Exploiting the growth promotion and induced resistance properties of Trichoderma hamatum for improved crop productivity.

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

With the human population projected to reach 9 billion by the year 2050, unsustainable demands will be placed on global agriculture to meet future food requirements. Improvements in crops yields that have traditionally relied on plant breeding and energy intensive agriculture are unlikely to meet these needs and therefore marginal land, previously considered unsuitable for agriculture, will need to be brought into cultivation. Much of this land will have sub-optimal fertility and nutrient-poor soils requiring substantial inputs of synthetic fertilizers to support sustainable crop production. However, significant increases in the price of fertilizers mean many farmers, particularly in developing countries, cannot afford such management practises. The spiraling financial burden of fertilizers, combined with growing public anxiety of the environmental and health impacts of synthetic chemical additives, means that alternative strategies for sustainable crop production need to be examined urgently. Trichoderma strains produce a diverse array of secondary metabolites and secreted proteins. Trichoderma isolates have been shown to activate broad spectrum immunity, ameliorate a wide range of abiotic stresses such as salinity and drought, improve photosynthetic efficiency, enhance nutrient uptake, and significantly increase nitrogen use efficiency in crops. These important attributes can all contribute to the enhanced plant growth characteristics often evident upon inoculation. The positive agronomic traits of growth promotion, enhanced tolerance to abiotic stress and broad spectrum enhanced systemic immunity afforded by many Trichoderma strains are striking and unique. The ability to exploit the signaling networks activated by Trichoderma to establish these agriculturally beneficial traits requires an understanding of both the bioactive inducing molecules and the signaling networks targeted by these activators. The rapid developments in genomic and analytical technologies means this is an opportune time to exploit the amazing chemical diversity of soil micro-organisms such as Trichoderma, with the objective of improving PGP and ISR on a range of agronomically important plant species. Research conducted at Exeter has identified a novel strain of the naturally occurring rhizosphere fungus Trichoderma hamatum (strain GD12) that improves crop productivity and imparts broad spectrum enhanced immunity to pathogens in the absence of costly fertilizers and environmentally damaging agrochemicals. At present, the mechanism underlying the plant-growth-promotion and enhanced systemic resistance phenomena are unknown and this proposal lays the foundations to address the molecular basis of the inducing bioactives and the plant response pathways targeted by these bioactives. This combined knowledge is important, and necessary to contemplate translating Trichoderma PGP and ISR into the agricultural arena. This multidisciplinary research programme will use a combination of genetics, genomics and metabolomics to unravel Trichoderma PGP/ISR bioactives, capture the plant transcriptional reprogramming induced by the bioactives and identify key components of the PGP, ISR signaling networks. We will use comparative transcriptomics to characterize signaling pathways in Arabidopsis and rice activated by Trichoderma inoculation. In combination with mutant Trichoderma and Arabidopsis lines compromised in phytohormone signaling we will identify candidate signaling components/pathways that contribute to PGP and ISR. We will undertake comparative metabolic profiling using liquid chromatography mass spectrometry to characterize the bioactives and test their efficacy on different plants with the objective of developing novel natural agrochemicals.

Technical Summary

Trichoderma strains produce a diverse array of secondary metabolites and secreted proteins meaning there is enormous agrochemical potential to exploit these key agronomic traits. Work at Exeter has disrupted hexosaminidase activity in T. hamatum, an enzyme involved in chitin de-polymerisation and release of sequestered nitrogen for plant growth, and an enzyme thought to be key to plant growth promotion (PGP) activity of a strain of the fungus (strain GD12). Contrary to our expectations, disruption of enzyme production dramatically enhanced PGP by the fungus leading to a further significant increase in plant growth. Strikingly, this mutant strain caused different PGP responses compared to GD12 on Arabidopsis mutants compromised in phytohormone signaling. What is clear from our preliminary studies, is that both transcriptional and genetic data indicate that T. hamatum responses differ from conventional JA/ET dependencies reported in the literature. Therefore, understanding the mechanistic basis of T. hamatum PGP/ISR is clearly of both fundamental and strategic interest. The aim of this proposal is to unravel the host signaling networks activated in systemic foliar tissue following T. hamatum infection. These pathways are expected to provide a mechanistic insight into both PGP potential and systemic enhanced resistance. In parallel we will undertake a detailed comparative metabolic profiling of the T. hamatum secretome to identify bioactive compounds responsible for PGP and ISR. We outline a multidisciplinary proposal that exploits the rapid developments in genomic and analytical technologies to (i) examine the nature of the bioactive inducers in the mutant and GD12 Trichoderma lines, (ii) identify the plant signaling networks activated by these bioactive compounds through comparative transcriptomics in Arabidopsis mutant lines, and between Arabidopsis and rice undergoing PGP/ISR responses and (iii) characterise key plant regulatory component of this pathway.

Planned Impact

There is a clear need to accelerate the translation of fundamental research into practice, the proposed project addresses the overarching BBSRC aim of delivering excellence with impact, and concentrates on an area of research under-developed in the current UK efforts towards improved food security via crop genetic improvement. A major positive benefit in pursuing natural elicitors and understanding their mode of action is that they should have generic inducing properties and subsequent developments are not dependent on major investment in a genomic research platform for each crop. The major aim of this research is to determine the contributory factors underpinning growth promotion and broad spectrum induced resistance activated by the rhizosphere fungus Trichoderma hamatum on a range of crop and experimental plants. Trichoderma strains have been shown to ameliorate a wide range of abiotic stresses such as salinity and drought, improve photosynthetic efficiency, enhance nutrient uptake, and significantly increase nitrogen use efficiency in crops. Recent research conducted in the co-I's research group has identified a novel strain of the naturally occurring rhizosphere fungus T. hamatum (strain GD12) that improves crop productivity in the absence of costly fertilizers and environmentally damaging agrochemicals. We wish to investigate how strain GD12 induces such benefits by focussing our study on the molecular and biochemical pathways (both the bioactive inducing molecules and the signalling networks targeted by these activators within the host plant) responsible for this phenomenon. Our goal is to provide the molecular foundations for exploiting this extraordinary capacity to improve crop growth and resistance to a broad spectrum of pathogens. We see this work as having a high probability of translational success and complementary to ongoing investment in genomic and breeding programmes for crop improvement within the UK. The approach is novel and quite different from other agricultural driven research in the UK. The long-term objective of the work is to exploit the growth promotion and induced resistance properties of T. hamatum in order to provide innovative approaches for improved crop productivity, leading to the development of sustainable agricultural practices. The work may lead to the generation of novel mechanisms of disease control in integrated pest management systems and the identification and exploitation of beneficial traits during plant breeding programmes. Emerging knowledge is expected to have tremendous biotechnological potential leading to increased crop productivity, improved sustainable resource management and the reduced environmental impact of agricultural practises (e.g. reduced use of synthetic fertilizer and pesticides). We therefore identify the agrochemical industry, plant breeders and the composting industry as key beneficiaries of this work.
 
Description We studied a novel Trichoderma strain, T. hamatum GD12 that uniquely processes
both plant growth promotion and biocontrol activities. We characterised these properties, undertook genome sequencing, assembly and annotation, transcriptional profiling and metabolic analysis. This integrated approach gave us unexpected new insight into both dual beneficial attributes of this saprophytic fungi, growth promotion and biocontrol. Strikingly we found that more than 40% of the GD12 genome was unique and not homologous to any of the 4 related sequenced Trichoderma genomes. this degree of divergence was unexpected, and highlighting the potential to generate novel anti-microbial and plant growth promotion compounds. This rapidly became the focus of the project. In the "GD12 unique" proportion of the genome, we have identified non-ribosomal peptide synthases and polyketide synthases, which have the potential to encode novel fungal secondary metabolites. We verified the "uniqueness" of GD12 by sequencing another local T. hamatum isolate (strain GD11) and demonstrated that it shares 98% identity with GD12. Within the genome we also predicted at least ~1,100 secreted proteins representing candidate effector proteins that are probably contributing to the beneficial traits of GD12. We undertook targeted metabolite profiling, and in the process developed novel algorithms for calling "features" (small molecules). Using the genome sequence we generated two mutants, one designed to remove genome-silencing repressors to release hidden cryptic chemistries that we predict were activated during biocontrol or plant growth promotion activities.
One mutant in particular secretes a range of potent antifungal metabolites that we are further investigating.
In summary, an integrated approach has identified remarkable potential for GD12 to synthesise novel chemistries that contribute to agronomically important traits. Metabolite profiling combined with targeted mutagenesis informed by our genome sequencing has enabled us to identify potentially important antifungal compounds that are effective not only against plant pathogens, but also medically important fungal pathogens.
Exploitation Route We have interest from two agrochemical companies after disclosing the results of the antifungal activities generated in the silencing suppressor mutant. We are in the process of holding a second round of discussions on further developing thiis line of research.

2019: Subsequent work funded by start-up monies and a BBSRC PhD studentship has moved this forward to identifying a HPLC-fraction of fungal supernatant that has powerful antifungal activity against both plant and human drug resistant fungi. This is being taken forward for purificaiton and NMR.
Sectors Agriculture, Food and Drink,Chemicals,Environment

 
Description We signed an NDA with Germains Seed Technology, a major seed improvement company and Zelam, a New Zealand based biologics company. We hve had discussions with Syngenta with respect to the bioactives identified and Elsoms regarding the potential to use as a seed coat.
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Beneficial impact of Trichoderma on clover-grassland
Amount £4,640 (GBP)
Organisation Stapledon Memorial Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2014 
End 12/2014
 
Description SouthWest Bio DTP
Amount £120,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2014 
End 09/2019
 
Description Warwick Impact Fund
Amount £41,961 (GBP)
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 11/2016 
End 07/2017
 
Title New algroithm to analyse untargetted mass spec data 
Description Yang, Z-R & Grant, M. (2012) An ultra-fast metabolite prediction algorithm. PLoS One 7: e39158 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact This was a much faster, less computational intensive method for aligning mass spectral data from LC-MS-MS small molecule profiling. 
 
Title Pipeline for analysis of discriminant ions in liquid chromatrography, mass spectrometry data 
Description Developed a computational pipeline to take mass spectra of Trichoderma, plant, plant pathogen samples, align data, determine discriminant ions, visualise data and interrogate publically available mass spectra databases. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? No  
Impact Has enhanced our ability to interrogate complex mass spectra datasets 
 
Title Genomic sequence of Trichoderma hamatum GD12 
Description A draft genome sequence of GD12 from 12 million pairs of Illumina GA2 paired-end 73-bp reads using Velvet 1.1.04. This yielded 2,770 scaffolds with a N50 length of 41.6 Kb. The total length of the assembly was 38.2 Mb. The whole genome shotgun data have been deposited at DDBJ/EMBL/GenBank under the accession ANCB00000000. Using FgenesH (trained on Neurospora crassa) we predicted 12,391 protein-coding genes in GD12. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact Highly downloaded dataset. 
 
Description Application of Trichoderma hamatum to waste reduction 
Organisation TEG Environmental Waste
Country United Kingdom 
Sector Private 
PI Contribution Collaboration with one of the largest waste disposal companies to add value to organic waste by amending with T. hamatum
Collaborator Contribution Carried out scoping studies to determine the benefits of Trichoderma hamatum ammendment to improving organic waste compostation as a potential route to add value.
Impact Not assessed as economically viable to pursue on a large scale.
Start Year 2011
 
Description Metabolic changes induced in Arabidopsis thaliana by purified plant growth promotion 
Organisation University of Naples
Country Italy 
Sector Academic/University 
PI Contribution Testing of 3 potential plant growth promoting compounds purified from Trichoderma harzianium on Arabidopsis thaliana.
Start Year 2011
 
Description Use of Trichoderma hamatum as an component of biologic enhancements 
Organisation Zelam Ltd
Country New Zealand 
Sector Private 
PI Contribution Established a collaboration with an SEM in New Zealand - Zelam Ltd - to look at incorporating T. hamatum into their plant growth enhancement solutions.
Start Year 2012
 
Title MTA agreement 
Description Collaborative research with industry 
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact Interest from both Syngenta and BASF
 
Title Aligning extracted LC-MS peak lists via density maximization 
Description Using the process of density maximisation to call replicated peaks in liquid chromatography mass spec spectral alignments and filling in missing data 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact Cited 8 times - not bad for a very specialist technique 
URL http://dx.doi.org/10.1007/s11306-011-0389-x
 
Title An ultra-fast metabolite prediction algorithm 
Description Developed an ultra-fast algorithm to predict metabolites (or features) in biological replicates of mass spec sepctral outputs 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact Difficult to determine 
 
Description BBC One Roadshow 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Public outreach activity In conjunction with the British Society of Plant Pathology supported the 'Plant Doctor' stand at the BBC One show Summer Festival in Weston Super Mare. Supported development of a "Plant Doctor" stand - developing visual materials for the general public to help with diagnosing plant diseases -- an area that has recently been highlighted as a skills deficit area in the UK

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Elsoms 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Presented results of Trichoderma hamatum plant growth promotion and biocontrol to selected employees of Elsom's Seeds in Spalding (5th December 2018)
Year(s) Of Engagement Activity 2018
 
Description International Industrial Innovation Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Midlands Engine Commonwealth Business Conference A collection of Warwickshire businesses linked to the local enterprise Partnership and a collection of Commonwealth Politicians/business leaders, including High Commissioners from London.Presented a talk on Food Security - one of the "left field" talks of the day to an audience primarily focussed on industrial innovation, robotics and artificial intelligence.
Year(s) Of Engagement Activity 2018
 
Description Invited Seminar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Seminar at Center of Excellence for Crop Protection, Lincoln University, NZ Presented project work to an audience of plant and animal scientists at the Centre for Crop Protection - one of the Centres for Excellence in New Zealand Seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2013
 
Description Kenilworth Agricultural Show 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Ran an educational demonstration at the Royal Kenilworth Agricultural Show
Year(s) Of Engagement Activity 2017
 
Description NWAFU 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Keynote presentation at the North West Agricultural and forestry University
Year(s) Of Engagement Activity 2018
 
Description Public Evening - Science on the Hill 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presented the hypothetical scenario - what plants could grow on Mars, and what would they look like (i.e. what adaptations are required for a life on Mars) as part of an extra-solar planet talk.
Year(s) Of Engagement Activity 2019
 
Description Public engagement evening - Food Security 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public Engagement evening consisting of 4 talks and 1 hour of demonstrations, hands on events and technology displays
Year(s) Of Engagement Activity 2017