• Potato Breeding at Oak Park 2000-2006

      Dowley, L.J.; Griffin, D. (Teagasc, 01/07/2009)
      The potato breeding programme at Oak Park was started in the 1960's and has consisted of a number of distinct phases. In the first phase the focus was on the evaluation of the main domestic and foreign varieties for suitability for the Irish market. This was followed by a breeding programme for the domestic market, with particular emphasis on the production of a blight resistant replacement for Kerr’s Pink. The emphasis then switched to breeding for the export market, with the focus on the UK and Mediterranean markets. Since then the breeding programme has been focused on both the domestic, processing and export markets. The process of breeding, testing and multiplying a new potato variety from the making of the initial cross until the new variety can be commercially grown takes about 15 years (see Appendix 1). This report covers the period 2001-2006 (RMIS NO 4720).
    • Exploration of flowering control in Lolium perenne L.

      Byrne, Stephen; Mur, Luis AJ; Donnisson, Iain; Guiney, Emma (Teagasc, 01/08/2009)
      Flowering or heading in Lolium perenne (perennial ryegrass) is induced by a period of vernalization, followed by long days at higher temperatures. When heading occurs there is a reduction in the feed quality of the forage and therefore extending the period of vegetative growth or eliminating heading during the growing season will improve the potential of perennial ryegrass in agriculture. Conversely, a better control of flowering time and increased heading will lead to higher seed yield for commercial producers. The aim of this project was to investigate the underlying genetic control of flowering time in perennial ryegrass. An F1 population was created by crosspollinating two lines with different heading dates and a genetic linkage map was constructed using Simple Sequence Repeat (SSR) markers. The population and genetic linkage map was then used to identify Quantitative Trait Loci (QTL) associated with heading date, spike length and spikelets per spike. A number of QTL were identified for all traits, some of which had not previously been identified in perennial ryegrass. A Suppression Subtractive Hybridization (SSH) study was also employed to identify genes differentially expressed between an extremely late flowering line and earlier flowering sibling line. Expression analysis of a number of identified genes through floral induction was performed using real time RT-PCR. This revealed a number of transcripts with expression profiles indicative of a role to play in floral induction.
    • Potential for gene-flow from cultivated Irish grasses and cereals

      Mullins, Ewen; Ryan, Eimear; Meade, Conor (Teagasc, 01/08/2009)
      The 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.
    • Assessing populations of the major cereal pathogens for reduced sensitivity to MBC, DMI and Strobilurin fungicides

      O’Sullivan, Eugene (Teagasc, 01/08/2009)
      Studies of eyespot populations in winter wheat crops in the period 2001 to 2003 showed that the R type (Tapesia acuformis) is the dominant strain comprising 77% of isolates. Over 90% of isolates were resistant to MBC fungicides, 53% showed reduced sensitivity to prochloraz and 22% reduced sensitivity to cyprodinil. A study of winter wheat crops in February and March 2003 showed that resistance to strobilurin fungicides was widespread in Mycosphaerella graminicola (Septoria tritici) populations. Resistance was found in all but one of 21 crops sampled, at levels ranging from 9% to 84% with an average of 48%. Subsequent studies of 27 crops over the summer of 2003 showed that strobilunin resistance increased from an average of 31% before the application of the second (T2) spray to an average of 73%, three to four weeks after the application of the final (T3) spray. Strobilurin resistance in M. graminicola remained high in 2004, ranging from 50% to 100% with an average of 83% in populations in winter wheat crops sampled in March. The effect of different fungicide programmes on resistance was studied during summer 2003 at two experimental sites. Levels of strobilurin resistance in M. graminicola populations increased during the summer, in unsprayed plots and plots treated with triazole fungicides as well as in those treated with strobiurin fungicides at both sites. M. graminicola populations in winter wheat crops sampled in 2003 and 2004 were predominantly resistant to MBC-generating fungicides and sensitive to the triazole-based fungicides. Studies of the barley leaf blotch pathogen Rhynchosporium secalis showed that resistance to MBC fungicides occurred in 20% of isolates collected from crops from 2001 to 2003, but all isolates were sensitive to triazole and strobilurin fungicides.
    • Evaluation of rape-seed oil production, extraction and use as fuel in modified diesel engines

      Rice, B.; Sustainable Energy Ireland (Teagasc, 01/09/2009)
      It is now well established that rape-seed oil can provide a sustainable source of renewable fuel for diesel engines. The main problem is a high viscosity and vaporisation temperature, which could lead to pumping, atomisation and combustion difficulties. These problems can be overcome in either of two ways: by further processing of the oil to improve its pumping and combustion properties (usually achieved by esterification and layer separation to produce biodiesel) or by some peripheral modifications to the engine to allow it to cope with the more viscous fuel. Engine conversion kits for this purpose are widely available. The second option has attractions in Ireland, at least in the short-term, for a number of reasons. Plants can be established quickly, and so could make an immediate if small contribution to the achievement of Ireland’s substitution target in the Transport Biofuels Directive (Commission of European Communities, 2003). The small operating scale of coldpressing oil extraction plants could be achieved without undue difficulty, and the capital investment required to launch such a project is relatively low. In the event of a biodiesel plant being established at some stage in the future, the option of sourcing some of the oil requirement from these extraction plants would still be available.
    • Technologies for restricting mould growth on baled silage

      O’Kiely, Padraig; Forristal, Dermot; O’Brien, Martin; McEniry, Joseph; Laffin, Christopher; Fuller, Hubert T.; Egan, Damian; Doohan, Fiona M.; Doyle, Evelyn M.; Clipson, Nicholas J.W.; McNally, Gerard M.; Small, Christopher M.; Nielsen, Kristian F.; Frisvad, Jens C. (Teagasc, 01/12/2007)
      Silage is made on approximately 86% of Irish farms, and 85% of these make some baled silage. Baled silage is particularly important as the primary silage making, storage and feeding system on many beef and smaller sized farms, but is also employed as a secondary system (often associated with facilitating grazing management during mid-summer) on many dairy and larger sized farms (O’Kiely et al., 2002). Previous surveys on farms indicated that the extent of visible fungal growth on baled silage was sometimes quite large, and could be a cause for concern. Whereas some improvements could come from applying existing knowledge and technologies, the circumstances surrounding the making and storage of baled silage suggested that environmental conditions within the bale differed from those in conventional silos, and that further knowledge was required in order to arrive at a secure set of recommendations for baled silage systems. This report deals with the final in a series (O’Kiely et al., 1999; O’Kiely et al., 2002) of three consecutive research projects investigating numerous aspect of the science and technology of baled silage. The success of each depended on extensive, integrated collaboration between the Teagasc research centres at Grange and Oak Park, and with University College Dublin. As the series progressed the multidisciplinary team needed to underpin the programme expanded, and this greatly improved the amount and detail of the research undertaken. The major objective of the project recorded in this report was to develop technologies to improve the “hygienic value” of baled silage.
    • Teagasc Research Report 2008, Crops Research Centre, Oak Park

      Crops Research Centre, Teagasc (Teagasc, 01/12/2008)
      This report details research projects at the Teagasc Crops Research Centre in 2008.
    • A temporal assessment of nematode community structure and diversity in the rhizosphere of cisgenic Phytophthora infestans-resistant potatoes

      Ortiz, Vilma; Phelan, Sinead; Mullins, Ewen; European Union; KBBE.2011.3.5-01 (Biomed Central, 01/12/2016)
      Background Nematodes play a key role in soil processes with alterations in the nematode community structure having the potential to considerably influence ecosystem functioning. As a result fluctuations in nematode diversity and/or community structure can be gauged as a ‘barometer’ of a soil’s functional biodiversity. However, a deficit exists in regards to baseline knowledge and on the impact of specific GM crops on soil nematode populations and in particular in regard to the impact of GM potatoes on the diversity of nematode populations in the rhizosphere. The goal of this project was to begin to address this knowledge gap in regards to a GM potato line, cisgenically engineered for resistance to Phytophthora infestans (responsible organism of the Irish potato famine causing late blight disease). For this, a 3 year (2013, 2014, 2015) field experimental study was completed, containing two conventional genotypes (cvs. Desiree and Sarpo Mira) and a cisgenic genotype (cv. Desiree + Rpi-vnt1). Each potato genotype was treated with different disease management strategies (weekly chemical applications and corresponding no spray control). Hence affording the opportunity to investigate the temporal impact of potato genotype, disease management strategy (and their interaction) on the potato rhizosphere nematode community. Results Nematode structure and diversity were measured through established indices, accounts and taxonomy with factors recording a significant effect limited to the climatic conditions across the three seasons of the study and chemical applications associated with the selected disease management strategy. Based on the metrics studied, the cultivation of the cisgenic potato genotype exerted no significant effect (P > 0.05) on nematode community diversity or structure. The disease management treatments led to a reduction of specific trophic groups (e.g. Predacious c–p = 4), which of interest appeared to be counteracted by a potato genotype with vigorous growth phenotype e.g. cv. Sarpo Mira. The fluctuating climates led to disparate conditions, with enrichment conditions (bacterial feeding c–p = 1) dominating during the wet seasons of 2014 and 2015 versus the dry season of 2013 which induced an environmental stress (functional guild c–p = 2) on nematode communities. Conclusions Overall the functional guild indices in comparison to other indices or absolutes values, delivered the most accurate quantitative measurement with which to determine the occurrence of a specific disturbance relative to the cultivation of the studied cisgenic P. infestans-resistant potatoes.
    • Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells

      Rathore, Deeraj Singh; Doohan, Fiona M.; Mullins, Ewen; Teagasc Walsh Fellowship Programme (Bio-protocol LLC, 05/10/2017)
      Ensifer 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.
    • Laboratory Development of a Passive Proportional Sampler for Overland FlowStudies in Agricultural Fields

      Ryan, T. Declan; Forristal, Dermot (Canadian Society for Bioengineering / Société Canadienne de Génie Agroalimentaire et de Bioingénierie, 06/02/2015)
      Water-quality in many rivers remains poor and needs to be improved. Diffuse pollution continues to cause difficulties. Some instruments are available which can monitor pollution of rivers from land. They allow measurement and sampling of overland flow (OLF), but they do not offer the precision required (proportional sampling and samples 0.1% of OLF). A laboratory unit was constructed to mimic instrument performance in the field. This was used to test three sampler designs. A V-notch weir was used in the first sampler and a Sutro weir in the second and third as this unit possessed a proportional discharge to head ratio, which the Vnotch weir did not have. Other parameters investigated included ground slope, sampler slope, pipe size and port location. The remaining issues of nozzle size (0.7, 1.0 and 2.0 mm), the number of 1.0 mm nozzles and the effect of aspiration were investigated. The arrangement with the Sutro weir and three 1.0 mm nozzles in series gave proportional discharge and the target low sampling rate of 0.1%. This will allow the calculation of sediment and chemical losses for the monitored area and will put the loss in context with other losses in a catchment.
    • Genome sequence of Ensifer adhaerens OV14 provides insights into its ability as a novel vector for the genetic transformation of plant genomes

      Rudder, Steven; Doohan, Fiona; Creevey, Christopher J.; Wendt, Toni; Mullins, Ewen; Science Foundation Ireland; 11/RFP.1/GEN/3420. (Biomed Central, 07/04/2014)
      Additional file 1:Figure S1. Circular representation of the four replicons of E. adhaerens OV14. Figure S2. Synteny plots showing total sequence of Ensifer adhaerens OV14 pOV14 (top bar) vs Agrobacterium tumefaciens C58 pTi (bottom bar), computed using DoubleACT version2 on tBLASTx setting with cut off set at 100. Visualised in Artemis ACT. Additional file 2; BLAST of Ensifer adhaerens OV14 replicons.xlsx. Excel file includes tables of BLAST search of individual Ensifer adhaerens OV14 replicons Additional file 3. BLAST of Agrobacterium tumefaciens C58 replicons.xlsx. Excel file includes tables of BLAST searches of individual Agrobacterium tumefaciens C58
    • Pathways for Nutrient Loss to Water; Slurry and Fertilizer Spreading

      Ryan, T. Declan; Holden, Nicholas M.; Carton, Owen T.; Fitzgerald, D.; Murphy, F.; Environmental Protection Agency (Teagasc, 08/07/2008)
      There are almost 150,000 farms in Ireland and these contribute substantial quantities of N and P to inland and coastal waters. Some of these nutrients are carried from wet soils by overland flow and by leaching from dry soils. Farm practice can reduce the loss from farms by judicious management of nutrients. Improvements are required to diminish export of nutrients without impairing operations on the farm. Literature regarding nutrient loss from agriculture was reviewed in this project and maps were prepared to predict best slurry spreading times around Ireland. Two further maps were prepared to show slurry storage requirement on farms.
    • Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites

      Foito, Alexandre; Hackett, Christine A; Stewart, Derek; Velmurugan, Janaki; Milbourne, Dan; Byrne, Stephen L; Barth, Susanne; Department of Agriculture, Food and the Marine, Ireland; RSF 06–346 (Biomed Central, 10/10/2017)
      Background Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. Results In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. Conclusions These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.
    • A hybrid next generation transcript sequencing-based approach to identify allelic and homeolog-specific single nucleotide polymorphisms in allotetraploid white clover

      Nagy, Istvan; Barth, Susanne; Mehenni-Ciz, Jeanne; Abberton, Michael T; Milbourne, Dan; Department of Agriculture, Food and the Marine, Ireland; RSF 07–566 (Biomed Central, 13/02/2013)
      Background: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a “core set” of ancestrally derived homeolog specific variants in white clover.
    • Effects of over-winter green cover on groundwater nitrate and dissolved organic carbon concentrations beneath tillage land

      Premrov, Alina; Coxon, Catherine E.; Hackett, Richard; Kirwan, Laura; Richards, Karl G. (Elsevier, 13/09/2012)
      Application of over-winter green cover (e.g. cover crops) as a measure for reducing nitrate losses from tillage land has been frequently investigated, especially in the unsaturated zone. Monitoring of groundwater is less common in these studies. Studies on groundwater responses to different land treatments can be challenging because they can be influenced by various conditions, such as recharge, seasonal variations, and aquifer properties, often occurring at different time scales than surface water processes. The aim of this study was to evaluate groundwater nitrate (NO3−single bondN) and dissolved organic carbon (DOC) concentration responses to different over-winter green covers: mustard, natural regeneration and no cover. A field experiment was designed and run for three years on tillage land underlain by a vulnerable sand and gravel aquifer in the south-east of Ireland. Results showed that over-winter green cover growth on tillage land can be an effective measure to reduce groundwater NO3−single bondN concentrations. A significant decrease in groundwater NO3−single bondN concentrations was observed under the mustard cover compared to no cover. All treatments, including no cover, showed a decline in groundwater NO3−single bondN concentrations over time. A significant increase in groundwater DOC was also observed under the mustard cover. Although the overall groundwater DOC concentrations were low, the increased DOC occurrence in groundwater should be accounted for in carbon balances and could potentially enhance groundwater denitrification in cases where aquifer conditions may favour it.
    • Exploiting the inter-strain divergence of Fusarium oxysporum for microbial bioprocessing of lignocellulose to bioethanol

      Ali, Shahin S; Khan, Mojibur; Fagan, Brian; Mullins, Ewen; Doohan, Fiona M (Biomed Central, 15/03/2012)
      Microbial bioprocessing of lignocellulose to bioethanol still poses challenges in terms of substrate catabolism. A targeted evolution-based study was undertaken to determine if inter-strain microbial variability could be exploited for bioprocessing of lignocellulose to bioethanol. The microorganism studied was Fusarium oxysporum because of its capacity to both saccharify and ferment lignocellulose. Strains of F. oxysporum were isolated and assessed for their genetic variability. Using optimised solid-state straw culture conditions, experiments were conducted that compared fungal strains in terms of their growth, enzyme activities (cellulases, xylanase and alcohol dehydrogenase) and yield of bioethanol and the undesirable by-products acetic acid and xylitol. Significant inter-strain divergence was recorded in regards to the capacity of studied F. oxysporum strains to produce alcohol from untreated straw. No correlation was observed between bioethanol synthesis and either the biomass production or microbial enzyme activity. A strong correlation was observed between both acetic acid and xylitol production and bioethanol yield. The level of diversity recorded in the alcohol production capacity among closely-related microorganism means that a targeted screening of populations of selected microbial species could greatly improve bioprocessing yields, in terms of providing both new host strains and candidate genes for the bioethanol industry.
    • Markers associated with heading and aftermath heading in perennial ryegrass full-sib families

      Arojju, Sai K; Barth, Susanne; Milbourne, Dan; Conaghan, Patrick; Velmurugan, Janaki; Hodkinson, Trevor R; Byrne, Stephen L; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; EU Marie-Sklodowska-Curie Fellowship; RSF 14/S/819; H2020-MSCA-IF: 658031 (Biomed Central, 16/07/2016)
      Background Heading and aftermath heading are important traits in perennial ryegrass because they impact forage quality. So far, genome-wide association analyses in this major forage species have only identified a small number of genetic variants associated with heading date that overall explained little of the variation. Some possible reasons include rare alleles with large phenotypic affects, allelic heterogeneity, or insufficient marker density. We established a genome-wide association panel with multiple genotypes from multiple full-sib families. This ensured alleles were present at the frequency needed to have sufficient statistical power to identify associations. We genotyped the panel via partial genome sequencing and performed genome-wide association analyses with multi-year phenotype data collected for heading date, and aftermath heading. Results Genome wide association using a mixed linear model failed to identify any variants significantly associated with heading date or aftermath heading. Our failure to identify associations for these traits is likely due to the extremely low linkage disequilibrium we observed in this population. However, using single marker analysis within each full-sib family we could identify markers and genomic regions associated with heading and aftermath heading. Using the ryegrass genome we identified putative orthologs of key heading genes, some of which were located in regions of marker-trait associations. Conclusion Given the very low levels of LD, genome wide association studies in perennial ryegrass populations are going to require very high SNP densities. Single marker analysis within full-sibs enabled us to identify significant marker-trait associations. One of these markers anchored proximal to a putative ortholog of TFL1, homologues of which have been shown to play a key role in continuous heading of some members of the rose family, Rosaceae.
    • EST-derived SSR markers used as anchor loci for the construction of a consensus linkage map in ryegrass (Lolium spp.)

      Studer, Bruno; Kolliker, Roland; Muylle, Hilde; Asp, Torben; Frei, Ursula; Roldan-Ruiz, Isabel; Barre, Philippe; Tomaszewski, Celine; Meally, Helena; Barth, Susanne; Skot, Leif; Armstead, Ian P; Dolstra, Oene; Lubberstedt, Thomas (Biomed Central, 16/08/2010)
      Background: Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm. Results: A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2. Conclusions: The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.
    • Generating Phenotypic Diversity in a Fungal Biocatalyst to Investigate Alcohol Stress Tolerance Encountered during Microbial Cellulosic Biofuel Production

      Hennessy, Rosanna C.; Doohan, Fiona; Mullins, Ewen; Department of Agriculture, Food and the Marine, Ireland; RSF 07 513 (PLOS, 16/10/2013)
      Consolidated 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.
    • Assessment of water-limited winter wheat yield potential at spatially contrasting sites in Ireland using a simple growth and development model

      Lynch, J. P.; Fealy, Reamonn; Doyle, D.; Black, L.; Spink, John; Department of Agriculture, Food and the Marine, Ireland (Teagasc (Agriculture and Food Development Authority), Ireland, 19/09/2017)
      Although Irish winter wheat yields are among the highest globally, increases in the profitability of this crop are required to maintain its economic viability. However, in order to determine if efforts to further increase Irish wheat yields are likely to be successful, an accurate estimation of the yield potential is required for different regions within Ireland. A winter wheat yield potential model (WWYPM) was developed, which estimates the maximum water-limited yield achievable, within the confines of current genetic resources and technologies, using parameters for winter wheat growth and development observed recently in Ireland and a minor amount of daily meteorological input (maximum and minimum daily temperature, total daily rainfall and total daily incident radiation). The WWYPM is composed of three processes: (i) an estimation of potential green area index, (ii) an estimation of light interception and biomass accumulation and (iii) an estimation of biomass partitioning to grain yield. Model validation indicated that WWYPM estimations of water-limited yield potential (YPw) were significantly related to maximum yields recorded in variety evaluation trials as well as regional average and maximum farm yields, reflecting the model’s sensitivity to alterations in the climatic environment with spatial and seasonal variations. Simulations of YPw for long-term average weather data at 12 sites located at spatially contrasting regions of Ireland indicated that the typical YPw varied between 15.6 and 17.9 t/ha, with a mean of 16.7 t/ha at 15% moisture content. These results indicate that the majority of sites in Ireland have the potential to grow high-yielding crops of winter wheat when the effects of very high rainfall and other stresses such as disease incidence and nutrient deficits are not considered.