Special Issue "Plant Virus Ecology and Biodiversity".

I thank all the teams of authors, the scientists who reviewed submitted manuscripts and made suggestions that improved the reports, and the editorial staff workers who put this special issue together [...].

Modeling of Mutational Events in the Evolution of Viruses.

Diverse studies of viral evolution have led to the recognition that the evolutionary rates of viral taxa observed are dependent on the time scale being investigated-with short-term studies giving fast substitution rates, and orders of magnitude lower rates for deep calibrations. Although each of these factors may contribute to this time dependent rate phenomenon, a more fundamental cause should be considered. We sought to test computationally whether the basic phenomena of virus evolution (mutation, replication, and selection) can explain the relationships between the evolutionary and phylogenetic distances. We tested, by computational inference, the hypothesis that the phylogenetic distances between the pairs of sequences are functions of the evolutionary path lengths between them. A Basic simulation revealed that the relationship between simulated genetic and mutational distances is non-linear, and can be consistent with different rates of nucleotide substitution at different depths of branches in phylogenetic trees.

Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases.

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

ICTV Virus Taxonomy Profile: Virgaviridae.

The family Virgaviridae is a family of plant viruses with rod-shaped virions, a ssRNA genome with a 3'-terminal tRNA-like structure and a replication protein typical of alpha-like viruses. Differences in the number of genome components, genome organization and the mode of transmission provide the basis for genus demarcation. Tobacco mosaic virus (genus Tobamovirus) was the first virus to be discovered (in 1886); it is present in high concentrations in infected plants, is extremely stable and has been extensively studied. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Virgaviridae, which is available at www.ictv.global/report/virgaviridae.

The U.S. Culture Collection Network Responding to the Requirements of the Nagoya Protocol on Access and Benefit Sharing.

The U.S. Culture Collection Network held a meeting to share information about how culture collections are responding to the requirements of the recently enacted Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (CBD). The meeting included representatives of many culture collections and other biological collections, the U.S. Department of State, U.S. Department of Agriculture, Secretariat of the CBD, interested scientific societies, and collection groups, including Scientific Collections International and the Global Genome Biodiversity Network. The participants learned about the policies of the United States and other countries regarding access to genetic resources, the definition of genetic resources, and the status of historical materials and genetic sequence information. Key topics included what constitutes access and how the CBD Access and Benefit-Sharing Clearing-House can help guide researchers through the process of obtaining Prior Informed Consent on Mutually Agreed Terms. U.S. scientists and their international collaborators are required to follow the regulations of other countries when working with microbes originally isolated outside the United States, and the local regulations required by the Nagoya Protocol vary by the country of origin of the genetic resource. Managers of diverse living collections in the United States described their holdings and their efforts to provide access to genetic resources. This meeting laid the foundation for cooperation in establishing a set of standard operating procedures for U.S. and international culture collections in response to the Nagoya Protocol.

Adaptation and Validation of E-Probe Diagnostic Nucleic Acid Analysis for Detection of Escherichia coli O157:H7 in Metagenomic Data from Complex Food Matrices.

The Centers for Disease Control and Prevention recently emphasized the need for enhanced technologies to use in investigations of outbreaks of foodborne illnesses. To address this need, e-probe diagnostic nucleic acid analysis (EDNA) was adapted and validated as a tool for the rapid, effective identification and characterization of multiple pathogens in a food matrix. In EDNA, unassembled next generation sequencing data sets from food sample metagenomes are queried using pathogen-specific sequences known as electronic probes (e-probes). In this study, the query of mock sequence databases demonstrated the potential of EDNA for the detection of foodborne pathogens. The method was then validated using next generation sequencing data sets created by sequencing the metagenome of alfalfa sprouts inoculated with Escherichia coli O157:H7. Nonspecific hits in the negative control sample indicated the need for additional filtration of the e-probes to enhance specificity. There was no significant difference in the ability of an e-probe to detect the target pathogen based upon the length of the probe set oligonucleotides. The results from the queries of the sample database using E. coli e-probe sets were significantly different from those obtained using random decoy probe sets and exhibited 100% precision. The results support the use of EDNA as a rapid response methodology in foodborne outbreaks and investigations for establishing comprehensive microbial profiles of complex food samples.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

Influences of Plant Species, Season and Location on Leaf Endophytic Bacterial Communities of Non-Cultivated Plants.

Bacteria are known to be associated endophytically with plants. Research on endophytic bacteria has identified their importance in food safety, agricultural production and phytoremediation. However, the diversity of endophytic bacterial communities and the forces that shape their compositions in non-cultivated plants are largely uncharacterized. In this study, we explored the diversity, community structure, and dynamics of endophytic bacteria in different plant species in the Tallgrass Prairie Preserve of northern Oklahoma, USA. High throughput sequencing of amplified segments of bacterial rDNA from 81 samples collected at four sampling times from five plant species at four locations identified 335 distinct OTUs at 97% sequence similarity, representing 16 phyla. Proteobacteria was the dominant phylum in the communities, followed by the phyla Bacteriodetes and Actinobacteria. Bacteria from four classes of Proteobacteria were detected with Alphaproteobacteria as the dominant class. Analysis of molecular variance revealed that host plant species and collecting date had significant influences on the compositions of the leaf endophytic bacterial communities. The proportion of Alphaproteobacteria was much higher in the communities from Asclepias viridis than from other plant species and differed from month to month. The most dominant bacterial groups identified in LDA Effect Size analysis showed host-specific patterns, indicating mutual selection between host plants and endophytic bacteria and that leaf endophytic bacterial compositions were dynamic, varying with the host plant's growing season in three distinct patterns. In summary, next generation sequencing has revealed variations in the taxonomic compositions of leaf endophytic bacterial communities dependent primarily on the nature of the plant host species.

Determinants of taxonomic composition of plant viruses at the Nature Conservancy's Tallgrass Prairie Preserve, Oklahoma.

The role of biotic and abiotic factors in shaping the diversity and composition of communities of plant viruses remain understudied, particularly in natural settings. In this study, we test the effects of host identity, location, and sampling year on the taxonomic composition of plant viruses in six native plant species [Ambrosia psilostachya (Asteraceae), Vernonia baldwinii (Asteraceae), Asclepias viridis (Asclepiadaceae), Ruellia humilis (Acanthaceae), Panicum virgatum (Poaceae) and Sorghastrum nutans (Poaceae)] from the Nature Conservancy's Tallgrass Prairie Preserve in northeastern Oklahoma. We sampled over 400 specimens of the target host plants from twenty sites (plots) in the Tallgrass Prairie Preserve over 4 years and tested them for the presence of plant viruses applying virus-like particle and double-stranded RNA enrichment methods. Many of the viral sequences identified could not be readily assigned to species, either due to their novelty or the shortness of the sequence. We thus grouped our putative viruses into operational viral taxonomic units for further analysis. Partial canonical correspondence analysis revealed that the taxonomic composition of plant viruses in the target species had a significant relationship with host species (P value: 0.001) but no clear relation with sampling site or year. Variation partitioning further showed that host identity explained about 2-5 per cent of the variation in plant virus composition. We could not interpret the significant relationship between virus composition and host plants with respect to host taxonomy or ecology. Only six operational viral taxonomic units had over 5 per cent incidence over a 4-year period, while the remainder exhibited sporadic infection of the target hosts. This study is the first of its kind to document the dynamics of the entire range of viruses in multiple plant species in a natural setting.

Evaluating the impacts of stressors of Pseudomonas syringae pathovar tomato on the effectiveness of multi-locus variable number tandem repeat analysis and multi-locus sequence typing in microbial forensic investigations.

BACKGROUND: Crops in the USA are vulnerable to natural and criminal threats because of their widespread cultivation and lack of surveillance, and because of implementation of growing practices such as monoculture. To prepare for investigation and attribution of such events, forensic assays, including determination of molecular profiles, are being adapted for use with plant pathogens. The use of multi-locus variable number tandem repeat (VNTR) analysis (MLVA) and multi-locus sequence typing (MLST) in investigations involving plant pathogens may be problematic because the long lag periods between pathogen introduction and discovery of associated disease may provide enough time for evolution to occur in the regions of the genome employed in each assay. Thus, more information on the stability of the loci employed in these methods is needed. RESULTS: The MLVA fingerprints and MLST profiles were consistent throughout the experiment, indicating that, using a specific set of primers and conditions, MLVA and MLST typing systems reliably identify P.s. tomato DC3000. This information is essential to forensic investigators in interpreting comparisons between MLVA and MLST typing profiles observed in P.s. tomato isolates. CONCLUSIONS: Our results indicate that MLVA and MLST typing systems, utilizing the specified primers and conditions, could be employed successfully in forensics investigations involving P.s. tomato. Similar experiments should be conducted in the field and with other high-consequence plant pathogens to ensure that the assays are reliable for pathogens infecting plants in their natural environment and for organisms that may display faster rates of mutation.

Metagenomic search strategies for interactions among plants and multiple microbes.

Plants harbor multiple microbes. Metagenomics can facilitate understanding of the significance, for the plant, of the microbes, and of the interactions among them. However, current approaches to metagenomic analysis of plants are computationally time consuming. Efforts to speed the discovery process include improvement of computational speed, condensing the sequencing reads into smaller datasets before BLAST searches, simplifying the target database of BLAST searches, and flipping the roles of metagenomic and reference datasets. The latter is exemplified by the e-probe diagnostic nucleic acid analysis approach originally devised for improving analysis during plant quarantine.

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis.

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.

Enhanced reliability and accuracy for field deployable bioforensic detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using razor ex technology and TaqMan quantitative PCR.

A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.

A multi-target, non-infectious and clonable artificial positive control for routine PCR-based assays.

Positive controls are essential for PCR reliability and are challenging to obtain for rare, exotic and/or emerging pathogens and pose biosafety risks if manufactured using infectious pathogens. Custom synthetic DNA inserts can be designed de novo in tandems of forward and reverse complement priming sequences to be inserted in circular plasmid vectors. To test this concept, artificial positive controls (APCs) for use in PCR were synthesized to contain primer sequences targeting four viruses (Barley yellow dwarf virus, Soilborne wheat mosaic virus, Triticum mosaic virus and Wheat streak mosaic virus) pathogenic to wheat and, as internal control, the plant mitochondrial nad5 gene. Thermodynamics and folding parameters of twenty-four APC inserts were assessed in silico. Two thermodynamically different APCs, designated optimal and sub-optimal, were cloned and tested using end point PCR. The optimal APC had a 100% amplification rate, while only 92% of virus-infected plant tissues, commonly used as reference positive controls, amplified. An array of APC priming sequences from different organisms and/or previously tested primers can be accommodated in a large and flexible number of positive control targets. APCs will streamline and standardize routine PCR, improve reliability and biosafety, and create opportunities for development and commercialization of new synthetic positive control sequences.

Biosecurity implications of new technology and discovery in plant virus research.

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.

E-probe Diagnostic Nucleic acid Analysis (EDNA): a theoretical approach for handling of next generation sequencing data for diagnostics.

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 selected 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 generated by a decoy set of e-probes (background signal). The E-probe Diagnostic Nucleic acid Analysis (EDNA) process provides the framework for a new sequence-based detection system that eliminates the need for assembly of NGS data.

Community terminal restriction fragment length polymorphisms reveal insights into the diversity and dynamics of leaf endophytic bacteria.

BACKGROUND: Plant endophytic bacteria play an important role benefiting plant growth or being pathogenic to plants or organisms that consume those plants. Multiple species of bacteria have been found co-inhabiting plants, both cultivated and wild, with viruses and fungi. For these reasons, a general understanding of plant endophytic microbial communities and their diversity is necessary. A key issue is how the distributions of these bacteria vary with location, with plant species, with individual plants and with plant growing season. RESULTS: Five common plant species were collected monthly for four months in the summer of 2010, with replicates from four different sampling sites in the Tallgrass Prairie Preserve in Osage County, Oklahoma, USA. Metagenomic DNA was extracted from ground, washed plant leaf samples, and fragments of the bacterial 16S rDNA genes were amplified for analysis of terminal restriction fragment length polymorphism (T-RFLP). We performed mono-digestion T-RFLP with restriction endonuclease DdeI, to reveal the structures of leaf endophytic bacterial communities, to identify the differences between plant-associated bacterial communities in different plant species or environments, and to explore factors affecting the bacterial distribution. We tested the impacts of three major factors on the leaf endophytic bacterial communities, including host plant species, sampling dates and sampling locations. CONCLUSIONS: Results indicated that all of the three factors were significantly related (α = 0.05) to the distribution of leaf endophytic bacteria, with host species being the most important, followed by sampling dates and sampling locations.

An analysis of the genomic variability of the phytopathogenic mollicute Spiroplasma kunkelii.

Corn stunt disease has become a factor limiting maize production in some areas of the Americas in recent years. Although resistant maize genotypes have been developed in the past, this resistance has been unstable over time or in some geographical locations. To better understand disease components that could affect the stability of host resistance, we assessed the genome variability of the etiologic agent, Spiroplasma kunkelii. Isolates were obtained from a number of areas, and characterized molecularly by amplification of several regions of the spiroplasma chromosome and sequencing of specific gene fragments. The degree of polymorphism between isolates of different geographic origins was low, and the level of genomic variability was similar within isolates of different countries. Polymorphism among isolates was found in viral insertions and in the sequence of Skarp, a gene that encodes a membrane protein implicated in attachment to insect cells. The results suggest that the genome composition of this species is highly conserved among isolates. Hence, it is unlikely that the instability of maize resistance is due to generation of new pathotypes of S. kunkelii. Instead, other components of this complex pathosystem could account for the breakdown of resistance.

A Support Vector Machine based method to distinguish proteobacterial proteins from eukaryotic plant proteins.

BACKGROUND: Members of the phylum Proteobacteria are most prominent among bacteria causing plant diseases that result in a diminution of the quantity and quality of food produced by agriculture. To ameliorate these losses, there is a need to identify infections in early stages. Recent developments in next generation nucleic acid sequencing and mass spectrometry open the door to screening plants by the sequences of their macromolecules. Such an approach requires the ability to recognize the organismal origin of unknown DNA or peptide fragments. There are many ways to approach this problem but none have emerged as the best protocol. Here we attempt a systematic way to determine organismal origins of peptides by using a machine learning algorithm. The algorithm that we implement is a Support Vector Machine (SVM). RESULT: The amino acid compositions of proteobacterial proteins were found to be different from those of plant proteins. We developed an SVM model based on amino acid and dipeptide compositions to distinguish between a proteobacterial protein and a plant protein. The amino acid composition (AAC) based SVM model had an accuracy of 92.44% with 0.85 Matthews correlation coefficient (MCC) while the dipeptide composition (DC) based SVM model had a maximum accuracy of 94.67% and 0.89 MCC. We also developed SVM models based on a hybrid approach (AAC and DC), which gave a maximum accuracy 94.86% and a 0.90 MCC. The models were tested on unseen or untrained datasets to assess their validity. CONCLUSION: The results indicate that the SVM based on the AAC and DC hybrid approach can be used to distinguish proteobacterial from plant protein sequences.

Population genetic analysis of grapevine fanleaf virus.

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.