Ewen Mullins

The wheat–Septoria conflict: a new front opening up?

In the utopic absence of abiotic and/or biotic stressors, attaining the predicted increase (up to 70%) in wheat demand by 2050 in response to global population trends is a challenge. This objective becomes daunting, however, when one factors in the continuous constraint on global wheat production posed by Septoria tritici blotch (STB) disease. This is because, despite resistant loci being identified, a deficit of commercially relevant STB-resistant wheat germplasm remains. The issue is further compounded for growers by the emergence and prevalence of fungicide-resistant/insensitive strains of the causative pathogen Zymoseptoria tritici (formerly known as Mycosphaerella graminicola/Septoria tritici). However, biotechnology-based research is providing new opportunities in this struggle. As the exome response of wheat to STB attack begins to be deciphered, genes intrinsic to resistant and susceptible phenotypes will be identified. Combined with the application of genome-editing techniques and a growing appreciation of the complexity of wheats and the dynamism of Z. triticis genome, the generation of resulting STB-resistant wheat varieties will counter the prevalent threat of STB disease in wheat-production systems.

Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

Exploiting the inter-strain divergence of Fusarium oxysporum for microbial bioprocessing of lignocellulose to bioethanol

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.

Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato.

In this study the effect of Agrobacterium tumefaciens genotype of two strains AGL1 and LBA4404 was investigated in regard to the propensity for backbone integration during the transformation of potato for blight tolerance conferred by the resistant to blight (RB) gene carried by the vector pCLDO4541. A PCR based walking approach was employed to identify left and right backbone sequences as well as for selected genes carried on the plasmid backbone. It was found that adjacent to the left border insertion site, the integration of backbone sequence was greater for AGL1 than for LBA4404; however, the opposite was observed with regards to the right border T-DNA junction. Considering both T-DNA borders LBA4404 was found to have a two fold greater integration potential for backbone than the AGL1. The possibility of only backbone integration in T-DNA negative plants was also investigated with the average rate of integration between the two strains calculated at 4.2% with LBA4404 recording a three fold greater occurrence of backbone integration than AGL1. In summary, evidence of Agrobacterium genotype dependency showed that LBA4404 has greater potential to integrate non-T-DNA vector sequence than AGL1 and this should be taken into account when utilising the listed A. tumefaciens genotypes in generating transgenic potato. Additionally, the application of a PCR and primer walking system proved to be reliable and allows for fine detailed studies of backbone sequence integration of transgenic plant.

GM Crop Cultivation in Ireland: Ecological and Economic Considerations

Like many states in the European Union, Ireland has yet to fully commit itself to genetically modified (GM) crop technology. The general position of the Irish Government is ‘positive but precautionary’. However, with the European-wide de-facto moratorium on commercial production of GM crops now ended, many strategically important decisions regarding the commercial deployment of such crops and their co-existence with conventional/organic crops need to be considered. To date, little research on the environmental impact of GM crops has been carried out in Ireland, and the provision of relevant local information lags far behind that available in other countries in the European Union. In this paper, we discuss much of the new ecological and economic data that have emerged since the moratorium on GM crops was introduced in 1998, assess the likely impacts of pest-oriented GM crops should they be introduced to Ireland and examine criteria for post-release monitoring. We also describe the likely commercial demand for these crops and the consequent priorities for ecological research. We argue that the impact of GM technology needs to be assessed in relation to the environmental impact of modern agriculture as a whole. Public unease in relation to this technology may be addressed if adequate resources are made available for independent Irish research on the issue.

Fungal-mediated consolidated bioprocessing: the potential of Fusarium oxysporum for the lignocellulosic ethanol industry

Microbial bioprocessing of lignocellulose to bioethanol still poses challenges in terms of substrate catabolism. The most important challenge is to overcome substrate recalcitrance and to thus reduce the number of steps needed to biorefine lignocellulose. Conventionally, conversion involves chemical pretreatment of lignocellulose, followed by hydrolysis of biomass to monomer sugars that are subsequently fermented into bioethanol. Consolidated bioprocessing (CBP) has been suggested as an efficient and economical method of manufacturing bioethanol from lignocellulose. CBP integrates the hydrolysis and fermentation steps into a single process, thereby significantly reducing the amount of steps in the biorefining process. Filamentous fungi are remarkable organisms that are naturally specialised in deconstructing plant biomass and thus they have tremendous potential as components of CBP. The fungus Fusarium oxysporum has potential for CBP of lignocellulose to bioethanol. Here we discuss the complexity and potential of CBP, the bottlenecks in the process, and the potential influence of fungal genetic diversity, substrate complexity and new technologies on the efficacy of CPB of lignocellulose, with a focus on F. oxysporum.

Engineering for disease resistance: persistent obstacles clouding tangible opportunities

The accelerating pace of gene discovery, coupled with novel plant breeding technologies, provides tangible opportunities with which to engineer disease resistance into agricultural and horticultural crops. This is especially the case for potato, wheat, apple and banana, which are afflicted with fungal and bacterial diseases that impact significantly on each crops economic viability. Yet public scepticism and burdensome regulatory systems remain the two primary obstacles preventing the translation of research discoveries into cultivars of agronomic value. In this perspective review, the potential to address these issues is explained, and specific opportunities arising from recent genomics-based initiatives are highlighted as clear examples of what can be achieved in respect of developing disease resistance in crop species. There is an urgent need to tackle the challenge of agrichemical dependency in current crop production systems, and, while engineering for disease resistance is possible, it is not the sole solution and should not be proclaimed as so. Instead, all systems must be given due consideration, with none dismissed in the absence of science-based support, thereby ensuring that future cropping systems have the necessary advantage over those pathogens that continue to inflict losses year after year.

Identification of Fusarium oxysporum genes associated with lignocellulose bioconversion competency.

Fusarium oxysporum can convert straw to ethanol via consolidated bioprocessing (CBP)—a two-stage process that firstly involves aerobic saccharification and thereafter an oxygen-limiting fermentation phase. The efficacy of CBP is dependent upon the fungal strain used. Using suppression subtractive hybridisation (SSH), a total of 210 transcripts were identified as being overexpressed in a high as compared to low efficacy CBP strain in the aerobic and oxygen-limiting growth stages on a straw/bran mix. These transcripts encode proteins assigned to various categories, including carbohydrate metabolism, energy, protein and sugar transport and detoxification. Real-time RT-PCR analysis of 12 transcripts, including an endoglucanase III (EGIII), a novel ricin toxin A1 chain-like protein (RTA1), and two unknown transcripts (JX308289 and JX308290) validated the SSH findings. Post-transcriptional silencing of EGIII, RTA1 and an unknown transcript JX308289 in F. oxysporum strain 11C significantly reduced the capacity of the fungus to produce ethanol from a straw/bran mix. Thus these and other genes identified in this study are likely factors that determine the efficacy of CBP and such genes can be used as candidates for enhancing microbial ethanol production from straw and other lignocellulosic substrates.

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

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.

Facilitating co-existence by tracking gene dispersal in conventional potato systems with microsatellite markers

Based on international findings, Irish co-existence guidelines for the cultivation of GM potato stipulate that an isolation distance of 20 m is required to minimize the spread of transgenic pollen in accordance with required labeling thresholds. As potato tolerant to Phytophthora infestans is the most applicable GM crop from an Irish context, we tested the efficacy of this isolation distance under Irish environmental conditions using the conventional variety Désirée as a pollen donor and the male-sterile variety British Queen as a pollen receptor. Gene flow was determined by scoring for berry presence on receptor plants and confirmed using a microsatellite marker system designed to assess paternity in F(1) seedlings. 99.1% of seedlings recovered were identified as having Désirée paternity. Whereas 19.9% (140/708) of total berries formed on receptor plants occurred at a distance of 21 m from the pollen source, only 4 of these berries bore viable true potato seed (TPS), from which 23 TPS germinated. TPS-bearing berry formation was negatively correlated with distance from the pollen source, and although overall distribution of berries and seeds was non-random across the plot, no significant correlation was evident with respect to wind direction. Microsatellite markers were also used to confirm that the foraging beetle Meligethes aeneus is a vector for the transmission of potato pollen, but a more detailed statistical analysis of this dataset was limited by inclement weather during the trial. To conclude, we recommend that a two-tiered system be established in regard to establishing isolation distances for the experimental trial and commercial cultivation of GM potato in Ireland, and that responsible crop management be adopted to minimize the establishment of TPS-derived volunteers, which we have noted will emerge through a rotation as a result of pollen-mediated gene flow.