Metagenomic Studies of Viruses in Weeds and Wild Plants: A Powerful Approach to Characterise Variable Virus Communities.

High throughput sequencing (HTS) has revolutionised virus detection and discovery, allowing for the untargeted characterisation of whole viromes. Viral metagenomics studies have demonstrated the ubiquity of virus infection - often in the absence of disease symptoms - and tend to discover many novel viruses, highlighting the small fraction of virus biodiversity described to date. The majority of the studies using high-throughput sequencing to characterise plant viromes have focused on economically important crops, and only a small number of studies have considered weeds and wild plants. Characterising the viromes of wild plants is highly relevant, as these plants can affect disease dynamics in crops, often by acting as viral reservoirs. Moreover, the viruses in unmanaged systems may also have important effects on wild plant populations and communities. Here, we review metagenomic studies on weeds and wild plants to show the benefits and limitations of this approach and identify knowledge gaps. We consider key genomics developments that are likely to benefit the field in the near future. Although only a small number of HTS studies have been performed on weeds and wild plants, these studies have already discovered many novel viruses, demonstrated unexpected trends in virus distributions, and highlighted the potential of metagenomics as an approach.

An ABCC-type transporter endowing glyphosate resistance in plants.

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter (EcABCC8) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species.

Phosphite: a novel P fertilizer for weed management and pathogen control.

The availability of orthophosphate (Pi) is a key determinant of crop productivity because its accessibility to plants is poor due to its conversion to unavailable forms. Weed's competition for this essential macronutrient further reduces its bio-availability. To compensate for the low Pi use efficiency and address the weed hazard, excess Pi fertilizers and herbicides are routinely applied, resulting in increased production costs, soil degradation and eutrophication. These outcomes necessitate the identification of a suitable alternate technology that can address the problems associated with the overuse of Pi-based fertilizers and herbicides in agriculture. The present review focuses on phosphite (Phi) as a novel molecule for its utility as a fertilizer, herbicide, biostimulant and biocide in modern agriculture. The use of Phi-based fertilization will help to reduce the consumption of Pi fertilizers and facilitate weed and pathogen control using the same molecule, thereby providing significant advantages over current orthophosphate-based fertilization.

Impact of Cropping Systems, Soil Inoculum, and Plant Species Identity on Soil Bacterial Community Structure.

Farming practices affect the soil microbial community, which in turn impacts crop growth and crop-weed interactions. This study assessed the modification of soil bacterial community structure by organic or conventional cropping systems, weed species identity [Amaranthus retroflexus L. (redroot pigweed) or Avena fatua L. (wild oat)], and living or sterilized inoculum. Soil from eight paired USDA-certified organic and conventional farms in north-central Montana was used as living or autoclave-sterilized inoculant into steam-pasteurized potting soil, planted with Am. retroflexus or Av. fatua and grown for two consecutive 8-week periods to condition soil nutrients and biota. Subsequently, the V3-V4 regions of the microbial 16S rRNA gene were sequenced by Illumina MiSeq. Treatments clustered significantly, with living or sterilized inoculum being the strongest delineating factor, followed by organic or conventional cropping system, then individual farm. Living inoculum-treated soil had greater species richness and was more diverse than sterile inoculum-treated soil (observed OTUs, Chao, inverse Simpson, Shannon, P < 0.001) and had more discriminant taxa delineating groups (linear discriminant analysis). Living inoculum soil contained more Chloroflexi and Acidobacteria, while the sterile inoculum soil had more Bacteroidetes, Firmicutes, Gemmatimonadetes, and Verrucomicrobia. Organically farmed inoculum-treated soil had greater species richness, more diversity (observed OTUs, Chao, Shannon, P < 0.05), and more discriminant taxa than conventionally farmed inoculum-treated soil. Cyanobacteria were higher in pots growing Am. retroflexus, regardless of inoculum type, for three of the four organic farms. Results highlight the potential of cropping systems and species identity to modify soil bacterial communities, subsequently modifying plant growth and crop-weed competition.

Shared and host-specific microbiome diversity and functioning of grapevine and accompanying weed plants.

Weeds and crop plants select their microbiota from the same pool of soil microorganisms, however, the ecology of weed microbiomes is poorly understood. We analysed the microbiomes associated with roots and rhizospheres of grapevine and four weed species (Lamium amplexicaule L., Veronica arvensis L., Lepidium draba L. and Stellaria media L.) growing in proximity in the same vineyard using 16S rRNA gene sequencing. We also isolated and characterized 500 rhizobacteria and root endophytes from L. draba and grapevine. Microbiome data analysis revealed that all plants hosted significantly different microbiomes in the rhizosphere as well as in root compartment, however, differences were more pronounced in the root compartment. The shared microbiome of grapevine and the four weed species contained 145 OTUs (54.2%) in the rhizosphere, but only nine OTUs (13.2%) in the root compartment. Seven OTUs (12.3%) were shared in all plants and compartments. Approximately 56% of the major OTUs (>1%) showed more than 98% identity to bacteria isolated in this study. Moreover, weed-associated bacteria generally showed a higher species richness in the rhizosphere, whereas the root-associated bacteria were more diverse in the perennial plants grapevine and L. draba. Overall, weed isolates showed more plant growth-promoting characteristics compared with grapevine isolates.

Using next-generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS)-based resistance in barnyard grass, a polyploid grass weed.

BACKGROUND: Next-generation sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance, yet their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data. RESULTS: Three PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide-resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing. CONCLUSION: This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the advantages of NGS compared with other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.

Accidental introductions are an important source of invasive plants in the continental United States.

PREMISE OF THE STUDY: Preventing new plant invasions is critical for reducing large-scale ecological change. Most studies have focused on the deliberate introduction of nonnatives via the ornamental plant trade. However, accidental introduction may be an important source of nonnative, invasive plants. METHODS: Using Web and literature searches, we compiled pathways of introduction to the United States for 1112 nonnative plants identified as invasive in the continental United States. We assessed how the proportion of accidentally and deliberately introduced invasive plants varies over time and space and by growth habit across the lower 48 states. KEY RESULTS: Deliberate introductions of ornamentals are the primary source of invasive plants in the United States, but accidental introductions through seed contaminants are an important secondary source. Invasive forbs and grasses are the most likely to have arrived accidentally through seed contaminants, while almost all nonnative, invasive trees were introduced deliberately. Nonnative plants invading eastern states primarily arrived deliberately as ornamentals, while a high proportion of invasive plants in western states arrived accidentally as seed contaminants. Accidental introductions may be increasing in importance through time. Before 1850, 10 of 89 (11%) of invasive plants arrived accidentally. After 1900, 20 of 65 (31%) arrived accidentally. CONCLUSIONS: Recently enacted screening protocols and weed risk assessments aim to reduce the number of potentially invasive species arriving to the United States via deliberate introduction pathways. Increasing proportions of accidentally introduced invasive plants, particularly associated with contaminated seed imports across the western states, suggest that accidental introduction pathways also need to be considered in future regulatory decisions.

Plant invasions across the Northern Hemisphere: a deep-time perspective.

Few invasion biologists consider the long-term evolutionary context of an invading organism and its invaded ecosystem. Here, I consider patterns of plant invasions across Eastern North America, Europe, and East/Far East Asia, and explore whether biases in exchanges of plants from each region reflect major selection pressures present within each region since the late Miocene, during which temperate Northern Hemisphere floras diverged taxonomically and ecologically. Although there are many exceptions, the European flora appears enriched in species well adapted to frequent, intense disturbances such as cultivation and grazing; the North American composite (Asteraceae) flora appears particularly well adapted to nutrient-rich meadows and forest openings; and the East Asian flora is enriched in shade-tolerant trees, shrubs, and vines of high forest-invasive potential. I argue that such directionality in invasions across different habitat types supports the notion that some species are preadapted to become invasive as a result of differences in historical selection pressures between regions.

Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne.

Antifreeze proteins (AFP) are an evolutionarily diverse class of stress response products best known in certain metazoans that adopt a freeze-avoidance survival strategy. The perennial ryegrass, Lolium perenne (Lp), cannot avoid winter temperatures below the crystallization point and is thought to use its LpAFP in a freeze-tolerant strategy. In order to examine properties of LpAFP in relation to L. perenne's life history, cDNA cloning, recombinant protein characterization, ice-binding activities, gene copy number, and expression responses to low temperature were examined. Transcripts, encoded by only a few gene copies, appeared to increase in abundance after diploid plants were transferred to 4°C for 1-2 days, and in parallel with the ice recrystallization inhibition activities. Circular dichroism spectra of recombinant LpAFP showed three clear folding transition temperatures including one between 10 and 15°C, suggesting to us that folding modifications of the secreted AFP could allow the targeted degradation of the protein in planta when temperatures increase. Although LpAFP showed low thermal hysteresis activity and partitioning into ice, it was similar to AFPs from freeze-avoiding organisms in other respects. Therefore, the type of low temperature resistance strategy adopted by a particular species may not depend on the type of AFP. The independence of AFP sequence and life-history has practical implications for the development of genetically-modified crops with enhanced freeze tolerance.

Low pesticide rates may hasten the evolution of resistance by increasing mutation frequencies.

At very low pesticide rates, a certain low proportion of pests may receive a sublethal dose, are highly stressed by the pesticide and yet survive. Stress is a general enhancer of mutation rates. Thus, the survivors are likely to have more than normal mutations, which might include mutations leading to pesticide resistance, both for multifactorial (polygenic, gene amplification, sequential allelic mutations) and for major gene resistance. Management strategies should consider how to eliminate the subpopulation of pests with the high mutation rates, but the best strategy is probably to avoid too low application rates of pesticides from the outset.

Glyphosate degradation in glyphosate-resistant and -susceptible crops and weeds.

High levels of aminomethylphosphonic acid (AMPA), the main glyphosate metabolite, have been found in glyphosate-treated, glyphosate-resistant (GR) soybean, apparently due to plant glyphosate oxidoreductase (GOX)-like activity. AMPA is mildly phytotoxic, and under some conditions the AMPA accumulating in GR soybean correlates with glyphosate-caused phytotoxicity. A bacterial GOX is used in GR canola, and an altered bacterial glyphosate N-acetyltransferase is planned for a new generation of GR crops. In some weed species, glyphosate degradation could contribute to natural resistance. Neither an isolated plant GOX enzyme nor a gene for it has yet been reported in plants. Gene mutation or amplification of plant genes for GOX-like enzyme activity or horizontal transfer of microbial genes from glyphosate-degrading enzymes could produce GR weeds. Yet, there is no evidence that metabolic degradation plays a significant role in evolved resistance to glyphosate. This is unexpected, considering the extreme selection pressure for evolution of glyphosate resistance in weeds and the difficulty in plants of evolving glyphosate resistance via other mechanisms.

Strigolactone deficiency confers resistance in tomato line SL-ORT1 to the parasitic weeds Phelipanche and Orobanche spp.

The parasitic flowering plants of the genera Orobanche and Phelipanche (broomrape species) are obligatory chlorophyll-lacking root-parasitic weeds that infect dicotyledonous plants and cause heavy economic losses in a wide variety of plant species in warm-temperate and subtropical regions. One of the most effective strategies for broomrape control is crop breeding for broomrape resistance. Previous efforts to find natural broomrape-resistant tomato (Solanum lycopersicon) genotypes were unsuccessful, and no broomrape resistance was found in any wild tomato species. Recently, however, the fast-neutron-mutagenized tomato mutant SL-ORT1 was found to be highly resistant to various Phelipanche and Orobanche spp. Nevertheless, SL-ORT1 plants were parasitized by Phelipanche aegyptiaca if grown in pots together with the susceptible tomato cv. M-82. In the present study, no toxic activity or inhibition of Phelipanche seed germination could be detected in the SL-ORT1 root extracts. SL-ORT1 roots did not induce Phelipanche seed germination in pots but they were parasitized, at the same level as M-82, after application of the synthetic germination stimulant GR24 to the rhizosphere. Whereas liquid chromatography coupled to tandem mass spectrometry analysis of root exudates of M-82 revealed the presence of the strigolactones orobanchol, solanacol, and didehydro-orobanchol isomer, these compounds were not found in the exudates of SL-ORT1. It can be concluded that SL-ORT1 resistance results from its inability to produce and secrete natural germination stimulants to the rhizosphere.