Understanding the impact of soil nitrogen on plant disease resistance

Lead Research Organisation: University of Oxford
Department Name: Plant Sciences

Abstract

Plants obtain most of the nitrogen they need for growth and metabolism in the form of inorganic nitrogen ions such as ammonium and nitrate. Nitrogen ions are absorbed by roots and used to make amino acids that can be transported throughout the plant. Plant growth and development is regulated by the availability of nitrogen in soil, and nitrogen is frequently a growth limiting nutrient in natural ecosystems. Soil nitrogen and plant growth can be increased by treating soil with nitrogenous fertilisers, but the level and type of nitrogen used must be carefully controlled. High levels of nitrogen, especially ammonium, are toxic to some plants and moderately high levels promote lush vegetative growth that is susceptible to pests and diseases. An additional source of concern is that plants do not take up all of the nitrogen that is applied as fertilisers. Excess fertilisers are costly for farmers and act as environmental pollutants that can promote algal blooms through run-off into lakes and rivers and disturb natural ecosystems. Furthermore, increases in diseases and pests in fertiliser treated plants may require additional applications of pesticides and fungicides, again at an added cost to farmers and the environment. In this project we are particularly concerned with the link between soil nitrogen and increased plant disease. Researchers have observed that high soil nitrogen results in increased levels of inorganic nitrogen ions in plant tissues and alterations to both primary and secondary metabolism. The increased pest and disease susceptibility observed in over-fertilized plants could be due to two processes. Firstly, alterations to plant metabolism may make more nutrients available to pathogens (disease causing organisms such as bacteria and fungi). Secondly, the complex biosynthetic pathways used to synthesise anti-microbial chemicals may be suppressed by high soil nitrogen, making plants less able to defend themselves against infection. Intriguingly, some changes in plant physiology caused by high soil nitrogen resemble those caused by pathogen infection, which suggests that pathogens produce chemicals that inhibit and alter plant nitrogen metabolism in order to promote pathogen growth. We will use the interaction of the bacterial plant pathogen Pseudomonas syringae pv. tomato with tomato and the model plant Arabidopsis thaliana to investigate the role of soil and leaf nitrogen in disease resistance. P. syringae pv. tomato colonises the spaces between plant cells, taking nutrients from the apoplastic fluid that surrounds plant cells. This bacterium uses secreted proteins, toxins and hormones to control plant metabolism and can reach levels of 10 million bacteria/cm2 in the leaves of susceptible plants. We aim to describe the effect of soil nitrogen concentration on disease resistance to P. s. pv. tomato, and to measure the composition of apoplastic fluid in healthy and infected plants. We will specifically examine whether apoplastic fluid from plants treated with high levels of nitrogen supports higher rates of bacterial multiplication, and whether bacteria induce changes in apoplastic fluid that promote bacterial multiplication. We will also examine whether and how soil nitrogen affects the ability of plants to defend themselves against pathogens. The results of these analyses will provide three clear benefits. Firstly, they will clearly describe the mechanistic link between soil nitrogen and disease resistance. Secondly, we will be able to use this information to design experiments that use apoplastic composition analyses to optimise fertiliser composition and application. Finally, we may be able to use pathogen-induced changes in apoplast composition as an early sign of infection, facilitating early intervention and disease prevention.

Technical Summary

High soil nitrogen causes increased levels of inorganic nitrogen ions in plant tissues and in apoplastic fluid, along with increased susceptibility to pests and pathogens. The increased disease susceptibility of plants exposed to excess nitrogen could be due to two factors. Firstly, alterations to primary metabolism may make more nutrients available to pathogens. Secondly, high nitrogen levels may suppress defence-associated signal transduction and secondary metabolism, making plants less able to defend themselves against infection. Intriguingly, some changes caused by high soil nitrogen resemble those caused by pathogen infection, which could indicate that pathogens specifically target plant nitrogen metabolism in order to inhibit plant defences and promote pathogen growth. We aim to use the interaction of the bacterial plant pathogen Pseudomonas syringae pv. tomato with tomato and with Arabidopsis thaliana to investigate the role of soil and apoplastic nitrogen in plant-pathogen interactions. We will describe the effect of soil nitrogen on the composition of apoplastic fluid and on disease resistance to P. s. pv. tomato. We will test the effect of specific bacterial pathogenicity factors on the composition of apoplastic fluid and examine whether and how soil nitrogen affects plant defence responses using metabolomic, transcriptomic and microscopic techniques. This project combines expertise on bacterial pathogenesis and plant defence responses (Oxford) with expertise on metabolomics and plant nitrogen metabolism (MeT-RO/Rothamsted). We will be able to use information from these analyses to identify apoplastic biomarkers that can be used to optimize fertiliser application, and to identify pathogen-induced changes in apoplastic composition, facilitating early intervention and disease prevention. Ultimately, we will be able to extend these analyses to other plant-pathogen systems, facilitating integrated, sustainable, low-input crop management.

Publications

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Rico A (2011) The metabolic interface between Pseudomonas syringae and plant cells. in Current opinion in microbiology

 
Description Plant growth is highly dependent on nitrogen availability. However, nitrogen is frequently a growth limiting nutrient in natural ecosystems. Soil nitrogen can be increased by application of nitrogenous fertilisers, but the level and type of nitrogen used must be carefully controlled. High levels of nitrogen are toxic to some plants and moderately high levels can promote lush vegetative growth or increased susceptibility to pests and diseases, resulting in reduced yield, and increased application of pesticides and fungicides. An additional source of concern is that plants do not take up all of the nitrogen that is applied as fertilisers. Excess fertilisers are costly for growers and act as environmental pollutants that can promote algal blooms through run-off into lakes and rivers and disturb natural ecosystems.

In this project we have investigated the link between soil nitrogen and plant disease, focusing on the effect of soil nitrogen and pathogen infection on plant metabolism. The increased disease susceptibility observed in plants treated with high levels of nitrogen could be linked to two processes. Firstly, alterations to plant metabolism may alter the nutrients available to pathogens (disease causing organisms). Secondly, the metabolic pathways used in plant defence responses to infection may be suppressed by high soil nitrogen, compromising plant defences.

We have studied the effect of nitrogen on the susceptibility of tomato plants to bacterial speck disease, which is caused by a bacterium known as Pseudomonas syringae pv. tomato (Pst). Pst colonises the spaces between plant cells, and grows using nutrients present in the apoplastic fluid that surrounds plant cells. The recommended level of nitrogen for greenhouse tomato production is typically 10-15 mM nitrogen. However, some studies have suggested that lowering nitrogen concentrations to 4-6 mM nitrogen may have a positive effect on fruit quality, with relatively little reduction in yield. We observed that tomato plants grown on 15 or 30 mM nitrogen showed significantly increased susceptibility to bacterial infection when compared to plants grown on 5 mM nitrogen. This indicates that lower nitrogen concentrations may not only benefit tomato growers and consumers by increasing fruit quality, reducing fertilizer costs and fertilizer-associated pollution, but also by increasing the resistance of tomato plants to Pst.

To understand the effect of nitrogen on disease resistance in greater depth, we studied the effect of nitrogen and Pst on the chemical composition of plant tissues. We also studied the ability of Pst to grow using nutrients present in apoplastic fluid, and showed that this bacterium is well-adapted to grow using nutrients that are abundant in the apoplast of healthy and infected plants. Both nitrogen treatment and pathogen infection had a significant, but distinct impact on the chemical composition of plant leaves. Notably, high nitrogen treatment significantly altered plant responses to Pst, resulting in increased accumulation of certain amino acids, sugars and other metabolites in the apoplastic fluid of Pst-infected plants. This suggests that nitrogen treatment may enhance mobilization of nutrients, particularly nitrogen compounds, to the apoplast to support pathogen growth. We were also able to identify chemicals that were specifically present in diseased plants, even when no symptoms were apparent, and to show that these chemicals accumulated to higher levels in high nitrogen treated plants. These chemicals may have an important, but as yet undetermined role in disease development, and may be useful biomarkers to detect the early stages of pathogen infection. Collectively, our results highlight the benefits of using moderate levels of fertilizer to achieve an optimal balance between yield, food quality and disease resistance, and suggest promising new directions for applying knowledge of plant biochemistry to improve disease control.
Exploitation Route This project has refined and developed methods for studying the metabolism of plant pathogens, the metabolic interface between host and pathogen and the impact of genotype-phenotype-environment interactions on plant health and disease resistance.

These approaches can be adapted to address a wide variety of questions in plant and microbial physiology. Citations for manuscripts arising from this project indicate that the results and methods developed in this project have proved to be of value for researchers working in a number of fields, including plant disease, plant development, plant nutrition, high-throughput phenotyping, analysis of metabolic networks and microbial physiology.
Sectors Agriculture, Food and Drink,Environment

 
Description The findings from this project have informed and been reported in multiple public engagement activities. Results obtained and methods developed in this study have been widely cited, indicating their take up and application by researchers studying plant disease, plant development, plant growth promotion, microbial physiology and plant nutrition.
First Year Of Impact 2007
Sector Agriculture, Food and Drink
Impact Types Societal

 
Description Magdalen College Scholarship
Amount £60,000 (GBP)
Organisation University of Oxford 
Department Magdalen College Oxford
Sector Academic/University
Country United Kingdom
Start 09/2009 
End 12/2013
 
Description NERC Studentship
Amount £60,000 (GBP)
Organisation Natural Environment Research Council 
Sector Public
Country United Kingdom
Start 09/2005 
End 09/2009
 
Description Royal Society International Exchanges Cost Share Award - Argentina
Amount £9,000 (GBP)
Funding ID IEC\R2\170201 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 12/2019
 
Title High-throughput phenotyping 
Description In the course of this project we developed new applications of Biolog Phenotype Microarray Technology, including a novel method for studying the metabolic processes that occur in cells growing in complex environments such as the plant apoplast. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact However, the research carried out in this proposal has helped to strengthen informal links between the PI, Biolog Inc., and Biolog's UK distributor Technopath. As a consequence the PI has been invited to speak about the research carried out in this proposal at Biolog-supported symposia held at the Veterinary Laboratories Agency and The University of Reading, and at the Florence Conference on Phenotype Microarray Analysis in September 2010, bringing the methods and ideas developed in this project to the attention of a wider audience. We have also provided specific advice on the use and development of Phenotype Microarray approaches to a number of different research groups across the UK and worldwide. 
 
Title Plant growth regimes and apoplast extraction techniques 
Description We have used 1H NMR, ESI-MS, GC-MS and HPLC to profile the composition of whole leaf tissue and apoplast extracts from healthy and pathogen-infected tomato plants grown in three nitrogen regimes. We have also used 1H NMR and ESI-MS to profile whole leaf tissue and apoplast extracts from healthy and pathogen infected Arabidopsis plants grown in three nitrogen regimes. In the course of this work we have developed protocols for hydroponic cultivation of tomato and Arabidopsis in varying nitrogen regimes and have refined and improved protocols for leaf apoplast extraction and analysis. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact The methods developed in this project were shared with collaborators at the University of the West of England, enabling them to obtain evidence for the potential existence of factors within the bean apoplast that affect gene loss and gain in Pseudomonas syringae, thereby leading to the breakdown of disease resistance. This led to a high profile publication: Godfrey et al. 2011. PLoS Pathog 7:e1002010 and a successful application for joint funding from the BBSRC. 
 
Description Analysis of plant hormones 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Staff at Oxford prepared biological material for analysis and collaborated with staff at Imperial in the analysis of results obtained.
Collaborator Contribution Staff at Imperial performed LC/MS/MS analysis of plant samples to quantitate plant hormones.
Impact Results generated as a result of this collaboration have been reported in scientific publications.
Start Year 2008
 
Description National Metabolomics Centre 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Staff at Oxford worked to optimise procedures for the collection of biological material for metabolomic analysis using MeTRO facilities and collaborated in the analysis and interpretation of results.
Collaborator Contribution MeTRO analysed the composition of biological samples using 1H NMR and ESI-MS, and developed statistical approaches for analysis of metabolomic data generated during this project.
Impact Staff at the University of Oxford and at Rothamsted Research have drawn on results generated during this project to inform public and industry engagement activities.
Start Year 2007
 
Description Royal Society Cost Share Award - Argentina 
Organisation National University of San Martin
Country Argentina 
Sector Academic/University 
PI Contribution We have initiated a new collaboration to study polyamine metabolism in endophytic bacterial plant pathogens and its significance in stress resistance and pathogenicity, funded by a Royal Society International Exchanges Cost Share Award.
Collaborator Contribution Participation in future research exchanges and collaborative work.
Impact This award has supported a research visit by a member of my group to Argentina to conduct collaborative research, and a visit by Dr. Andres Garriz to Oxford in 2018. There are no publications or further awards arising as yet.
Start Year 2018
 
Description Artist in Residence - John Thomson 
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 n 2005 artist John Thomson was artist in residence at the Oxford Botanic Garden. Gail Preston provided the main point of contact for John with the Plant Science Department, and discussions about plant-microbe interactions and biological networks inspired some of the work he exhibited at the Oxford University Museum of Natural History between March and May 2006 (http://www.thomsonart.co.uk/exhibitions/disney_meets_dna.shtml). Work from John's residency was also exhibited in 2007 at Christ Church College, and Dr. Preston assisted Mr. Thomson with the preparation of programme and exhibit notes.

The activity led to exhibits at the University Museum of Natural History and Christ Church College that raised the profile of plant science and plant-microbe interactions and stimulated interest.
Year(s) Of Engagement Activity Pre-2006,2006,2007
URL http://thomsonart.co.uk/category/exhibitions/
 
Description National Metabolomics Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The National Metabolomics Centre, at which the metabolite profiling work carried out in this project was performed, receives many visitors, including the public, on open Rothamsted open days (previous open weekend October, 2006; next open day May 2010) and researchers linked to this grant at Rothamsted have given public lectures (for example, Dr Jane Ward 'Beneficial Phytochemicals in Cereal Grains and Other Foods' - Rothamsted Open Meeting, Nov 2008). Organised visits of parties of students range from local schools to universities. More specialist groups of scientists and policy makers, many from overseas regularly visit the metabolomics centre on an ad hoc basis, recent examples are a Chinese delegation interested in policy on registration of genetically modified crops, a Turkish group setting up a metabolomics centre in Ankara and the Worshipful Company of Scientific Instrument Makers. Such visits and interactions have raised awareness of plant science and the role of metabolomics approaches in studying bioscience. and encouraged the extension of the methods used in this project to diverse research questions.

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Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Questioning Evolution - Exhibition at Oxford Botanic Garden 
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 The principal investigator curated an exhibition on plant science research at the University of Oxford to accompany an installation of the BBSRC-sponsored "Questioning Evolution" exhibit at the Botanic Garden. This sparked interest in diverse areas of plant science.

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Year(s) Of Engagement Activity 2009
 
Description School visits 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact The principal investigator has given presentations at schools in the UK and France, which raised awareness of plant and microbial science for students who had received little formal training in these areas.

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Year(s) Of Engagement Activity Pre-2006,2008,2009
 
Description Science for Gardeners 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Seminars offered within the series encouraged questions and discussion and increased awareness of microbiology and plant science.

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Year(s) Of Engagement Activity Pre-2006,2006,2007,2008