Potential for gene-flow from cultivated Irish grasses and cereals

End of project reportThe importance of gene movement from cultivated plants has been highlighted in regard to minimising the movement of seed and/or pollen between GM and non-GM crops (i.e. gene flow). Although ryegrass covers in excess of 90% of Ireland’s agricultural area, very little is known about gene flow from ryegrass populations from an Irish context. The goal of this project was to address this lack of data by measuring the degree of pollen-mediated gene-flow between two Lolium spp. in a field environment. Ryegrass (esp. Lolium perenne) was selected because as the dominant pasture grass it is critical for the livestock industry as well as being a current target for novel improvements. The results from this research indicate that the potential for pollen-mediated gene flow from perennial ryegrass decreases exponentially with increased distance from the pollen source, with hybridisation events recorded out at 192m. In parallel to this research, a separate study was conducted to assess the degree of genetic diversity within feral and wild Lolium spp across Ireland and also within the important crop weed Avena fatua (‘wild oats’); thereby providing an insight into the degree of historic gene flow that has occurred within each species and in regard to the latter, identifying the potential for non-native A. fatua to colonise the Irish agrienvironment

Exploring the utility of Brachypodium distachyon as a model pathosystem for the wheat pathogen Zymoseptoria tritici

peer-reviewedBackground Zymoseptoria tritici, the causative organism of Septoria tritici blotch disease is a prevalent biotic stressor of wheat production, exerting substantial economic constraints on farmers, requiring intensive chemical control to protect yields. A hemibiotrophic pathogen with a long asymptomless phase of up to 11 days post inoculation (dpi) before a rapid switch to necrotrophy; a deficit exists in our understanding of the events occurring within the host during the two phases of infection. Brachypodium distachyon has demonstrated its potential as a model species for the investigation of fungal disease resistance in cereal and grass species. The aim of this study was to assess the physical interaction between Z. tritici (strain IPO323) and B. distachyon and examine its potential as a model pathosystem for Z. tritici. Results Septoria tritici blotch symptoms developed on the wheat cultivar Riband from 12 dpi with pycnidial formation abundant by 20 dpi. Symptoms on B. distachyon ecotype Bd21-1 were visible from 1 dpi: characteristic pale, water soaked lesions which developed into blotch-like lesions by 4 dpi. These lesions then became necrotic with chlorotic regions expanding up to 7 dpi. Sporulation on B. distachyon tissues was not observed and no evidence of fungal penetration could be obtained, indicating that Z. tritici was unable to complete its life cycle within B. distachyon ecotypes. However, observation of host responses to the Z. tritici strain IPO323 in five B. distachyon ecotypes revealed a variation in resistance responses, ranging from immunity to a chlorotic/necrotic phenotype. Conclusions The observed interactions suggest that B. distachyon is an incompatible host for Z. tritici infection, with STB symptom development on B. distachyon comparable to that observed during the early infection stages on the natural host, wheat. However first visible symptoms occurred more rapidly on B. distachyon; from 1 dpi in comparison to 12 dpi in wheat. Consequently, we propose that the interaction between B. distachyon and Z. tritici as observed in this study could serve as a suitable model pathosystem with which to investigate mechanisms underpinning an incompatible host response to Z. tritici.Teagasc Walsh Fellowship Programm

Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

peer-reviewedConsolidated bioprocessing (CBP) of lignocellulosic biomass offers an alternative route to renewable energy. The crop pathogen Fusarium oxysporum is a promising fungal biocatalyst because of its broad host range and innate ability to co-saccharify and ferment lignocellulose to bioethanol. A major challenge for cellulolytic CBP-enabling microbes is alcohol inhibition. This research tested the hypothesis that Agrobacterium tumefaciens - mediated transformation (ATMT) could be exploited as a tool to generate phenotypic diversity in F. oxysporum to investigate alcohol stress tolerance encountered during CBP. A random mutagenesis library of gene disruption transformants (n=1,563) was constructed and screened for alcohol tolerance in order to isolate alcohol sensitive or tolerant phenotypes. Following three rounds of screening, exposure of select transformants to 6% ethanol and 0.75% n-butanol resulted respectively in increased (≥11.74%) and decreased (≤43.01%) growth compared to the wild –type (WT). Principal component analysis (PCA) quantified the level of phenotypic diversity across the population of genetically transformed individuals and isolated candidate strains for analysis. Characterisation of one strain, Tr. 259, ascertained a reduced growth phenotype under alcohol stress relative to WT and indicated the disruption of a coding region homologous to a putative sugar transporter (FOXG_09625). Quantitative PCR (RT-PCR) showed FOXG_09625 was differentially expressed in Tr. 259 compared to WT during alcohol-induced stress (P<0.05). Phylogenetic analysis of putative sugar transporters suggests diverse functional roles in F. oxysporum and other filamentous fungi compared to yeast for which sugar transporters form part of a relatively conserved family. This study has confirmed the potential of ATMT coupled with a phenotypic screening program to select for genetic variation induced in response to alcohol stress. This research represents a first step in the investigation of alcohol tolerance in F. oxysporum and has resulted in the identification of several novel strains, which will be of benefit to future biofuel research.Funding provided through the Irish Department of Agriculture and Food's Research Stimulus Fund (Project Code RSF 07 513

An insight into the impact of arable farming on Irish biodiversity: A scarcity of studies hinders a rigorous assessment

peer-reviewedTo help understand and counteract future agronomic challenges to farmland biodiversity, it is essential to know how present farming practices have affected biodiversity on Irish farms. We present an overview of existing research data and conclusions, describing the impact of crop cultivation on biodiversity on Irish arable farms. An extensive literature review clearly indicates that peer-reviewed publications on research conducted in Ireland on this topic are quite scarce: just 21 papers investigating the effect of conventional crop cultivation on Irish biodiversity have been published within the past 30 years. Principally, these studies have concluded that conventional crop cultivation has had an adverse impact on biodiversity on Irish farms, with 15 of the 21 studies demonstrating negative trends for the taxa investigated. Compared to other EU states, the relative dearth of baseline data and absence of monitoring programmes designed to assess the specific impacts of crop cultivation on Irish biodiversity highlight the need to develop long-term research studies. With many new challenges facing Irish agriculture, a research programme must be initiated to measure current levels of biodiversity on arable land and to assess the main farming ‘pressures’ causing significant biodiversity loss or gains in these systems.This work was funded under the EPA ERTDI Research Programme (Grant 2006-B-MS-46)

Communicating the risks of genetically modified organisms: lessons learnt from an Irish field of cisgenic potatoes

peer-reviewedAs plant scientists we are all too familiar with the generic commentary that is often associated with the development or use of genetically modified organisms (GMOs) in agricultural systems, but through fact-driven communication, constructive engagement can be achieved. The EU-funded ‘AMIGA’ project, one element of which involved assessing the impact of a GM potato (previously engineered for late blight resistance using cisgenics) with field trials in Ireland, provides a valuable case study in how this can come about. The experiences detailed highlight important lessons learnt relating to the presentation of scientific evidence in a non-scientific format and the necessity for greater integration of biological and social sciences to support the participation of biological researchers in public engagement exercises.European Commission in the Framework programme

Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells

peer-reviewedEnsifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of crop species. In this protocol, we adapted a previously published account that describes the use of Arabidopsis thaliana roots to investigate the interaction of A. thaliana and Agrobacterium tumefaciens. In our laboratory, we routinely use A. thaliana root explants to examine the factors that enhance the utility of EMT. In addition, the E-ART protocol can be used to study the transcriptional response of E. adhaerens and host plant following exposure to explant tissue, the transformability of different Ensifer adhaerens strains/mutants as well as testing the susceptibility of A. thaliana mutant lines as a means to decipher the mechanisms underpinning EMT

Characterization of Potato Virus Y Isolates and Assessment of Nanopore Sequencing to Detect and Genotype Potato Viruses

peer-reviewPotato virus Y (PVY) is the most economically important virus infecting cultivated potato (Solanum tuberosum L.). Accurate diagnosis is crucial to regulate the trade of tubers and for the sanitary selection of plant material for propagation. However, high genetic diversity of PVY represents a challenge for the detection and classification of isolates. Here, the diversity of Irish PVY isolates from a germplasm collection and commercial sites was investigated using conventional molecular and serological techniques. Recombinant PVY isolates were prevalent, with PVYNTNa being the predominant genotype. In addition, we evaluated Nanopore sequencing to detect and reconstruct the whole genome sequence of four viruses (PVY, PVX, PVS, PLRV) and five PVY genotypes in a subset of eight potato plants. De novo assembly of Nanopore sequencing reads produced single contigs covering greater than 90% of the viral genome and sharing greater than 99.5% identity to the consensus sequences obtained with Illumina sequencing. Interestingly, single near full genome contigs were obtained for different isolates of PVY co-infecting the same plant. Mapping reads to available reference viral genomes enabled us to generate near complete genome sequences sharing greater than 99.90% identity to the Illumina-derived consensus. This is the first report describing the use of Oxford Nanopore’s MinION to detect and genotype potato viruses. We reconstructed the genome of PVY and other RNA viruses; indicating the technologies potential for virus detection in potato production systems, and for the study of genetic diversity of highly heterogeneous viruses such as PVY

Assessing the Economical and Environmental Impact of Cultivating Genetically Modified (GM) crops in Ireland

End of Project ReportAt present, there is no GM crop cultivation in Ireland. This could change in the near future however, following the inclusion of several GM maize varieties on the EU Common Seed Catalogue in 2004. Before an Irish GM tillage sector develops, information must be provided to farmers/regulators in regard to the potential economic impact of the technology and the environmental issues associated with GM crops. This project (RMIS 5211) has examined: 1. The economic cost-benefit of cultivating several GM crops (Phytophthora resistant potato, Septoria resistant wheat, Rhynchosporium resistant barley, Fusarium resistant wheat and herbicide tolerant sugar beet) 2. The environmental issue of gene flow by modelling the propensity of seven crop species (wheat, barley, sugar beet, oilseed rape, maize, potato and ryegrass) to spread their genetic material (be it GM/non-GM) through pollen/seed-mediated gene flow. The cost-benefit analysis specifically examined the impact of reduced chemical input and indicated that each GM crop tested would be more cost efficient than their conventional equivalent. Inputting the regimes and subsequent costs for the 2002 and 2003 growing season into the analysis, farmers would have returned a greater cost savings in 2002 for each of the GM crops, with the exception of potato. While a significant increase in gross margin was recorded for all GM crops, the greatest savings (€ha-1) occurred in the case of herbicide tolerant sugar beet in the absence (9.8% saving) or presence (23.2% saving) of a yield effect. Modelling a crop’s propensity to spread its genetic material (‘gene flow’) was achieved through the creation of a composite gene flow index (GFI) model. Taking into account both pollen and seed mediated data, presence/absence of interfertile wild relatives and current farming practises, a GFI value was returned for each crop. Unless the GM event altered the seed/pollen production of the crop, it can be anticipated that the same GFI value will apply to a GM/non-GM variety of the particular crop. Crops that returned the highest GFI values were ryegrass, oilseed rape and sugar beet. Importantly, a high GFI score does not imply the prohibition of GM varieties of that crop. Rather, it highlights those crops that possess a higher propensity for gene flow and thus require greater management precautions in light of coexistence regulations. To facilitate the provision of this and other relevant research information, a website (www.gmoinfo.ie) has been provided to further public understanding of the issues. Structured in a non-scientific format, this resource will be updated on a regular basis in response to public requests for further information and with research findings from the risk assessment programme at Oak park

Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions

peer-reviewedThe importance of geographic location and annual variation on the detection of differences in the rhizomicrobiome caused by the genetic modification of maize (Bt-maize, event MON810) was evaluated at experimental field sites across Europe including Sweden, Denmark, Slovakia and Spain. DNA of the rhizomicrobiome was collected at the maize flowering stage in three consecutive years and analyzed for the abundance and diversity of PCR-amplified structural genes of Bacteria, Archaea and Fungi, and functional genes for bacterial nitrite reductases (nirS, nirK). The nirK genes were always more abundant than nirS. Maize MON810 did not significantly alter the abundance of any microbial genetic marker, except for sporadically detected differences at individual sites and years. In contrast, annual variation between sites was often significant and variable depending on the targeted markers. Distinct, site-specific microbial communities were detected but the sites in Denmark and Sweden were similar to each other. A significant effect of the genetic modification of the plant on the community structure in the rhizosphere was detected among the nirK denitrifiers at the Slovakian site in only one year. However, most nirK sequences with opposite response were from the same or related source organisms suggesting that the transient differences in community structure did not translate to the functional level. Our results show a lack of effect of the genetic modification of maize on the rhizosphere microbiome that would be stable and consistent over multiple years. This demonstrates the importance of considering annual variability in assessing environmental effects of genetically modified crops

Insights into the transcriptomic response of the plant engineering bacterium Ensifer adhaerens OV14 during transformation

peer-reviewedThe ability to engineer plant genomes has been primarily driven by the soil bacterium Agrobacterium tumefaciens but recently the potential of alternative rhizobia such as Rhizobium etli and Ensifer adhaerens OV14, the latter of which supports Ensifer Mediated Transformation (EMT) has been reported. Surprisingly, a knowledge deficit exists in regards to understanding the whole genome processes underway in plant transforming bacteria, irrespective of the species. To begin to address the issue, we undertook a temporal RNAseq-based profiling study of E. adhaerens OV14 in the presence/absence of Arabidopsis thaliana tissues. Following co-cultivation with root tissues, 2333 differentially expressed genes (DEGs) were noted. Meta-analysis of the RNAseq data sets identified a clear shift from plasmid-derived gene expression to chromosomal-based transcription within the early stages of bacterium-plant co-cultivation. During this time, the number of differentially expressed prokaryotic genes increased steadily out to 7 days co-cultivation, a time at which optimum rates of transformation were observed. Gene ontology evaluations indicated a role for both chromosomal and plasmid-based gene families linked specifically with quorum sensing, flagellin production and biofilm formation in the process of EMT. Transcriptional evaluation of vir genes, housed on the pCAMBIA 5105 plasmid in E. adhaerens OV14 confirmed the ability of E. adhaerens OV14 to perceive and activate its transcriptome in response to the presence of 200 µM of acetosyringone. Significantly, this is the first study to characterise the whole transcriptomic response of a plant engineering bacterium in the presence of plant tissues and provides a novel insight into prokaryotic genetic processes that support T-DNA transfer