.
... In particular, metagenomics is a valuable NGS technique for detecting unidentified plant viruses, especially in asymptomatic hosts where traditional methods may not easily detect them. Metagenomics sequences all genetic material in a sample, providing a comprehensive snapshot of the viral community present [14]. ...
Background
Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran.
Results
Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5’-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3’. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain.
Conclusions
Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.
... The process of metagenomic analysis typically involves extracting genetic material from plant samples, sequencing it using next-generation sequencing technologies, and utilizing computational methods to analyze the resulting data [134]. This analysis provides information about the diversity of viral pathogens (virome) present in the sample, their genetic composition, and their potential interactions with host plants and other microbes [135]. Although each method has significant drawbacks, when combined, these findings highlight how little we actually know about plant viruses and highlight the need for more thorough research. ...
Recent advancements in molecular biology have revolutionized plant disease diagnosis and management. This review focuses on disease diagnosis through serological techniques, isothermal amplification methods, CRISPR-based approaches, and management strategies using RNA-based methods. Exploring high-throughput sequencing and RNA interference (RNAi) technologies like host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), this review delves into their potential. Despite the precision offered by RNAi in pest and pathogen management, challenges such as off-target effects and efficient dsRNA delivery persist. This review discusses the significance of these strategies in preventing aphid-mediated plant virus transmission, emphasizing the crucial role of meticulous dsRNA design for effective viral RNA targeting while minimizing harm to plant RNA. Despite acknowledged challenges, including off-target effects and delivery issues, this review underscores the transformative potential of RNA-based strategies in agriculture. Envisaging reduced pesticide dependency and enhanced productivity, these strategies stand as key players in the future of sustainable agriculture.
... Metagenomics based on HTS can be used to detect known viruses and discover novel plant viruses in nature [57]. Therefore, in this study we utilized a meta-transcriptomic approach to investigate the virome associated with black spots on hibiscus leaves (RsTHr) collected from Risaralda in Colombia. ...
Hibiscus is not native to Colombia but well suited to its arid soil and dry climates. A single hibiscus plant from Risaralda, showing black spots on upper and lower sides of its leaves, was collected for virome analysis using meta-transcriptomic high-throughput sequencing technology. Bioinformatic analysis identified 12.5% of the total reads in the Ribo-Zero cDNA library which mapped to viral genomes. BLAST searches revealed the presence of carlavirus, potexvirus, and of known members of the genera Betacarmovirus, Cilevirus, Nepovirus, and Tobamovirus in the sample; confirmed by RT-PCR with virus-specific primers followed by amplicon sequencing. Furthermore, in silico analysis suggested the possibility of a novel soymovirus, and a new hibiscus strain of citrus leprosis virus C2 in the mixed infection. Both RNA dependent RNA polymerase and coat protein gene sequences of the potex and carla viruses shared less than 72% nucleotide and 80% amino acid identities with any alphaflexi- and betaflexi-virus sequences available in GenBank, identifying three novel carlavirus and one potexvirus species in the Hibiscus rosa-sinensis plant. The detection of physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus are also new reports from Colombia. Overall, the meta-transcriptome analysis identified the complex virome associated with the black spot symptoms on hibiscus leaves and demonstrated the diversity of virus genera tolerated in the mixed infection of a single H. rosa-sinensis plant.
... The use of specific virus detection methods, e.g., serology or PCR, is laborious and time-consuming, especially in a plant harboring a large number of viruses, viroids, and phytoplasmas, and cannot provide a clear overview of the plant's virome [2]. Over the past decade, a new era in virology has begun with the employment of high-throughput sequencing (HTS) [10]. It does not require prior knowledge of the viruses, so the identification of viruses/viroids is achieved only by determining the sequence of small RNAs [11]. ...
A comprehensive study on the whole spectrum of viruses and viroids in five Iranian grapevine cultivars was carried out using sRNA libraries prepared from phloem tissue. A comparison of two approaches to virus detection from sRNAome data indicated a significant difference in the results and performance of the aligners in viral genome reconstruction. The results showed a complex virome in terms of viral composition, abundance, and richness. Thirteen viruses and viroids were identified in five Iranian grapevine cultivars, among which the grapevine red blotch virus and grapevine satellite virus were detected for the first time in Iranian vineyards. Grapevine leafroll-associated virus 1 (GLRaV1) and grapevine fanleaf virus (GFLV) were highly dominant in the virome. However, their frequency and abundance were somewhat different among grapevine cultivars. The results revealed a mixed infection of GLRaV1/grapevine yellow speckle viroid 1 (GYSVd1) and GFLV/GYSVd1 in grapevines that exhibited yellows and vein banding. We also propose a threshold of 14% of complete reconstruction as an appropriate threshold for detection of grapevine viruses that can be used as indicators for reliable grapevine virome profiling or in quarantine stations and certification programs.
... High-throughput sequencing (HTS) coupled with homology-dependent annotation (e.g., BLAST search) forms a classic combination that underpins viral metagenomic studies, enabling the dissection of constituents and structures of the virosphere (Roossinck et al. 2015;Shi et al. 2018;Shi et al. 2016). Homology found among conserved proteins shared by diverse viruses has significantly contributed to discovery of novel viruses, tracing the phylogenetic lineages and evolution, as well as the restructuring of the taxonomy (Koonin et al. 2020). ...
Viruses with split genomes are classified as being either segmented or multipartite based on whether their genomic segments occur within a single virion or across different virions. Despite variations in number and sequence during evolution, the genomic segments of many viruses are conserved within the untranslated regions (UTRs). In this study, we present a methodology that combines RNA sequencing with iterative BLASTn of UTRs (UTR-iBLASTn) (https://github.com/qq371260/Iterative-blast-v.1.0) to identify new viral genomic segments. Some novel multipartite-like viruses related to the phylum Kitrinoviricota were annotated using sequencing data from field plant samples and public databases. We identified potentially plant-infecting jingmen-related viruses (order Amarillovirales) and jivi-related viruses (order Martellivirales) with at least six genomic components. The number of RNA molecules associated with a genome of the novel viruses in the families Closteroviridae, Kitaviridae, and Virgaviridae within the order Martellivirales reached five. Several of these viruses seem to represent new taxa at the subgenus, genus, and family levels. The diversity of novel genomic components and the multiple duplication of proteins or protein domains within single or multiple genomic components emphasize the evolutionary roles of reassortment and recombination (horizontal gene transfer), and genetic deletion. The relatively conserved UTRs at the genome level might explain the relationships between monopartite and multipartite viruses, as well as how subviral agents such as defective RNAs and satellite viruses can coexist with their helper viruses.
... Amongst the five species of Fusarium, the species F. proliferatum was dominant, accounting for 89.2% out of all Fusarium reads. Other species of Fusarium found were F. graminearum (9.74%), F. oxysporum (0.89%), F. fujikuroi (0.1%) and F. poae (0.03%). to balance in a plant's life [25]. Beneficial or mutualistic symbioses of viruses with various host organisms, including bacteria, insects, fungi and plants have been discovered and reported [26]. ...
Global food production is challenged by plant pathogens that cause significant crop losses. Fungi, bacteria, and viruses have long threatened sustainable and profitable agriculture. The danger is even higher in vegetatively propagated horticultural crops, such as garlic. Currently, quarantine, rouging infected plants, and control of natural vectors are used as the main means of disease and pest control in garlic crops. Agricultural biotechnology, meristem-tip culture, and cryotherapy offer solutions for virus eradication and for the multiplication of ‘clean stocks’, but at the same time, impact the symbiotic and beneficial components of the garlic microbiome. Our research involves the first metatranscriptomic analysis of the microbiome of garlic bulb tissue, PCR analyses, and a biological assay of endophytes and pathogens. We have demonstrated that in vitro sanitation methods, such as shoot tip culture or cryotherapy can alter the garlic microbiome. Shoot tip culture proved ineffective in virus elimination, but reduced bacterial load and eliminated fungal infections. Conversely, cryotherapy was efficient in virus eradication but demolished other components of the garlic microbiome. Garlic plants sanitized by cryotherapy exhibited a lower survival rate, and a longer in vitro regeneration period. The question arises whether total eradication of viruses, at the expense of other microflora, is necessary, or if a partial reduction in the pathogenic load would suffice for sanitized garlic production. We explore this question from both scientific and commercial perspectives.
... HTS technologies allow a generic approach to virus identification that does not require any prior knowledge on the targeted pathogens but can deliver a species/strain-specific result (Pecman et al., 2017). The only limitation is the completeness of the reference database against which the obtained sequences are compared and the selection and adaptation of the appropriate protocols for enrichment and sequencing of viromes for complete identification of the species composition in the analysed samples (Hasiów-Jaroszewska et al., 2021;Maree et al., 2018;Massart et al., 2014;Roossinck et al., 2015). ...
... Plant viruses are perilous pathogens causing massive destruction of productivity in horticultural crops. Based on genome composition, plant viruses are grouped into five major categories: double-stranded RNA (dsRNA) viruses, positive sense single-stranded RNA (+ssRNA) viruses, negative sense singlestranded RNA (-ssRNA) viruses, single-stranded DNA (ssDNA) viruses and double-stranded DNA (dsDNA) viruses (Roossinck et al., 2015). Researchers have employed two main strategies to design sgRNAs for engineering virus-resistant plants. ...
Environmental changes and increasing population are major concerns for crop production and food security as a whole. To address this, researchers had focussed on the improvement of cereals and pulses and have made considerable progress till the beginning of this decade. However, cereals and pulses together, without vegetables and fruits, are inadequate to meet the dietary and nutritional demands of human life. Production of good quality vegetables and fruits is highly challenging owing to their perishable nature and short shelf life as well as abiotic and biotic stresses encountered during pre- and post-harvest. Genetic engineering approaches to produce good quality, to increase shelf life and stress-resistance, and to change the time of flowering and fruit ripening by introducing foreign genes to produce genetically modified crops were quite successful. However, several biosafety concerns, such as the risk of transgene-outcrossing, limited their production, marketing, and consumption. Modern genome editing techniques, like the CRISPR/Cas9 system, provide a perfect solution in this scenario, as it can produce transgene-free genetically edited plants. Hence, these genetically edited plants can easily satisfy the biosafety norms for crop production and consumption. This review highlights the potential of the CRISPR/Cas9 system for the successful generation of abiotic and biotic stress resistance and thereby improving the quality, yield, and overall productivity of vegetables and fruits.
... Measuring the toxicity of pesticides to aquatic organisms is an important method to evaluate their safety (Tian et al., 2005) (Roossinck et al., 2015, Roossinck & Condit, 2013. Traditional methods to control plant virus diseases depend on antiviral agents, such as ningnanmycin and ribavirin, and plant immunity inducers, such as amino oligosaccharide, oligosaccharide chain protein, LNT, methiadinil and plant immunityinducing proteins (Ryu et al., 2017;Su et al., 2016;Yang et al., 2017;Dewen et al., 2017). ...
Control of plant virus diseases largely depends on the induced plant defence achieved by the external application of synthetic chemical inducers with the ability to modify defence-signalling pathways. However, most of the molecular mechanisms underlying these chemical inducers remain unknown. Here, we developed a chitosan-coated lentinan-loaded hydrogel and discovered how it protects plants from different virus infections. The hydrogel was synthesized by coating chitosan on the surface of the calcium alginate-lentinan (LNT) hydrogel (SL-gel) to form a CSL-gel. CSL-gels exhibit the capacity to prolong the stable release of lentinan and promote Ca 2+ release. Application of CSL-gels on the root of plants induces broad-spectrum resistance against plant viruses (TMV, TRV, PVX and TuMV). RNA-seq analysis identified that Nicotiana benthamiana calmodulin-like protein gene 3 (NbCML3) is upregulated by the sustained release of Ca 2+ from the CSL-gel, and silencing and overexpression of NbCML alter the susceptibility and resistance of tobacco to TMV. Our findings provide evidence that this novel and synthetic CSL-gel strongly inhibits the infection of plant viruses by the sustainable release of LNT and Ca 2+. This study uncovers a novel mode of action by which CSL-gels trigger NbCML3 expression through the stable and sustained release of Ca 2+ .
... They are divided into six main groups based on the characteristics of their genomes: single-stranded DNA (ssDNA), reverse-transcribing viruses, double--stranded RNA (dsRNA), negative sense single--stranded RNA (ssRNA), and positive sense singlestranded RNA (ssRNA+) viruses. The double-stranded DNA (dsDNA) group does not include plant viruses (Roossinck et al. 2015). RNA genomes are present in the majority of plant viruses. ...
The field of plant pathology has adopted targeted genome editing technology as one of its most crucial and effective genetic tools. Due to its simplicity, effectiveness, versatility, CRISPR together with CRISPR-associated proteins found in an adaptive immune system of prokaryotes have recently attracted the interest of the scientific world. Plant disease resistance must be genetically improved for sustainable agriculture. Plant biology and biotechnology have been transformed by genome editing, which makes it possible to perform precise and targeted genome modifications. Editing offers a fresh approach by genetically enhancing plant disease resistance and quickening resistance through breeding. It is simpler to plan and implement, has a greater success rate, is more adaptable and less expensive than other genome editing methods. Importantly CRISPR/Cas9 has recently surpassed plant science as well as plant disease. After years of research, scientists are currently modifying and rewriting genomes to create crop plants which are immune to particular pests and diseases. The main topics of this review are current developments in plant protection using CRISPR/Cas9 technology in model plants and commodities in response to viral, fungal , and bacterial infections, as well as potential applications and difficulties of numerous promising CRISPR/Cas9-adapted approaches.
... Moreover, interactions between viruses, microbes, and hosts can create complex dynamics (Knief et al., 2012;Van Belleghem et al., 2018;Rodriguez et al., 2019) in which viruses may serve as resident regulators impacting hosts or their endophytic populations to alter net secondary metabolite biosynthesis (Roossinck et al., 2015;Seo and Kweon, 2019). Using whole-genome shotgun genomics (WGS) can reveal hidden viral diversity within plants (Forero-Junco et al., 2021). ...
Introduction:
Viruses generally cause disease, but some viruses may be beneficial as resident regulators of their hosts or host microbiomes. Plant-associated viruses can help plants survive by increasing stress tolerance or regulating endophytic communities. The goal of this study was to characterize endophytic virus communities in banana and plantain (Musa spp.) genotypes, including cultivated and wild species, to assess virome repertoires and detect novel viruses.
Methods:
DNA viral communities were characterized by shotgun sequencing of an enriched endosphere extract from leaves and roots or corm of 7 distinct Musa genotypes (M. balbisiana, Thai Black, M. textilis, M. sikkimensis, Dwarf Cavendish, Williams Hybrid, and FHIA-25 Hybrid).
Results:
Results showed abundant virus-like contigs up to 108,191 bp long with higher relative abundance in leaves than roots. Analyses predicted 733 phage species in 51 families, with little overlap in phage communities among plants. Phage diversity was higher in roots and in diploid wild hosts. Ackermanniviridae and Rhizobium phage were generally the most abundant taxa. A Rhizobium RR1-like phage related to a phage of an endophytic tumor-causing rhizobium was found, bearing a holin gene and a partial Shiga-like toxin gene, raising interest in its potential to regulate endophytic Rhizobiaceae. Klebsiella phages were of interest for possible protection against Fusarium wilt, and other phages were predicted with potential to regulate Erwinia, Pectobacterium, and Ralstonia-associated diseases. Although abundant phage-containing contigs were functionally annotated, revealing 1,038 predicted viral protein domains, gene repertoires showed high divergence from database sequences, suggesting novel phages in these banana cultivars. Plant DNA viruses included 56 species of Badnavirus and 26 additional non-Musa plant viruses with distributions that suggested a mixture of resident and transient plant DNA viruses in these samples.
Discussion:
Together, the disparate viral communities in these plants from a shared environment suggest hosts drive the composition of these virus communities. This study forms a first step in understanding the endophytic virome in this globally important food crop, which is currently threatened by fungal, bacterial, and viral diseases.
... (iii) little to no a priori genetic information about the pathogen causing the plant disease is required (Quince et al., 2017;Sekse et al., 2017;Semenov, 2021;Aragona et al., 2022); and (iv) the technique allows for the recovery of genomes from yet uncultured microorganisms (Duan et al., 2009;Piombo et al., 2021). Especially for detection of viral plant pathogens, metagenomic approaches are useful, as viruses do not have universal genes that are targeted in amplicon sequencing, and as mentioned above, the metagenomic approach doesn't require prior knowledge, enabling detection of plant pathogens that are yet unknown (Adams et al., 2009;Roossinck et al., 2015;Adams and Fox, 2016). Despite these advantages, the regular use of shotgun metagenomics for plant pathogen detection is still not widespread. ...
Plant pathogenic microorganisms cause substantial yield losses in several economically important crops, resulting in economic and social adversity. The spread of such plant pathogens and the emergence of new diseases is facilitated by human practices such as monoculture farming and global trade. Therefore, the early detection and identification of pathogens is of utmost importance to reduce the associated agricultural losses. In this review, techniques that are currently available to detect plant pathogens are discussed, including culture-based, PCR-based, sequencing-based, and immunology-based techniques. Their working principles are explained, followed by an overview of the main advantages and disadvantages, and examples of their use in plant pathogen detection. In addition to the more conventional and commonly used techniques, we also point to some recent evolutions in the field of plant pathogen detection. The potential use of point-of-care devices, including biosensors, have gained in popularity. These devices can provide fast analysis, are easy to use, and most importantly can be used for on-site diagnosis, allowing the farmers to take rapid disease management decisions.
... to use other types of nucleic acid templates, such as total RNA, double-stranded RNA, polyadenylated RNA, or virion-associated nucleic acids purified from virus-like particles, to identify the possible existence of other underlying viral pathogens (Roossinck et al., 2015). The viral genome of the members in the genus Polerovirus in the family Solemoviridae contains a polycistronic, positivesense RNA of 4-6 kb. ...
Lily (Lilium) is an important bulbous perennial herb that is frequently infected by one or more viruses. To investigate the diversity of lily viruses, lilies with virus-like symptoms in Beijing were collected to perform small RNA deep sequencing. Then, the 12 complete and six nearly full-length viral genomes, including six known viruses and two novel viruses were determined. Based on sequence and phylogenetic analyses, two novel viruses were considered to be members of the genera Alphaendornavirus (Endornaviridae) and Polerovirus (Solemoviridae). These two novel viruses were provisionally named lily-associated alphaendornavirus 1 (LaEV-1) and lily-associated polerovirus 1 (LaPV-1). Based on sequence, phylogenetic and recombination analyses, strawberry latent ringspot virus (SLRSV) in the genus Stralarivirus (Secoviridae) was identified for the first time in China, and shown to exhibit the highest nucleotide (nt) diversity among the available full-length SLRSV genome sequences, with the highest identities of 79.5% for RNA1 and 80.9% for RNA2. Interestingly, the protease cofactor region in RNA1 was 752 aa in length, whereas those of the other 27 characterized isolates ranged from 700-719 aa in length. The genome sequences of lily virus A (Potyvirus), lily virus X (Potexvirus), and plantago asiatica mosaic virus (Potexvirus) exhibited varying degrees of sequence diversity at the nucleotide level compared with their corresponding characterized isolates. In addition, plantago asiatica mosaic virus (PlAMV) tended to cluster on a host species-basis. One identified lily mottle virus (Potyvirus) isolate was detected as a recombinant, and which clustered in a different group with four other isolates. Seven identified lily symptomless virus (Carlavirus) isolates, including one recombinant, were clustered into three clades. Our results revealed the genetic diversity of lily-infecting viruses, and sequence insertion, host species and recombination are factors that likely contribute to this diversity. Collectively, our results provide useful information regarding the control of viral disease in lily.
... The advent of high throughput sequencing revolutionized the plant virus disease diagnosis, especially in virus detection and new virus discovery (Radford et al. 2012;Wu et al. 2015). Sequencing of ribo-depleted total RNA, small RNA, double-stranded RNA, or enriched viral DNA allowed the discovery of plant viruses regardless its type of nucleotide genome, and with this method, hundreds of plant viruses had been characterized (Roossinck et al. 2015;Massart et al. 2014). Recently, a badnavirus referred as jujube mosaicassociated virus (JuMaV) was identified from a Chinese jujube tree showing mosaic and malformation symptoms in the leaves (Du et al. 2017). ...
Background
Jujube is an economically important fruit tree and native to China. Viral disease is a new threat to jujube production, and several new viruses have been identified infecting jujube plants. During our field survey, jujube mosaic disease was widely distributed in Beijing, but the associated causal agents are still unknown.
Methods
Small RNA deep sequencing was conducted to identify the candidate viruses associated with jujube mosaic. Further complete genome sequences of the viruses were cloned, and the genomic characterization of each virus was analyzed. The field distribution of these viruses was further explored with PCR/RT-PCR detection of field samples.
Results
Mixed infection of four viruses was identified in a plant sample with the symptom of mosaic and leaf twisting, including the previously reported jujube yellow mottle–associated virus (JYMaV), persimmon ampelovirus (PAmpV), a new badnavirus tentatively named jujube-associated badnavirus (JaBV), and a new secovirus tentatively named jujube-associated secovirus (JaSV). PAmpV-jujube was 14,093 nt in length with seven putative open reading frames (ORFs) and shared highest (79.4%) nucleotide (nt) sequence identity with PAmpV PBs3. Recombination analysis showed that PAmpV-jujube was a recombinant originating from plum bark necrosis stem pitting-associated virus isolates nanjing (KC590347) and bark (EF546442). JaBV was 6449 bp in length with conserved genomic organization typical of badnaviruses. The conserved RT and RNAse H region shared highest 67.6% nt sequence identity with jujube mosaic-associated virus, which was below the 80% nt sequence identity value used as the species demarcation threshold in Badnavirus. The genome of JaSV composed of two RNA molecules of 5878 and 3337 nts in length, excluding the polyA tails. Each genome segment contained one large ORF that shared homology and phylogenetic identity with members of the family Secoviridae. Field survey showed JYMaV and JaBV were widely distributed in jujube trees in Beijing.
Conclusion
Two new viruses were identified from jujube plants, and mixed infections of JYMaV and JaBV were common in jujube in Beijing.
... The monitoring and early detection of plant pathogens from various habitats has been successfully done using metagenomics. Metagenomic studies on gut microbiota of different herbivores and rodents identified many plant viruses (Roossinck et al. 2015). Beneficial soil microbes improve plant health and growth through nutrient and vitamin supply, enhance developmental process and protect host from virulence factors. ...
The interaction between microbes and plants in rhizospheric environment is evident regarding sustainable development in agriculture. Microbes are involved in various metabolic activities in plant systems, which in turn help in plant health improvement. Eventually, plant-microbe interactions are connected with biogeochemical cycles. In this context, metagenomic study helps us to survey the microbial diversity in their natural niches, especially in rhizospheric regions. Noticeably, a diverse group of bacteria, fungi, and archaea are likely to be involved in plant growth promoting (PGP) activities. Variation in microbial communities in the rhizosphere depends on various parameters, such as soil organic matter, plant genotype, plant exudates, crop rotation, soil pH, nutrient cycling, etc. Some abiotic factors and chemical fertilizers have negative impact on crop productivity, influencing sustainable development of environment. Despite having negative impacts from climate change, microbes cope with this altered scenario and try to adjust themselves successfully and consequently promote plant growth by nutrient acquisition and stress tolerance approaches. Therefore, climate change has appeared as a big threat to the agricultural sector in recent past and this might be persistent in near future. However, the conservation of microbial diversity in the rhizospheric regions appears as one of the most promising options for long-term environmental sustainability.
... The use of high-throughput sequencing (HTS) has revolutionized virus discovery and resulted in an unprecedented flow of novel viral sequences being recognized in association with all environments and organisms, including plants [1][2][3]. Despite these tremendous advances, HTS-based virus discovery is still not a fully straightforward process and a number of pitfalls have been identified [4][5][6]. ...
Jiviruses are a group of recently described viruses characterized with a tripartite genome and having affinities with Virgaviridae (RNA1 and 2) and Flaviviridae (RNA3). Using a combination of high-throughput sequencing, datamining and RT-PCR approaches, we demonstrate here that in grapevine samples infected by grapevine-associated jivivirus 1 (GaJV-1) up to 7 additional molecules can be consistently detected with conserved 5′ and 3′ non-coding regions in common with the three previously identified GaJV-1 genomic RNAs. RNA4, RNA5, RNA6, RNA7, RNA8 and RNA10, together with a recombinant RNArec7-8, are all members of a family sharing a previously non recognized conserved protein domain, while RNA9 is part of a distinct family characterized by another conserved motif. Datamining of pecan (Carya illinoinensis) public transcriptomic data allowed the identification of two further jiviviruses and the identification of supplementary genomic RNAs with homologies to those of GaJV-1. Taken together, these results reshape our vision of the divided genome of jiviviruses and raise novel questions about the function(s) of the proteins encoded by jiviviruses supplementary RNAs.
... Plant viruses are among the most common pathogens and cause hazardous diseases in a variety of economically important crops. There are five main groups based on viral genomes characters: sense-single-stranded-RNA (ssRNA+), antisense-single stranded-RNA (asRNA-), single-stranded-DNA (ssDNA), double-stranded-DNA (dsDNA), and doublestranded-RNA (dsRNA) viruses [130]. A rolling-circle amplification system is required to replicate the virus genome through recombination-mediated duplication or by a ds-DNA replicative form [131]. Their genome holds a mutual fragment of 220 bp, which is prearranged in one (A, monopartite) or two (A and B, bipartite) constituents [132]. ...
Crossbreeding, mutation breeding, and traditional transgenic breeding take much time to improve desirable characters/traits. CRISPR/Cas-mediated genome editing (GE) is a game-changing tool that can create variation in desired traits, such as biotic and abiotic resistance, increase quality and yield in less time with easy applications, high efficiency, and low cost in producing the targeted edits for rapid improvement of crop plants. Plant pathogens and the severe environment cause considerable crop losses worldwide. GE approaches have emerged and opened new doors for breeding multiple-resistance crop varieties. Here, we have summarized recent advances in CRISPR/Cas-mediated GE for resistance against biotic and abiotic stresses in a crop molecular breeding program that includes the modification and improvement of genes response to biotic stresses induced by fungus, virus, and bacterial pathogens. We also discussed in depth the application of CRISPR/Cas for abiotic stresses (herbicide, drought, heat, and cold) in plants. In addition, we discussed the limitations and future challenges faced by breeders using GE tools for crop improvement and suggested directions for future improvements in GE for agricultural applications, providing novel ideas to create super cultivars with broad resistance to biotic and abiotic stress.
... A viral metagenomics approach makes it possible to identify both already known as well as highly divergent viral genomes in wild plants. At present, four main classes of nucleic acids including total RNA or DNA, virion-associated nucleic acids (VANA) purified from virus-like particles, double-stranded RNAs (dsRNA), and virus-derived small interfering RNAs (siRNAs) are used for plant viral metagenomic approach [2,3], they have their own advantages and disadvantages such as the total RNA/DNA approach is simplest, but it can obtain a very high proportion of non-virus sequences, viruses in thus samples may not be detected because of their low titers; although the process of the VANA approach is cumbersome, it can simultaneous detect both RNA and DNA viruses which are encapsulated; the dsRNA approach can give a very in-depth analysis of virus-specific sequences, but the main weakness of this approach is not suit for the detection of single negative strand RNA viruses and DNA viruses; the siRNA approach is very sensitive for detecting both known and unknown viruses within single plants, but this approach may miss persistent viruses and may be difficult to accurately assemble novel viruses. We combined and simplified the total RNA/DNA approach and the VANA approach for the virome study from clinical samples and animal tissue or fecal samples in our previous studies. ...
Background
Since viral metagenomic approach was applied to discover plant viruses for the first time in 2006, many plant viruses had been identified from cultivated and non-cultivated plants. These previous researches exposed that the viral communities (virome) of plants have still largely uncharacterized. Here, we investigated the virome in 161 species belonging to 38 plant orders found in a riverside ecosystem.
Results
We identified 245 distinct plant-associated virus genomes (88 DNA and 157 RNA viruses) belonging to 27 known viral families, orders, or unclassified virus groups. Some viral genomes were sufficiently divergent to comprise new species, genera, families, or even orders. Some groups of viruses were detected that currently are only known to infect organisms other than plants. It indicates a wider host range for members of these clades than previously recognized theoretically. We cannot rule out that some viruses could be from plant contaminating organisms, although some methods were taken to get rid of them as much as possible. The same viral species could be found in different plants and co-infections were common.
Conclusions
Our data describe a complex viral community within a single plant ecosystem and expand our understanding of plant-associated viral diversity and their possible host ranges.
Plants are continuously attacked by a wide range of disease-causing microorganisms, which damage crops and threaten agricultural sustainability. To deal with this situation, crop varieties that are resistant to biotic stresses have been created by altering their genomes. Plant genome editing with designed nucleases makes it easier to characterize and quantify cells as well as to understand their structural and functional dynamics. The scientific community is well aware of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated system) as a highly effective tool for genome editing. It is an RNA-guided repetitive DNA spacer sequence that is primarily found in archaea and bacterial cells in the form of adaptive immunity. The physicochemical and functional characterization of these gene-centric CRISPR/Cas genetic scissors, which has revolutionized microarray’s limitations, has benefited the production of stress-tolerant plant varieties. The class 2 CRISPR-Cas system’s Cas9 and Cas12 proteins cut double-stranded DNA, whereas Cas13 targets mRNA and promises a repair mechanism to accurately manipulate the targeted gene. The interdependent basic links of the redesigned heteroduplex structure along with its stochastic omic studies provide a complete understanding and redefining of the gene ontology (GO) for genetic engineering. This chapter will discuss RNA-guided endonuclease-induced technologies and data-driven from the interactomics of plant cells to manage the complex regulatory and metabolic processes in plants to achieve resistance against biotic stress.
World's population is elevating at an alarming rate thus, the rising demands of producing crops with better adaptability to biotic and abiotic stresses, superior nutritional as well as morphological qualities, and generation of high‐yielding varieties have led to encourage the development of new plant breeding technologies. The availability and easy accessibility of genome sequences for a number of crop plants as well as the development of various genome editing technologies such as zinc finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) has opened up possibilities to develop new varieties of crop plants with superior desirable traits. However, these approaches has limitation of being more expensive as well as having complex steps and time‐consuming. The CRISPR/Cas genome editing system has been intensively studied for allowing versatile target‐specific modifications of crop genome that fruitfully aid in the generation of novel varieties. It is an advanced and promising technology with the potential to meet hunger needs and contribute to food production for the ever‐growing human population. This review summarizes the usage of novel CRISPR/Cas genome editing tool for targeted crop improvement in stress resistance, yield, quality and nutritional traits in the desired crop plants.
Medicinal Plants: Microbial Interactions, Molecular Techniques, and Therapeutic Trends is a comprehensive exploration of the fascinating world of medicinal plants, their therapeutic advancements, and the application of molecular techniques to unlock their full potential. This book is structured into three illuminating sections, each shedding light on different facets of this rapidly developing field.
Section 1: Exploring Plant-Microbe Interactions
Covers the relationship between microbes and plants, the historical context and the pivotal role of microbes in shaping the future of medicinal plants. Discover the diverse array of bacteria associated with these plants and grasp their significance in enhancing the medicinal value of plants.
Section 2: Harnessing Molecular Techniques
Covers cutting-edge molecular techniques such as genome editing and modern breeding methods to optimize the genetic traits of medicinal plants. By understanding these techniques, readers will learn how to enhance plant growth, yield and quality.
Section 3: Nanotechnology for Therapeutic Enhancement
Covers nanotechnology and its transformative impact on medicinal plants. The section highlights emerging nano-engineering technology that can revolutionize the therapeutic properties of these plants.
Medicinal Plants: Microbial Interactions, Molecular Techniques, and Therapeutic Trends is a book for Interdisciplinary readers: students, scientists, academics, and industry professionals alike.
Whether you're a student, scientist, academic, or industry professional, this book is your gateway to the evolving world of plant-based medicine.
Plant-infecting RNA viruses from 30 families and floating genera, as well as a great number of uncultured as yet-unclassified plant-associated viruses have been described. Even so, the plant RNA virosphere is still underexplored. RNA extracted from enriched virus particles of 50 L water samples from the Teltow Canal and the Havel River in Berlin, Germany, was sequenced using Illumina next-generation sequencing. Sequences were searched for plant viruses with BLAST and DIAMOND. Phylogenetic analyses were conducted with IQ-TREE 2. Altogether, 647 virus sequences greater than 1 kb were detected and further analyzed. These data revealed the presence of accepted and novel viruses related to Albetovirus, Alphaflexiviridae, Aspiviridae, Bromoviridae, Endornaviridae, Partitiviridae, Potyviridae, Solemoviridae, Tombusviridae and Virgaviridae. The vast majority of the sequences were novel and could not be taxonomically assigned. Several tombus- and endorna-like viruses make use of alternative translation tables that suggest unicellular green algae, ciliates, or diplomonades as their hosts. The identification of 27 albeto-like satellite viruses increases available sequence data five-fold. Sixteen new poty-like viruses align with other poty-like viruses in a link that combines the Astroviridae and Potyviridae families. Further, the identification of viruses with peptidase A6-like and peptidase A21-like capsid proteins suggests horizontal gene transfer in the evolution of these viruses.
Numerous species of plant viruses are naturally transmitted by insect vectors, mainly homopterans like aphids and whiteflies. Depending on the vector specificity and the mode of transmission, different durations of the periods for acquisition, retention, and inoculation are required for a successful transmission. Therefore, the experimental setup to perform controlled transmission experiments under laboratory conditions involves handling the vector organisms and managing the times for the different steps of the process to optimize and standardize the results. This chapter describes some basic procedures that can be applied to vector-mediated transmission experiments with selected viruses using aphids or whiteflies and different host plants.
Crop stress due to various biotic (insect pests and disease pathogens) and/or abiotic stressors (drought, heat, cold, salinity, nutrient toxicity, etc.) declines crop yields and threatens global food security. Abiotic stress factors contribute up to 51–82%, pests up to 10–28%, and plant pathogens up to 16% in potential crop yield losses globally on an annual basis. CRISPR/Cas is a current frontline revolutionary site-specific genome editing technology in the field of agriculture with wider applicability. Traits of simplicity, robustness, inexpensiveness, high accuracy, and preciseness of versatile CRISPR/Cas toolbox have given new dimensions to research in the arena of plant biology for the manipulation of plant genomes. CRISPR/Cas has laid foundation for designing of plants with genetic improvements for desired novel traits of resilience against both the biotic and abiotic stressors to improve adaptability and increase in yield. This chapter focuses on the classes, mechanism of action, and success stories of the abiotic and biotic climate-resilient crop plants designed by CRISPR/Cas genome editing toolsets.
Saffron (Crocus sativus L.) is one of the world's most expensive medicinal and aromatic plants, which is extensively grown in Iran. Viral diseases are important causes of serious losses and quality decline in saffron production worldwide. To investigate the saffron viromes in the major commercial saffron fields in Iran, we used RNA deep sequencing and RT-PCR and found almost complete sequences of three potyviruses, including an unrecorded virus. Assays showed the presence of turnip mosaic virus (TuMV), saffron latent virus (SaLV), and an unrecorded potyvirus, which has been tentatively named saffron yellow mosaic virus (SYMV). There was no evidence of recombination. The viral genome of the SYMV-IR isolate was 9541 nucleotides long (excluding poly(A) tail) and showed conserved potyvirus-specific patterns and motifs. It featured two open reading frames, one of which encoded a polyprotein comprised of 3097 amino acids and the other encoded a PIPO protein of 64 amino acids. Phylogenetic analyses and sequence comparisons supported that SYMV is a distinct potyvirus most closely related to members of the bean yellow mosaic virus group (with 54–63% nucleotide identity across the genome). According to our knowledge, this is the first instance of mixed infections of potyviruses associated with Iranian saffron plants naturally. The results expand our understanding of the diversity of viruses that inflict a severe limitation on the commercial production of saffron in Iran. This has important implications for saffron breeding programs globally.
Control of plant virus diseases largely depends on the induced plant defence achieved by the external application of synthetic chemical inducers with the ability to modify defence‐signalling pathways. However, most of the molecular mechanisms underlying these chemical inducers remain unknown. Here, we developed a chitosan‐coated lentinan‐loaded hydrogel and discovered how it protects plants from different virus infections. The hydrogel was synthesized by coating chitosan on the surface of the calcium alginate‐lentinan (LNT) hydrogel (SL‐gel) to form a CSL‐gel. CSL‐gels exhibit the capacity to prolong the stable release of lentinan and promote Ca ²⁺ release. Application of CSL‐gels on the root of plants induces broad‐spectrum resistance against plant viruses (TMV, TRV, PVX and TuMV). RNA‐seq analysis identified that Nicotiana benthamiana calmodulin‐like protein gene 3 ( NbCML3 ) is upregulated by the sustained release of Ca ²⁺ from the CSL‐gel, and silencing and overexpression of NbCML alter the susceptibility and resistance of tobacco to TMV. Our findings provide evidence that this novel and synthetic CSL‐gel strongly inhibits the infection of plant viruses by the sustainable release of LNT and Ca ²⁺ . This study uncovers a novel mode of action by which CSL‐gels trigger NbCML3 expression through the stable and sustained release of Ca ²⁺ .
The large genetic and structural divergences between plants and fungi may hinder the transmission of viruses between these two kingdoms to some extent. However, recent accumulating evidence from virus phylogenetic analyses and the discovery of naturally occurring virus cross-infection suggest the occurrence of past and current transmissions of viruses between plants and plant-associated fungi. Moreover, artificial virus inoculation experiments showed that diverse plant viruses can multiply in fungi and vice versa. Thus, virus cross-infection between plants and fungi may play an important role in the spread, emergence, and evolution of both plant and fungal viruses and facilitate the interaction between them. In this review, we summarize current knowledge related to cross-kingdom virus infection in plants and fungi and further discuss the relevance of this new virological topic in the context of understanding virus spread and transmission in nature as well as developing control strategies for crop plant diseases.
Expected final online publication date for the Annual Review of Virology, Volume 10 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Plantago asiatica mosaic virus (PlAMV) infects ornamental lilies and causes severe necrotic symptoms. PlAMV has also been isolated from several perennial weeds, including Plantago asiatica. While multiple isolates from cultivated lilies have been identified and shown to share a high degree of sequence identity with one another, there have been few studies of PlAMV isolates from weeds. Furthermore, a comprehensive phylogenetic analysis of PIAMV isolates from both lilies and weeds has not been performed. Here, we performed phylogenetic and population genetic analyses of the full-length genome sequences of 36 PlAMV isolates, including 14 new isolates obtained in this study. These analyses suggested intraspecific diversification of PlAMV isolates into five clades, including a clade consisting of ornamental lily isolates (Lily clade) and four other clades consisting of isolates from weeds or medicinal plants. The specific clade membership identified by the phylogenetic analysis was mainly correlated with the geographical location of the members of each clade. All PlAMV isolates except those in the Lily clade had a high level of genetic diversity. The Lily clade had a higher level of genetic differentiation from the other clades. The neutrality test suggested that genetic drift contributed to shaping PlAMV isolates in the Lily clade. Infectivity tests with infectious clones of the representative PIAMV isolates to four plant species, including lilies and P. asiatica, showed that isolates in different phylogenetic clades had variation in infectivity to the subset of plant species.
The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.
Healthy agroecosystems are dependent on a complex web of factors and inter-species interactions. Flowers are hubs for pathogen transmission, including the horizontal or vertical transmission of plant-viruses and the horizontal transmission of bee-viruses. Pollination by the European honey bee (Apis mellifera) is critical for industrial fruit production, but bees can also vector viruses and other pathogens between individuals. Here, we utilized commercial honey bee pollination services in blueberry (Vaccinium corymbosum) farms for a metagenomics-based bee and plant virus monitoring system. Following RNA sequencing, viruses were identified by mapping reads to a reference sequence database through the bioinformatics portal Virtool. In total, 29 unique plant viral species were found at two blueberry farms in British Columbia (BC). Nine viruses were identified at one site in Ontario (ON), five of which were not identified in BC. Ilarviruses blueberry shock virus (BlShV) and prune dwarf virus (PDV) were the most frequently detected viruses in BC but absent in ON, while nepoviruses tomato ringspot virus and tobacco ringspot virus were common in ON but absent in BC. BlShV coat protein (CP) nucleotide sequences were nearly identical in all samples, while PDV CP sequences were more diverse, suggesting multiple strains of PDV circulating at this site. Ten bee-infecting viruses were identified, with black queen cell virus frequently detected in ON and BC. Area-wide bee-mediated pathogen monitoring can provide new insights into the diversity of viruses present in, and the health of, bee-pollination ecosystems. This approach can be limited by a short sampling season, biased towards pollen-transmitted viruses, and the plant material collected by bees can be very diverse. This can obscure the origin of some viruses, but bee-mediated virus monitoring can be an effective preliminary monitoring approach.
Sequence-based approaches and detection technologies have revolutionized plant disease diagnostics in the last few decades, opening the door to highly selective and specific diagnostic assays. However, diagnostic assay development and validation remains hindered by many factors, which would be ameliorated by research prioritization and coordination. Massive amounts of sequence data, in the forms of evolutionary informative markers and now whole genomes, are available yet not easily accessible or properly organized to guide efficient assay development. For many key pathogen groups, particularly those that are not easily culturable, data is scant. Identification of pathogen-specific sequences in microbial genomes, the ultimate diagnostic markers, requires massive data processing, and is slowed by a lack of quality data curation pipelines that efficiently translate data to problem solving. New technologies have the potential to allow detection at extremely low titer, but many of these have not yet found their way into diagnostic labs, much less the field. Here we review the current status of diagnostics readiness in select groups of pathogens and pathosystems and define research priorities to fill knowledge and resource gaps to support scientists engaged in diagnostics. These priorities include improved coordination of data generation and curation and validation of new technologies, such as LAMP, MiFi®, RPA and CRISPR-Cas, to accelerate the implementation of more specific and sensitive assays. Finally, novel approaches such as detection of volatiles and microbiome analyses, have the potential to compliment pathogen diagnostics focusing on specific pathogens.
Viral diseases cause the significant loss of crop yield and a marked deterioration of its quality annually. The main directions of plant protection against viral infections are: the rehabilitation of seeds by isolating and cultivating the apical meristem in vitro, the generation of transgenic plants resistant to viral infections using genes of specific and nonspecific defense, use of plant protection preparations of a chemical and biological nature against viral vectors, the application of inductors of plant resistance, etc. Thus, there is a need to look for efficient and eco-friendly measures which can prevent viral diseases from spreading. The use of endophytes for these purposes is of great interest. Endophytes are ubiquitous microorganisms, detectable in almost all plant species. Endophytes are currently regarded as the overall pools of microorganisms, including the bacteria living in plant tissues; they considerably influence the formation of plant phenotype and its adaptation to changing environmental conditions. In this review, we discuss the mechanisms of plant interactions with endophytic microorganisms. We propose the development of a microbiome using endophytic strains of microorganisms which are known to have complex biocidal activity against causal agents of infections and harmful insects as well as the ability to display viricidal properties directly and to induce plant protective reactions along with the promotion of growth and productivity is proposed as a promising approach of plant protection against viruses. At the same time, endophytes are considered delivery vectors of genes capable of blocking the reproduction of viruses in plant tissues.
Background:
Maize-infecting viruses are known to inflict significant agronomic yield loss throughout the world annually. Identification of known or novel causal agents of disease prior to outbreak is imperative to preserve food security via future crop protection efforts. Toward this goal, a large-scale metagenomic approach utilizing high throughput sequencing (HTS) was employed to identify novel viruses with the potential to contribute to yield loss of graminaceous species, particularly maize, in North America.
Results:
Here we present four novel viruses discovered by HTS and individually validated by Sanger sequencing. Three of these viruses are RNA viruses belonging to either the Betaflexiviridae or Tombusviridae families. Additionally, a novel DNA virus belonging to the Geminiviridae family was discovered, the first Mastrevirus identified in North American maize.
Conclusions:
Metagenomic studies of crop and crop-related species such as this may be useful for the identification and surveillance of known and novel viral pathogens of crops. Monitoring related species may prove useful in identifying viruses capable of infecting crops due to overlapping insect vectors and viral host-range to protect food security.
Most studies of plant viruses have focused on the acute viruses that cause disease in crop and ornamental plants. These viruses are transmitted horizontally, often by insect vectors, and are occasionally transmitted vertically. Although known for at least four decades, the persistent viruses of plants are very poorly studied. These viruses were previously called “cryptic” because they did not appear to illicit any symptoms in infected plants (Boccardo et al., 1987). Persistent plant viruses are not known to be transmitted horizontally, although phylogenetic evidence suggests some level of transmission (Roossinck, 2010). They are vertically transmitted at nearly 100% levels through both ova and pollen (Valverde and Gutierrez, 2007). They have been identified in metagenomic studies by their similarity to known persistent viruses, and because they lack any movement protein, a feature of all known acute viruses that must move through the plant plasmodesmata to establish a systemic infection. Persistent viruses do not move between plant cells, but rather infect every cell and move by cell division.
Most plant persistent viruses have double-stranded (ds) RNA genomes, and encode only an RNA dependent RNA polymerase (RdRp) and a coat protein. Of the well-characterized persistent plant viruses, those in the Endornaviridae are the exception. These viruses have a single-stranded (ss) RNA genome, based on their RdRp, and encode a large polyprotein that does not have any apparent coat protein, but encodes a number of additional domains that appear to be derived from diverse sources (Roossinck et al., 2011). They are usually found as dsRNA replicative intermediates.
Viruses of fungi have very similar lifestyles to plant persistent viruses, and several virus families are shared between plants and fungi. Phylogenetic evidence indicates that virus transmission has occurred within and between the two kingdoms (Roossinck, 2010; Roossinck et al., 2011).
Fungal viruses are even less well-studied than plant viruses, and the diversity of these viruses remains mostly unknown. A majority of known fungal viruses have dsRNA genomes, some have ssRNA genomes, and a few examples of DNA viruses are known (Yu et al., 2010). Recently a negative sense ssRNA virus was characterized from a fungus (Liu et al., 2014). Similar to plant viruses, most fungal viruses have been studied in the context of pathogenic fungi. The discovery of the hypovirulence phenotype of Cryphonectria hypovirus 1 that suppresses the disease phenotype of the chestnut blight fungus led to a search for other examples that could be exploited to mitigate the effects of plant pathogenic fungi [reviewed in Dawe and Nuss (2013)].
Densoviruses (DVs) infect arthropods and belong to the Parvoviridae family. Here, we report the complete coding sequence of a novel DV isolated from the plant Hordeum marinum (Poaceae) by viral metagenomics, and we confirmed reamplification by PCR. Phylogenetic analyses showed that this novel DV is related
to the genus Iteradensovirus.
Significance
Knowledge of ancient viruses is limited due to their low concentration and poor preservation in ancient specimens. Using a viral particle-associated nucleic acid enrichment approach, we genetically characterized one complete DNA and one partial RNA viral genome from a 700-y-old fecal sample preserved in ice. Using reverse genetics, we reconstituted the DNA virus, which replicated and systemically spread in a model plant species. Under constant freezing conditions, encapsidated viral nucleic acids may therefore be preserved for centuries. Our finding indicates that cryogenically preserved materials can be repositories of ancient viral nucleic acids, which in turn allow molecular genetics to regenerate viruses to study their biology.
Internal necrosis of carrot has been observed in UK carrots for at least 10 years, and has been anecdotally linked to virus infection. In the 2009 growing season some growers had up to 10% of yield with these symptoms. Traditional diagnostic methods are targeted towards specific pathogens. By using a metagenomic approach with high throughput sequencing technology, other, as yet unidentified causes of root necrosis were investigated. Additionally a statistical analysis has shown which viruses are most closely associated with disease symptoms. Carrot samples were collected from a crop exhibiting root necrosis (102 Affected: 99 Unaffected) and tested for the presence of the established carrot viruses: Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV), Carrot red leaf associated viral RNA (CtRLVaRNA) and Parsnip yellow fleck virus (PYFV). The presence of these viruses was not associated with symptomatic carrot roots either as single viruses or in combinations. A sub-sample of carrots of mixed symptom status was subjected to MiSeq sequencing. The results from these tests suggested Carrot yellow leaf virus (CYLV) was associated with symptomatic roots. Additionally a novel Torradovirus, a novel Closterovirus and two novel Betaflexiviradae related plant viruses were detected. A specific diagnostic test was designed for CYLV. Of the 102 affected carrots, 98% were positive for CYLV compared to 22% of the unaffected carrots. From these data we conclude that although we have yet to practically demonstrate a causal link, CYLV appears to be strongly associated with the presence of necrosis of carrots.
The genome of yam virus X (YVX), a new member of the genus Potexvirus from yam (Dioscorea trifida), was completely sequenced. Structural and phylogenetic analysis showed that the closest relative of YVX is nerine virus X. A prevalence study found YVX only in plants maintained in Guadeloupe and showed that it also infects members of the complex D. cayenensis rotundata. This study provides evidence for the existence of two additional potexviruses, one of which infects D. nummularia in Vanuatu and the other, D. bulbifera and D. rotundata in Haiti and D. trifida and D. rotundata in Guadeloupe. This work also shows that existing potexvirus-specific degenerate primers targeting the ORF1-encoded polymerase domain are well suited for the identification of the three potexviruses reported here.
As cuisine becomes globalized, large volumes of fresh produce are traded internationally. The potential exists for pathogens infecting fresh produce to hitchhike to new locations and perhaps to establish there. It is difficult to identify them using traditional methods if pathogens are novel, scarce, and/or unexpected. In an attempt to overcome this limitation, we used high-throughput sequencing technology as a means of detecting all RNA viruses infecting garlic (Allium sativum L.) bulbs imported into Australia from China, the USA, Mexico, Argentina and Spain, and those growing in Australia. Bulbs tested were grown over multiple vegetative generations and all were stably infected with one or more viruses, including two species not previously recorded in Australia. Present in various combinations from 10 garlic bulbs were 41 virus isolates representing potyviruses (Onion yellow dwarf virus, Leek yellow stripe virus), carlaviruses (Shallot latent virus, Garlic common latent virus) and allexiviruses (Garlic virus A, B, C, D, and X), for which 19 complete and 22 partial genome sequences were obtained, including the first complete genome sequences of two isolates of GarVD. The most genetically distinct isolates of GarVA and GarVX described so far were identified from Mexico and Argentina, and possible scenarios explaining this are presented. The complete genome sequence of an isolate of the potexvirus Asparagus virus 3 (AV3) was obtained in Australia from wild garlic (A. vineale L.), a naturalized weed. This is first time AV3 has been identified from wild garlic and the first time it has been identified beyond China and Japan. The need for routine generic diagnosis and appropriate legislation to address the risks to primary production and wild plant communities from pathogens spread through the international trade in fresh produce is discussed.
Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.
Environmental DNA sequencing has revealed the expansive biodiversity of microorganisms and clarified the relationship between host-associated microbial communities and host phenotype. Shotgun metagenomic DNA sequencing is a relatively new and powerful environmental sequencing approach that provides insight into community biodiversity and function. But, the analysis of metagenomic sequences is complicated due to the complex structure of the data. Fortunately, new tools and data resources have been developed to circumvent these complexities and allow researchers to determine which microbes are present in the community and what they might be doing. This review describes the analytical strategies and specific tools that can be applied to metagenomic data and the considerations and caveats associated with their use. Specifically, it documents how metagenomes can be analyzed to quantify community structure and diversity, assemble novel genomes, identify new taxa and genes, and determine which metabolic pathways are encoded in the community. It also discusses several methods that can be used compare metagenomes to identify taxa and functions that differentiate communities.
Microbes are the most abundant biological entities on earth, therefore, studying them is important for understanding their roles in global ecology. The science of metagenomics is a relatively young field of research that has enjoyed significant effort since its inception in 1998. Studies using next-generation sequencing techniques on single genomes and collections of genomes have not only led to novel insights into microbial genomics, but also revealed a close association between environmental niches and genome evolution. Herein, we review studies investigating microbial genomics (largely in the marine ecosystem) at the individual and community levels to summarize our current understanding of microbial ecology in the environment.
In the past decade the concept of plant viruses as strictly disease-causing entities has been challenged. While the most well-studied and obvious interactions between plants and viruses are related to disease, there are several examples of mutualistic relationships between plants and viruses, both indirect and direct. These mutualistic interactions have not been fully explored, and many questions remain unanswered. One problem is the lack of knowledge of plant viruses in nature. Metagenomic surveys have estimated that only a small fraction of virus species are known. Additionally, globalization has led to the increased movement of plant material and virus movement. As viruses move from one area to another, new potential hosts offer the possibility of new interactions, both negative and positive.
Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.
The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales.
Complete genome sequences of two new isolates of narcissus late season yellows virus (NLSYV) from Australia were compared with the other NLSYV genome from China and with two complete genomes of isolates designated narcissus yellow stripe virus (NYSV), one from Australia and the other from China. On the basis of symptoms on natural and experimental host species, and genome sequence identity, the isolates could either be classified as closely related members of three different species or placed together in one taxon. Options for classification of these potyvirus isolates are discussed.
Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with 'potexvirus-like' replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative proteins are distant from plant viruses. DOSV is not readily classified in current lower order virus taxa.
The complete genome sequence of a tobamovirus was determined from a wild plant of yellow tailflower (Anthocercis littorea, family Solanaceae) that exhibited mild mottling and chlorosis on the leaves. The virus induced severe symptoms including systemic necrosis when inoculated to plants of three other solanaceous species. The viral genome was resequenced after passage in Nicotiana benthamiana. The two genomes were 6379 nucleotides in length, and they differed by three nucleotides. Phylogenetic analysis and the deduced architecture of the genome place the virus, provisionally named yellow tailflower mild mottle virus, with other tobamoviruses that infect solanaceous hosts.
The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.
Pepper mild mottle virus (PMMoV) is a plant virus that has been recently proposed as a potential indicator of human fecal
contamination of environmental waters; however, information on its geographical occurrence in surface water is still limited.
We aimed to determine the seasonal and geographic occurrence of PMMoV in drinking water sources all over Japan. Between July
2008 and February 2011, 184 source water samples were collected from 30 drinking water treatment plants (DWTPs); viruses from
1 to 2 liters of each sample were concentrated by using an electronegative membrane, followed by RNA extraction and reverse
transcription. Using quantitative PCR, PMMoV was detected in 140 (76%) samples, with a concentration ranging from 2.03 × 103 to 2.90 × 106 copies/liter. At least one of the samples from 27 DWTPs (n = 4 or 8) was positive for PMMoV; samples from 10 of these DWTPs were always contaminated. There was a significant difference
in the occurrence of PMMoV among geographical regions but not a seasonal difference. PMMoV was frequently detected in samples
that were negative for human enteric virus or Escherichia coli. A phylogenetic analysis based on the partial nucleotide sequences of the PMMoV coat protein gene in 12 water samples from
9 DWTPs indicated that there are genetically diverse PMMoV strains present in drinking water sources in Japan. To our knowledge,
this is the first study to demonstrate the occurrence of PMMoV in environmental waters across wide geographical regions.
The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.
Human activity is causing new encounters between viruses and plants. Anthropogenic interventions include changing land use, decreasing biodiversity, trade, the introduction of new plant and vector species to native landscapes, and changing atmospheric and climatic conditions. The discovery of thousands of new viruses, especially those associated with healthy-appearing native plants, is shifting the paradigm for their role within the ecosystem from foe to friend. The cost of new plant virus incursions can be high and result in the loss of trade and/or production for short or extended periods. We present and justify three recommendations for plant biosecurity to improve communication about plant viruses, assist with the identification of viruses and their impacts, and protect the high economic, social, environmental, and cultural value of our respective nations' unique flora: 1) As part of the burden of proof, countries and jurisdictions should identify what pests already exist in, and which pests pose a risk to, their native flora; 2) Plant virus sequences not associated with a recognized virus infection are designated as "uncultured virus" and tentatively named using the host plant species of greatest known prevalence, the word "virus," a general location identifier, and a serial number; and 3) Invest in basic research to determine the ecology of known and new viruses with existing and potential new plant hosts and vectors and develop host-virus pathogenicity prediction tools. These recommendations have implications for researchers, risk analysts, biosecurity authorities, and policy makers at both a national and an international level.
We determined the complete sequence and organization of the genome of a putative member of the genus Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range.
During a large scale "non a priori" survey in 2010 of South African plant-infecting single stranded DNA viruses, a highly divergent geminivirus genome was isolated from a wild spurge, Euphorbia caput-medusae. In addition to being infectious in E. caput-medusae, the cloned viral genome was also infectious in tomato and Nicotiana benthamiana. The virus, named Euphorbia caput-medusae latent virus (EcmLV) due to the absence of infection symptoms displayed by its natural host, caused severe symptoms in both tomato and N. benthamiana. The genome organization of EcmLV is unique amongst geminiviruses and it likely expresses at least two proteins without any detectable homologues within public sequence databases. Although clearly a geminivirus, EcmLV is so divergent that we propose its placement within a new genus that we have tentatively named Capulavirus. Using a set of highly divergent geminiviruses genomes, it is apparent that recombination has likely been a primary process in the genus-level diversification of geminiviruses. It is also demonstrated how this insight, taken together with phylogenetic analyses of predicted coat protein and replication associated protein (Rep) amino acid sequences indicate that the most recent common ancestor of the geminiviruses was likely a dicot-infecting virus that, like modern day mastreviruses and becurtoviruses, expressed its Rep from a spliced complementary strand transcript.
A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.
To identify the causal agent of citrus vein enation disease, we examined by deep sequencing (Solexa-Illumina) the small RNA (sRNA) fraction from infected and healthy Etrog citron plants. Our results showed that virus-derived sRNAs (vsRNAs): (i) represent about 14.21% of the total sRNA population, (ii) are predominantly of 21- and 24-nt with a biased distribution of their 5' nucleotide and with a clear prevalence of those of (+) polarity, and (iii) derive from all the viral genome, although a prominent hotspot is present at a 5'-proximal region. Contigs assembled from vsRNAs showed similarity with luteovirus sequences, particularly with Pea enation mosaic virus, the type member of the genus Enamovirus. The genomic RNA (gRNA) sequence of a new virus, provisionally named Citrus vein enation virus (CVEV), was completed and characterized. The CVEV gRNA was found to be single-stranded, positive-sense, with a size of 5,983 nts and five open reading frames. Phylogenetic comparisons based on amino acid signatures of the RNA polymerase and the coat protein clearly classifies CVEV within the genus Enamovirus. Dot-blot hybridization and RT-PCR tests were developed to detect CVEV in plants affected by vein enation disease. CVEV detection by these methods has already been adopted for use in the Spanish citrus quarantine, sanitation and certification programs.
Viruses have generally been studied either as disease-causing infectious agents that have a negative impact on the host (most eukaryote-infecting viruses), or as tools for molecular biology (especially bacteria-infecting viruses, or phage). Virus ecology looks at the more complex issues of virus-host-environment interactions. For plant viruses this includes studies of plant virus biodiversity, including viruses sampled directly from plants and from a variety of other environments; how plant viruses impact species invasion; interactions between plants, viruses and insects; the large number of persistent viruses in plants that may have epigenetic effects; and viruses that provide a clear benefit to their plant hosts (mutualists). Plants in a non-agricultural setting interact with many other living entities such as animals, insects, and other plants, as well as their physical environment. Wild plants are almost always colonized by a number of microbes, including fungi, bacteria and viruses. Viruses may impact any of these interactions [1].
Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3' proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5' transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ∼500nt from the 3' terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5' transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5' of PLRV genomic RNA.
The complete nucleotide sequence of a sweet potato virus, first identified two decades ago as virus "C-6", was determined in this study. Sequence similarity and phylogenetic analysis clearly place it as a member of a distinct species within the genus Carlavirus, family Betaflexiviridae. Its genome structure was typical for that of other carlaviruses except that the ORF for the cysteine-rich protein was replaced by an ORF encoding a predicted protein with no similarity to any known protein.
Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput
viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco,
CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families
infecting vertebrates, invertebrates, and plants (BLASTx E score, <10−4), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus)
basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized.
Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily
were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible
vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related
to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity
found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses.
An isolate of a new virus, Caladenia virus A (CalVA), was identified infecting Australian terrestrial orchids. The complete genome of 9,847 nucleotides encodes 11 gene products typical of most members of the family Potyviridae. Sequence comparisons of the polyprotein revealed that CalVA shared highest sequence identity (37.5-39.6 %) with members of the genus Poacevirus. Although a vector for CalVA was not identified, a mite transmission motif was present in the helper component protease, indicating that, like other poaceviruses, mites may transmit it. CalVA is the only proposed member of the genus Poacevirus not isolated from a poaceous host.
The phylogenetic diversity of an oligotrophic marine picoplankton community was examined by analyzing the sequences of cloned ribosomal genes. This strategy does not rely on cultivation of the resident microorganisms. Bulk genomic DNA was isolated from picoplankton collected in the north central Pacific Ocean by tangential flow filtration. The mixed-population DNA was fragmented, size fractionated, and cloned into bacteriophage lambda. Thirty-eight clones containing 16S rRNA genes were identified in a screen of 3.2 x 10(4) recombinant phage, and portions of the rRNA gene were amplified by polymerase chain reaction and sequenced. The resulting sequences were used to establish the identities of the picoplankton by comparison with an established data base of rRNA sequences. Fifteen unique eubacterial sequences were obtained, including four from cyanobacteria and eleven from proteobacteria. A single eucaryote related to dinoflagellates was identified; no archaebacterial sequences were detected. The cyanobacterial sequences are all closely related to sequences from cultivated marine Synechococcus strains and with cyanobacterial sequences obtained from the Atlantic Ocean (Sargasso Sea). Several sequences were related to common marine isolates of the gamma subdivision of proteobacteria. In addition to sequences closely related to those of described bacteria, sequences were obtained from two phylogenetic groups of organisms that are not closely related to any known rRNA sequences from cultivated organisms. Both of these novel phylogenetic clusters are proteobacteria, one group within the alpha subdivision and the other distinct from known proteobacterial subdivisions. The rRNA sequences of the alpha-related group are nearly identical to those of some Sargasso Sea picoplankton, suggesting a global distribution of these organisms.
Many plants harbor persistent cytoplasmic viruses that are not transmitted horizontally and do not move from cell to cell. These viruses have extensive longevity within individual plant cultivars. Based on phylogenetic evidence they appear to undergo rare transmission events between plants and fungi. Very few functions have been attributed to persistent viruses in plants, but their longevity and protection from the plant’s immune system suggest that they provide a selective advantage for their hosts, at least under some conditions. In addition, some persistent plant virus sequences have been found in plant genomes and are expressed as functional genes. Hence, rather than simply molecular hitchhikers, they may be cytoplasmic epigenetic elements that could provide genetic information to their plant hosts.
No abstract is available for this article.
The 9573-nucleotide genome of a potyvirus was sequenced from a Coriandrum sativum plant from India with viral symptoms. On analysis, this virus was shown to have greater than 85 % nucleotide sequence identity to vanilla distortion mosaic virus (VDMV). Analysis of the putative coat protein sequence confirmed that this virus was in fact VDMV, with greater than 91 % amino acid sequence identity. The genome appears to encode a 3083-amino-acid polyprotein potentially cleaved into the 10 mature proteins expected in potyviruses. Phylogenetic analysis confirmed that VDMV is a distinct but ungrouped member of the genus Potyvirus.
Viruses use diverse strategies for their replication, related in part to the genome structure (double-stranded or single-stranded; positive sense or negative sense; RNA or DNA). During each round of replication, mutations are introduced in the viral genome and the mode of replication (stamping machine and geometric replication) may affect the population dynamics of the progeny virus. Our understanding of the relationships among genome strandedness, mode of replication and the population variation is still limited. Here we will review what is known about virus replication by stamping machine or geometric modes, and how that relates to the biology of single stranded versus double stranded RNA genomes. We will present how this may affect the mutation frequency and population dynamics. Finally the potential importance of the population dynamics in acute viruses and persistent viruses will be discussed.
Small interfering RNA deep sequencing (SRDS) was used to detect viruses in 23 sweetpotato plants, collected from various locations in Tanzania. Alignment of small RNA reads using a MAQ program recovered genomes of viruses from five families, namely Geminiviridae (2), Closteroviridae (1), Betaflexiviridae (1), Caulimoviridae (1) and Potyviridae (1). This was in agreement with the variation of symptoms observed on sweetpotato plants in fields and screen house, which included leaf curl, vein yellowing, chlorosis, stunted growth and brown blotches. PCR was also used to confirm the occurrence of viruses associated with leaf curl and symptomless infections. A complete genome (2768 nucleotides) was obtained for a sweepovirus that was 89.9% identical to the strain of Sweet potato leaf curl Sao Paulo virus (SPLCSPV; Begomovirus) reported in South Africa. Sweepoviruses are known to undergo frequent recombinations and evidence for this was found in the SPLCSPV sequence studied. The SRDS-based results indicated occurrence of the poorly studied Sweet potato badnavirus B (SPBV-B) and Sweet potato badnavirus A (collectively known as Sweet potato pakakuy virus; SPPV; Caulimoviridae) in sweetpotato plants in Tanzania. A 5′-end partial sequence (3065 nucleotides), encoding hypothetical, movement and coat proteins, was obtained and found to be 86.3% and 73.1% identical to SPBV-B and SPBV-A, respectively. A survey for the distribution of SPPV and Sweet potato symptomless mastrevirus 1 (SPSMV-1) showed that these viruses were wide spread and co-infecting sweetpotato plants in Tanzania. The importance of East Africa as a hot spot for the diversity and evolution of sweet potato viruses is discussed.
The ability to provide a fast, inexpensive and reliable diagnostic for any given viral infection is a key parameter in efforts to fight and control these ubiquitous pathogens. The recent developments of high-throughput sequencing (also called Next Generation Sequencing - NGS) technologies and bioinformatics have drastically changed the research on viral pathogens. It is now raising a growing interest for virus diagnostics. This review provides a snapshot vision on the current use and impact of high throughput sequencing approaches in plant virus characterization. More specifically, this review highlights the potential of these new technologies and their interplay with current protocols in the future of molecular diagnostic of plant viruses. The current limitations that will need to be addressed for a wider adoption of high-throughput sequencing in plant virus diagnostics are thoroughly discussed.
Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered from various environments. Our results indicate a single event of capsid protein (CP) gene capture from an RNA virus in the history of this virus group. The domestication of the CP gene was followed by an unprecedented recurrent replacement of the Rep genes in CHIVs with distant counterparts from diverse ssDNA viruses. We suggest that parasitic and symbiotic interactions between unicellular eukaryotes were central for the emergence of CHIVs and that such turbulent evolution was primarily dictated by incongruence between the CP and Rep proteins.
Plant biosecurity requires rapid identification of pathogenic organisms. While there are many pathogen-specific diagnostic assays, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitations during assembly and similarity searching of sequence data which extend the time needed to make a diagnostic decision. To minimize the amount of bioinformatic processing time needed, unique pathogen-specific sequences (termed e-probes) were designed to be used in searches of unassembled, non-quality checked, sequence data. E-probes have been designed and tested for several select phytopathogens, including an RNA virus, a DNA virus, bacteria, fungi, and an oomycete, illustrating the ability to detect several diverse plant pathogens. E-probes of 80 or more nucleotides in length provided satisfactory levels of precision (75%). The number of e-probes designed for each pathogen varied with the genome size of the pathogen. To give confidence to diagnostic calls, a statistical method of determining the presence of a given pathogen was developed, in which target e-probe signals (detection signal) are compared to signals generate by a decoy set of e-probes (background signal). The E-probe Diagnostic Nucleic acid Assay (EDNA) process provides the framework for a new sequence-based detection system that eliminates the need for assembly of NGS data.
A new panicovirus was identified from a metagenomic survey of plant viruses in the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, USA. Thin paspalum asymptomatic virus (TPAV) was isolated from Paspalum setaceum var. muehlenbergii. The complete 4195 nt sequence encodes six proteins of 44, 109, 8, 6.8, 26, and 15 kDa, whose genes are similar in size and location to those of panicum mosaic virus (PMV) and cocksfoot mild mosaic virus (CMMV). Infectious transcripts made from cDNAs of the 2005 isolate were used for initial host range analyses. Of the 11 tested grasses, only Paspalum setaceum (var. stramineum), Setaria italica, and Setaria viridis were hosts, producing asymptomatic infections, and Nicotiana benthamiana was not infected. Protein and RNA alignments indicate that TPAV is more closely related to PMV than CMMV. RNA alignments and structure predictions indicate that TPAV and CMMV have conserved eight structural elements previously predicted or analyzed for PMV. The initial TPP specimen also contained RNAs related to alphacryptoviruses. Partial sequences of RNA1 (∼35%) and RNA2 (∼40%) of putative thin paspalum cryptic virus were obtained. These represent the first sequences for a putative grass-infecting alphacryptovirus.
The diagnosis of novel unidentified viral plant diseases can be problematic, as the conventional methods such as real‐time PCR or ELISA may be too specific to a particular species or even strain of a virus, whilst alternatives such as electron microscopy (EM) or sap inoculation of indicator species do not usually give species level diagnosis. Next‐generation sequencing (NGS) offers an alternative solution where sequence is generated in a non‐specific fashion and identification is based on similarity searching against GenBank. The conventional and NGS techniques were applied to a damaging and apparently new disease of maize, which was first identified in Kenya in 2011. ELISA and TEM provided negative results, whilst inoculation of other cereal species identified the presence of an unidentified sap transmissible virus. RNA was purified from material showing symptoms and sequenced using a Roche 454 GS‐FLX+. Database searching of the resulting sequence identified the presence of Maize chlorotic mottle virus and Sugarcane mosaic virus, a combination previously reported to cause maize lethal necrosis disease. Over 90% of both viral genome sequences were obtained, allowing strain characterization and the development of specific real‐time PCR assays which were used to confirm the presence of the virus in material with symptoms from six different fields in two different regions of Kenya. The availability of these assays should aid the assessment of the disease and may be used for routine diagnosis. The work shows that next‐generation sequencing is a valuable investigational technique for rapidly identifying potential disease‐causing agents such as viruses.
The complete genomic RNA sequences of the tymovirus isolates Hu and Col from potato which originally had been considered to be strains of the same virus species, i.e. Andean potato latent virus (APLV), were determined by siRNA sequencing and assembly, and found to share only c. 65% nt sequence identity. This result together with those of serological tests and comparisons of the coat protein gene sequences of additional tymovirus isolates from potato suggest that the species Andean potato latent virus should be subdivided into two species, i.e. APLV and Andean potato mild mosaic virus (APMMV). Primers were designed for the broad specificity detection of both viruses.
The complete genome sequences of RNA1 and RNA2 of the oca strain of the potato virus arracacha virus B were determined using next-generation sequencing. The RNA1 molecule is predicted to encode a 259-kDa polyprotein with homology to proteins of the cheraviruses apple latent spherical virus (ALSV) and cherry rasp leaf virus (CRLV). The RNA2 molecule is predicted to encode a 102-kDa polyprotein which also has homology to the corresponding protein of ALSV and, to a lesser degree, CRLV (30 % for RNA1, 24 % for RNA2). Detailed analysis of the genome sequence confirms that AVB is a distinct member of the genus Cheravirus.
DESPITE the importance of cyanobacteria in global primary
productivity1and of heterotrophic bacteria in the consumption of
organic matter in the sea2, the causes of their mortality,
particularly the cyanobacteria, are poorly understood. It is usually assumed
that mortality is due to protozoan grazing3,4 rather than to
viral infection, probably because abundances of phage and host in nature are
presumed to be low5. Previously, either very few marine
bacteriophages have been found by plaque assays6–9, or
viruses have been simply observed10–12or
counted13,14 by transmission electron microscopy, with the
assumption that 'phage-looking' forms are locally active bacteriophages. Here
we report not only high viral abundance in the ocean but also counts of
bacteria and cyanobacteria in the final irreversible stage of lytic infection.
The latter counts are necessary to evaluate mortality, because the sources,
hosts, viability and ages of observed free viruses are unknown; even finding
viruses attached to cells does not prove successful infection. Up to 7% of the
heterotrophic bacteria and 5% of the cyanobacteria from diverse marine
locations contained mature phage; interpretation via culture data indicates
that up to 70% of the prokaryotes could be infected. These data demonstrate the
existence of a significant new pathway of carbon and nitrogen cycling in marine
food webs and have further implications for gene transfer between marine
organisms.
Four species of Diuris temperate terrestrial orchids from wild and captive populations were tested for the presence of polyadenylated RNA viruses. The genomes of three exotic viruses were determined: two potyviruses, Bean yellow mosaic virus and Ornithogalum mosaic virus, and the polerovirus Turnip yellows virus. The genomes of five indigenous viruses were detected, including four novel species. They were the potyvirus Blue squill virus A, another potyvirus, two proposed capilloviruses, and a partitivirus. Partitivirus infection is of interest as this group of viruses is also associated with endophytic fungi (mycorrhizae) that are necessary for the germination, growth, development of many terrestrial orchids. Sequence divergence data indicate post-european, pre-european, and endemic origins for these viruses via inoculum from introduced and native plants. The implications of the findings of this study for orchid conservation, and particularly reintroduction programs where viruses may be spread inadvertently to wild populations from infected propagation sources, are discussed.
Viral metagenomics is the study of viruses in environmental samples, using next generation sequencing that produces very large data sets. For plant viruses, these studies are still relatively new, but are already indicating that our current knowledge grossly underestimates the diversity of these viruses. Some plant virus studies are using thousands of individual plants so that each sequence can be traced back to its precise host. These studies should allow for deeper ecological and evolutionary analyses. The finding of so many new plant viruses that do not cause any obvious symptoms in wild plant hosts certainly changes our perception of viruses and how they interact with their hosts. The major difficulty in these (as in all) metagenomic studies continues to be the need for better bioinformatics tools to decipher the large data sets. The implications of this new information on plant viruses for international agriculture remain to be addressed. Expected final online publication date for the Annual Review of Genetics Volume 46 is November 02, 2012. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing (YVC) disease of lemon trees, reported in Pakistan, India and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads was used to construct the whole genome of CYVCV, consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ~74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative RT-PCR on YVC symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of YVC disease and represents a new species in the genus Mandarivirus.