Final Report Summary - ROOTOPOWER (Empowering root-targeted strategies to minimize abiotic stress impacts on horticultural crops)
Executive Summary:
'A paradigm shift to root system engineering'.
After more than fifty years of crop improvement principally selecting for above ground traits, scientists now perceive root system engineering as an opportunity to integrate new approaches to maintain sustainable crop production under changing environmental conditions while minimizing the demand for new resources. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource capture and plant development under adverse conditions.
'ROOTOPOWER: understanding the power of root traits'.
ROOTOPOWER aims to develop a multidisciplinary suite of new tools targeted to the root system to enhance agronomical stability and sustainability of dicotyledonous crops under multiple and combined stress conditions. Central to our approach is the use of tomato as a model species since it can be very easily grafted, (and indeed is usually grafted in commercial protected cropping). This surgical technique attaches genetically different shoot and root systems, allowing precise assessment of the effect of altering root traits on crop performance independently of shoot traits, since the scion (shoot) is constant. Many physiological studies have employed grafting between different genotypes of dicotyledonous species (or even between species) to improve our understanding of root-to-shoot signalling or to alter shoot phenotype. This project has been analyzing and exploiting the natural genetic variability existing in wild-relative tomato species (used as rootstocks) and their beneficial interactions with natural soil microorganisms (arbuscular mycorrhizal fungi AMF, and plant growth promoting rhizobacteria, PGPR). This project has obtained genetic information and physiological understanding of mechanisms vital for high-performing root systems. The key research challenges approached by ROOTOPOWER have been therefore:
• To identify stress-resistant root systems and rhizosphere microorganisms (and their synergisms) for enhanced resistance to individual and combined abiotic stresses.
• To understand the underlying genetic and physiological mechanisms, which are potentially fundamental to all crops, and readily exploited in dicotyledonous crops.
This project has firstly identified genetic variation and quantitative trait loci (QTL) that allow tomato roots to confer crop resistance to a range of abiotic stresses, alone or in association with arbuscular mycorrhizal fungi (AMF) and/or plant growth promoting rhizobacteria (PGPR). This approach is establishing the physiological mechanisms and signalling processes conferred by key QTLs, and identifying candidates for the causative genes by obtaining and evaluating near isogenic lines (NILs) for selected QTLs. Primarily, ROOTOPOWER evaluated rootstocks of a unique recombinant inbred line population (RIL) from a cross between Solanum lycopersicum var. cerasiforme and S. pimpinellifolium for their performance under multiple abiotic stresses and for their interaction with AMF and PGPR. This project conducted detailed analysis of the underlying rootstock-derived molecular, physiological and morphological mechanisms that influence plant growth, fruit yield and quality under suboptimal conditions. Several combinations of below-ground abiotic stresses have been addressed in the project: salinity, water stress, soil compaction and low fertilizer (N, P, K) input.
Project Context and Objectives:
The strategic aim of ROOTOPOWER is to help crop producers and breeders deal with the predicted impacts of climate change and to overcome the consequences of unsustainable agricultural practices that are causing soil degradation and depleting natural resources. ROOTOPOWER seeks to improve crop stress resistance and develop more resource-efficient crops, allowing more efficient use of dwindling water and phosphorus resources, and to reduce excessive use of nitrogen fertilizers (which have a high carbon footprint). Without this research there will be a trend for below-ground abiotic stresses to decrease plant growth and development throughout Europe leading to serious crop yield losses.
'Taking advantage of a multidisciplinary approach'.
Since ROOTOPOWER aims to understand crop responses to multiple below-ground stresses (including root stress perception and communication to influence shoot growth and development), it has been analysing the relationships between root genotype and shoot performance. Beyond root and rootstock-influenced shoot phenotyping, the consortium has identified QTL and candidate genes (fine mapping, transcriptomics and RT-PCR) for rootstock-mediated abiotic stress resistances and interactions with beneficial rhizosphere fungi and bacteria that will be directly suitable for breeding programs. High throughput comprehensive hormonal and ionome analyses have been performed in root and leaf xylem sap to identify and quantify the signals involved in root perception and response to individual and combined stresses and selected biological interactions. Regulatory effects of root genotype and rhizosphere microbiology on shoot photosynthesis, organ growth and development, carbon, nitrogen, phosphorous, potassium accumulation and water fluxes have been identified. Data from analyses of the genome, transcriptome, hormone, ionome and phenome are being processed through advanced statistical and modelling approaches to define the mechanistic links between genes and trait responses to stress. Such understanding will enable delivery of specific combinations of root and biota-traits for use in root-targeted breeding strategies.
'Analysis of abiotic factors'.
The project has been focused on specific abiotic stress factors in particular salinity and water stress, soil compaction and low fertilizer (N, P, K) input, and indirectly apply extreme temperatures (by evaluating crops grown in different locations and growing seasons in commercial-scale experiments). This choice is fully justified since these factors are of great relevance for determining worldwide agricultural productivity, including horticultural crops such as tomato. It is widely accepted that climate changes occurring in the years ahead will be characterized by an unpredictable increase of extreme temperatures and frequency and intensity of precipitation and intensity of drought periods. This uneven distribution of water and temperature in space and time, in addition to unsustainable agricultural practises resulting in soil degradation and water contamination (eg. excessive irrigation causing nutrient leaching from crops, or irrigation with poor quality water causing salinity) will also contribute to the overall scarcity of suitable agricultural land.
'Analysis of rhizosphere biotic interactions'.
Major groups of rhizosphere organisms known to improve crop resource capture and enhance stress resistance include rhizobia that fix nitrogen in the legume nodule; mycorrhizal fungi that enhance water and nutrient uptake, and plant growth promoting rhizobacteria that have multiple beneficial impacts including changing root system architecture and root hormone status. Although not a legume, tomato is a suitable model species to investigate the genetics, genomics and signalling processes involved in the root associations with AMF and PGPR and the subsequent effect on root and shoot development under abiotic stress conditions. ROOTOPOWER has considered both how these organisms affect the root system (and thereby shoot processes) but also how root exudation to the rhizosphere affects colonisation of rhizosphere organisms.
The scientific objectives of the project are:
• To identify new QTLs, candidate genes and novel signalling processes involved in root specific responses of tomato to six individual abiotic stresses including 3 major nutrient deficiencies (e.g. moderate salinity, restricted soil water availability, moderate soil impedance, decreased nutrient supply: N, P, K), and some naturally occurring stress combinations
• To generate new genetic, genomic and physiological information, on the basis of the genetic variability of the P-RIL population, regarding the capacity or root colonisation by beneficial rhizosphere microorganisms (AMF and PGPR).
• To link the generated genetic, physiological and agronomical knowledge through the modelling of genetic and environmental dependencies of root system architecture.
• To evaluate selected and combined genetic plant material and microorganisms in controlled multi-stress environments and real Northern and Southern European cropping conditions.
• To understand biological interactions between roots and rhizosphere microorganisms (AMF and PGPR), and to identify at least one root genetic marker for each microorganism and the associated root-signalling mechanisms.
• To understand the genetic controls of different types of root-to-shoot signalling (hydraulic, ionomic and hormonal) that positively affect shoot performance under the above described abiotic stress conditions.
At the technological level, project objectives are:
• To deliver to the scientific community at least two contrasting tomato lines used as rootstocks for their induced differential shoot response to i) moderate salinity, (ii) decreased soil water availability (iii) moderate soil impedance, (iv) decreased nutrient supply (N, P, K) and their combinations, and (v) for their capacity to associate beneficial fungi and bacteria.
• To deliver to the plant breeding community between 8 and 20 tomato lines (P-RILs) harbouring dominant and additive traits for different individual and combined abiotic stresses, some of them with direct commercial interest.
• To deliver strategies for root-targeted, marker-assisted selection for resistance to individual and combined stresses by identifying DNA-based genetic markers and an understanding of the additive effects and epistatic interactions of multiple QTLs.
• To deliver to the scientific community at least 2 pairs of near isogenic lines (NILs) for at least 4 selected QTLs conferring resistance rootstock-mediated resistance to below-ground abiotic stresses.
• To build and deliver a xylem profiling model (hormones + nutrients) that will provide physiological markers to assist root-targeted breeding for resistance to individual and combined stresses.
• To develop a predictive model of root-mediated plant response to the above described abiotic stresses.
• To develop innovative methods for testing biotic interactions (eg. root vs PGPR) and for root phenotyping (e.g. changing root system architecture in response to different stimuli).
• To propose new targets for the development of root-targeted breeding strategies in crop-species for abiotic stress resistance.
• To propose new sustainable strategies of crop management on the basis of the availability of more resource efficient root systems and associated microorganisms.
• To develop a commercial product for abiotic stress alleviation based on AMF/PGPR.
Dissemination and technology transfer objectives include:
• Reporting our results via publications (at least 10) in high impact scientific journals.
• Disseminate novel scientific advances via PI attendance at scientific and trade, national and international conferences (eg. International Society for Horticultural Science meetings) and our interactions with the advisory board and the international Solanaceae community.
• Disseminate science at a general public level through school activities, and general public targeted information in order to convey to the public opinion clear notions on abiotic stress impacts on horticultural crops.
• To demonstrate rootstock- and biota-mediated benefits of a sustainable modern agriculture (via our field-scale trials at grower sites) to a wide community of horticultural farmers within (at least) The Netherlands, Turkey and Spain.
• To train a PhD student or postdoctoral researcher in rootstock breeding, providing a career opportunity of a further contract with the SME breeding company.
• To assist the SME breeding company to start a rootstock breeding programme for some selected abiotic stress conditions on the basis of the knowledge and tools issued from ROOTOPOWER.
• To organise a workshop on phenotyping at the outset of the project to standardise data collection across consortium partners.
Relevance to the addressed topic
Elucidating root perception and response to below-ground abiotic stresses comprises phytohormone, hydraulic signalling, complete ionome, transcriptome and candidate genes analyses at local (within the root), long-distance (root-shoot communication assayed via transport fluids) and systemic (shoot) levels in the whole RIL population and in selected contrasting lines. Analysis of relationships between signalling and soil biophysical variables under controlled uniform conditions (eg. soil matric potential, soil strength, and nutrient status) has been used to determine whether these relationships can predict signalling responses to heterogeneity and combinations of abiotic stresses. The predictive value of relationships developed under root-constrained conditions, and their moderation by developmental responses of unconstrained root systems, has been tested by upscaling experiments to the field. Root interactions with the biotic environment has been measured via rhizosphere colonization by introduced biotic inoculants (AMF and PGPR), the analysis of root exudates (defined as chemicals released by roots into the rhizosphere) and root xylem sap in response to inoculation, and root system architecture. The project relies on interdisciplinary approaches integrating different expertise within the consortium composition.
Project Results:
Although a better statement of the main results of the project will be possible upon a more comprehensive analysis of the huge amount of data obtained, below there is a description of the main scientific and technological results of the ROOTOPOWER project that have been highlighted so far in relation to the foreseen objectives.
• To identify new QTLs, candidate genes and novel signalling processes involved in root specific responses of tomato to six individual abiotic stresses including 3 major nutrient deficiencies (e.g. moderate salinity, restricted soil water availability, moderate soil impedance, decreased nutrient supply: N, P, K), and some naturally occurring stress combinations
'QTL detection for rootstock effects under individual and combined stresses'.
The EU ROOTOPOWER project (Grant # 289365) is exploiting the natural genetic variability existing in wild-relative tomato species (used as rootstocks) to enhance agronomic stability of tomato by maximising water and nutrient capture and sensing and adjusting plant growth and shoot physiology to six abiotic stresses (drought, salinity, high impedance and nutrient - NPK - deficits). For this purpose, a commercial tomato cultivar was grafted onto 144 different rootstocks: six accessions from S. lycopersicum and S. pimpinellifolium, selected for drought tolerance (sourced from AVRDC); nine introgression lines from S. lycopersicum x S. pennellii and x S. habrochaites, selected for high root/shoot ratio, salinity and drought tolerances (sourced from TGRC); and a population of 129 recombinant inbred lines derived from S. lycopersicum x S. pimpinellifolium (sourced from IVIA). Since grafting allows precise assessment of the effect of altering root traits on general crop performance, the production of a huge amount of uniform grafted plants to delivery in eight phenotyping sites across Europe has been a key milestone of the project. More than 150.000 datapoints have been obtained below-, between- and above-ground to unravel rootstock-specific QTLs, physiological mechanisms and candidate genes for high-performing root systems. Although the results reveal a rootstock-induced (1.5-5.2-fold) variability in shoot vegetative biomass across different environments, the lack of correlation between them suggests important genotypic x environment interactions. However, stronger correlations in selected lines for different abiotic stresses suggest that common trait(s) might be responsible for rootstock performance under different environments, as supported by genetic and physiological studies.
The phenotyping of those populations under individual stresses has allowed the identification not only of contrasting rootstock lines than can increase or decrease the plant performance respect to the ungrafted scion variety, but also of lines showing a better performance under low P and K nutrition compared to normal fertilization, which is of high interest for further studies and rootstock development for a more sustainable agriculture.
To integrate the tolerance response of the grafted tomato plants to the different stress factors tested separately and the composition of the extracted root xylem sap in each experiment, the phenotype data collected from 8 experiments in five different countries across Europe (Spain, Turkey, Netherlands, Belgium and United Kingdom) were used for genetic analysis. A total of 170 significant QTLs were identified for evaluated traits, with five particularly QTL-rich linkage groups.
Under moderate salinity (75 mM NaCl), only two fruit yield QTLs were detected and in different regions that the previous fruit yield QTLs detected under high salinity (125 mM NaCl). This result suggests that the detection of rootstock-dependent fruit yield QTLs highly depends on the level of salinity. In fact, the heritability estimates at both levels were different at high and moderate salinity levels, respectively, suggesting that the higher the salinity level is, the higher the heritability of the rootstock-dependent fruit yield seems to be. However, two salt tolerance QTLs at the vegetative stage did not co-locate with fruit yield QTLs at the same salinity level, suggesting that both vegetative and reproductive growth under salinity can be differentially affected by the rootstock. Interestingly, the majority of QTLs related to Na+ concentration of the leaf and xylem sap (under control, salinity, drought, low P) were detected on the same chromosome where HKT HKT1 (codifying for a Na+ transporter) genes are located.
Most relevant QTLs related to tolerance to P deprivation were linked to tolerance QTLs to low N. Moreover, most QTLs related to drought tolerance under field conditions were linked to a QTL for number of penetrating roots through geotex T1000 and a low N tolerance QTL. A low K tolerance QTL was detected at the same region of chromosome 9 (chr9) than a QTL for leaf area under low N and a drought tolerance QTL.
A set of linked QTLs controlling the concentrations of numerous cations in the xylem sap under drought, was detected on chr8, linked to no drought tolerance QTL but to two salt tolerance QTLs. Several QTLs controlling jasmonic acid concentration under both low P and low N were detected on chr9. Up to 14 QTLs controlling the cytokinin trans-zeatin concentration were detected on different linkage groups, suggesting an important role for this hormone in the rootstock-mediated responses under stress conditions. Other QTLs were detected for other hormones such as abscisic and salicylic acids
As far as genetic studies are concerned most of them are preliminary and need to be complemented with further data and analyses. There were RILs that, as rootstocks, increased fruit yield and quality of the commercial variety used as scion. QTL analysis of rootstock-mediated scion nutrition is a powerful forward genetic approach to identify wild genes for rootstock breeding. These results could have an impact on the characterization and utilization of germplasm in rootstock breeding programs of horticultural and fruit crops towards the sustainability of agriculture using marginal water.
• To deliver to the scientific community at least two contrasting tomato lines used as rootstocks for their induced differential shoot response to i) moderate salinity, (ii) decreased soil water availability (iii) moderate soil impedance, (iv) decreased nutrient supply (N, P, K) and their combinations, and (v) for their capacity to associate beneficial fungi and bacteria.
Under field conditions, 42% of the experimental rootstocks performed better (increased shoot fresh weight) under drought conditions. Similarly, under drought in glasshouse conditions, 45% of the lines showed increased biomass compared to self-grafted controls. Under salt stress in glasshouse experiments, 20% of the lines had a higher shoot fresh weight, with some lines reaching a 24% increase. Moreover inbred tomato rootstocks had improved vegetative growth and nutrient use efficiency under single (between 11% and 67% for the self-grafted variety) and combined (up to 100% for the self-grafted variety) abiotic stress conditions (salinity/drought and NPK nutrient deficiencies). Interestingly, some rootstocks improved shoot growth more under low nutrient supply than under conventional nutrition, which is of great interest for increasing sustainability. Finally, improved rootstock association with beneficial rhizosphere microorganisms detected in some lines (an order of magnitude increase in colonisation) will contribute to additional positive effects on alleviating stress penalties on yield.
• To deliver to the plant breeding community between 8 and 20 tomato lines (P-RILs) harbouring dominant and additive traits for different individual and combined abiotic stresses, some of them with direct commercial interest.
• To deliver strategies for root-targeted, marker-assisted selection for resistance to individual and combined stresses by identifying DNA-based genetic markers and an understanding of the additive effects and epistatic interactions of multiple QTLs.
• To propose new targets for the development of root-targeted breeding strategies in crop-species for abiotic stress resistance.
'QTL validation, fine mapping and analysis of candidate genes'.
A fruit yield QTL gFW9.1 reported before this project was chosen to be validated by the production and phenotyping of lines that differ by a single chromosomal region (near-isogenic lines, NILs) at the genomic region where it was detected in the tomato RIL population. For this purpose, a set of NILs around this QTL were obtained and then a high salt tolerance experiment had to be carried out to find out the physical interval (defined by the significant differences among the NILs) where gFW9.1 fine-maps. Rootstock QTL gFW9.1 was previously reported on chr9, and many additional rootstock QTL were identified on chr9. In order to identify candidate genes on chr9 that might underlie the QTL gFW9.1 and other QTL on the same region, a pair of NILs derived from the Solanum lycopersicum var cerasiforme (E9) x S. pimpinellifolium (L5) recombinant inbred line population that differ in a large section of chr9 was evaluated. For the transcriptome analysis, grafted NILs were grown in a control treatment and a Low Phosphorus + Drought combined stress treatment.
A total of 80 genes that were significantly differentially expressed in roots (when comparing NILs) and that also located to chr9 were identified; these all represent candidate genes that could be causally related to chr9 QTL effects. Within this group of genes, two genes can be highlighted: (1) a gene that is predicted to have a non-functional allele in L5, annotated as a transmembrane protein of unknown function; and (2) a UBX domain-containing protein, whose expression level shows a significant interaction between genotype and treatment. It belongs to the large family of proteins which are involved in the substrate recruitment for directed protein degradation that could have a role in abiotic stress resistance.
For the remaining genes within the group of 80, specific genes can be highlighted based on relevant annotation, extent of up- or down-regulation, or statistical robustness, but, to further refine the list of candidate genes, additional validation and fine-mapping of QTL is required to narrow the locus of interest.
These results provide a list of candidate genes on chr9 that could be causative genes underlying the QTL gFW9.1 or indeed any QTL found within the region of chr9 that differs between NILs. Since the gFW9.1 QTL was not detected in the experiments designed to fine map it, further work is needed to validate and to map it to a smaller interval, and then to narrow the list of candidate genes.
'Combined stress-induced genes'.
Data from root transcriptome study also highlights genes that are induced by the Low P + Drought combined stress treatment, irrespective of the genotype, and this may provide information about how tomato roots respond to multistress treatments. Many genes related to phosphate and nitrogen metabolism and to drought and root-related processes have been highlighted. Additional systematic analysis of gene expression patterns in relation to functional annotation will be undertaken to understand how the plants have responded to the Low P + Drought combined stress.
The most prominent genes upregulated by the Low P + Drought combined stress treatment (irrespective of genotype) were the four copies of the proteinase inhibitor genes on chr9. Homologous protease inhibitor genes in the related potato (Solanum tuberosum) were previously reported to have high expression under cold stress. Also of note are several pyridoxal phosphate phosphatase genes whose homologous genes are known to be induced by phosphate starvation in Arabidopsis. The most strongly downregulated genes include genes related to nitrate transport and a nitrate reductase, and also nodulin-like genes which are believed to be involved in amino acid transport. This, combined with the upregulation of proteinase inhibitor genes, support a shift in N metabolism. A more detailed analysis of the functional annotation of these differentially expressed genes may be helpful to understand how tomato roots respond to this common combined stress in the field.
• To deliver to the scientific community at least 2 pairs of near isogenic lines (NILs) for at least 4 selected QTLs conferring resistance rootstock-mediated resistance to below-ground abiotic stresses.
'Validation of new QTLs and analysis of candidate genes in ‘high ABA’ rootstocks'.
A cluster of eleven new QTL detected on chr9 that influence rootstock performance were investigated using a collection of eleven near-isogenic lines (NILs) designed for validation and mapping of QTL. The NILs were grown in root penetration assays in tubes, in drought experiments in polytunnels, and under low nitrogen conditions and data was collected on growth, xylem sap hormones and gas-exchange.
When used as rootstocks in these experiments, the different NILs often differentially influenced scion phenotypes in statistically significant ways that are dependent on their genotypes. For example, differences due to rootstock NIL genotype gave over 50% difference in shoot dry weights under drought conditions, double the number of penetrating roots, or 25% increase in fruit yield. However, there were no systematic differences between NILs related to their genotypic differences on chr9 that allowed the validation of any QTL in a simple single QTL model. More complex models are required to explain the results.
There are many explanations for the inability to validate QTL, for example: the QTLs were too weak to be detected with the level of replication available; they have too strong a genotype x environmental interaction; they are artefactual due to random variation in the data; they rely on epistatic interactions that are missing in the NIL lines; additional unknown residual heterozygous regions were segregating in NILs that confounded the chr9 effects. Further work is required to understand the genetic basis for the differential effects of the NILs.
Since there were no validated QTL that could be investigated by transcriptomics, the project microarray resources were used to investigate transgenic rootstocks that increased fruit yield under mild salinity conditions. The transgenic rootstocks overexpress a tomato gene encoding a rate-limiting enzyme in abscisic acid biosynthesis and are previously known to exhibit increased accumulation of abscisic acid in xylem sap; the rootstocks were shown to increase fruit yield of grafted scions in conditions of mild salinity stress. Transcriptomic data highlighted a number of classes of genes that are differentially expressed due to the presence of the LeNCED1 transgene; notably several specific aquaporin genes are either up or down regulated, ethylene signalling related genes are upregulated, and many pathogenesis related genes are downregulated. This data set can be used to understand how abscisic acid influences rootstock impact on scion performance and to identify a list of candidate genes involved.
• To generate new genetic, genomic and physiological information, on the basis of the genetic variability of the P-RIL population, regarding the capacity or root colonisation by beneficial rhizosphere microorganisms (AMF and PGPR).
• To understand biological interactions between roots and rhizosphere microorganisms (AMF and PGPR), and to identify at least one root genetic marker for each microorganism and the associated root-signalling mechanisms.
'Rootstock variation in AMF/PGPR dependency and plant growth promotion'.
The results obtained clearly demonstrate a variation in the colonisation performance of the different tomato RILs under in vivo and in vitro culture conditions, as well as a differential effect of the microbial inoculants (AMF Rhizophagus intrarradices or PGPR Variovorax paradoxus) on plant performance, depending on the RIL involved. Genetic analysis determined heritability for root capacity to be colonized by AMF and by PGPR, but no QTL was detected above significant threshold with the linkage map (158 loci) used as background in the project. Since root colonization by AMF appears as a heritable trait, further analysis will be required to locate putative genes involved. A comprehensive metabolomic analysis of signalling molecules involved in AMF colonisation revealed significant differences between contrasting lines. Interestingly, many of the molecules belong to the xenobiotics and tyrosine metabolism. Similarly, transcriptomic analysis revealed important differences in the gene expression profile between contrasting lines used as rootstocks, helping to explain their differential response to drought stress in presence or in absence of the biota AMF and PGPR. Protein abundance and phosphorylation status was analysed for aquaporins PIP1 and PIP2, although no clear relation was found with rootstock-mediated increase in root hydraulic conductivity and plant performance. However, many other differentially expressed genes were related to KEGG pathways involved in several metabolic processes that could be considered as candidate genes underlying the rootstock x biota mediated plant performance under water stress: amino acid biosynthesis, amino sugar and nucleotide metabolism, purine and pyrimidine metabolism, glycerophospholipid metabolism and carbon fixation in photosynthetic organisms, phosphatidylinositol signaling system or phenylpropanoid biosynthesis, among others. The information generated in ROOTOPOWER regarding genetics, signalling molecules and candidate genes involved in plant microbe interactions and resistance could be considered at the cutting edge of this scientific field and could serve to develop more efficient rootstocks with improved capacity to interact with soil biota and to increase plant performance under abiotic stresses.
• To develop a commercial product for abiotic stress alleviation based on AMF/PGPR.
'Validation of AMF production process in crop field conditions and technology transfer on commercial AMF strain'.
The production of mycorrhizal (AMF) inoculum has been optimize and validated at the field scale in ROOTOPOWER. The first production was done from in vitro starter inoculum and then enriched gradually to generate a sufficient volume of concentrated AMF inoculum. Several parameters were tested, such as the ability of propagules to germinate in different substrates (sand or vermiculite) or the influence of the host plant. Finally, a new AMF product based on pure mycorrhizal root powder was developed at one of the specialized SME partner, showing a significantly increase of the mycorrhizal development compared to indigenous AMF strains. The data set obtained under the frame of the project has allowed development of a concentrated mycorrhizal inoculum, able to generate mycorrhiza under greenhouse and field conditions while decreasing the amount of inoculum compared to classic crude inoculum use. Based on the best results obtained all along the ROOTOPOWER project with the R. irregularis MUCL41833 strain, a mass production was set up with a commercial strain of the same species, whose propagule richness was increased by 305 fold under vermiculite substrate (root powder prepared and mixed back in the remaining substrate) compared to initial capacity. In the next years, it is expected that an ultra-rich mycorrhizal inoculum (able to inoculate crop fields with several billions propagules per hectare) will be brought to market, which constitutes a significant step forward regarding the current available offers.
• To evaluate selected and combined genetic plant material and microorganisms in controlled multi-stress environments and real Northern and Southern European cropping conditions.
'Physiological and genetic interactions conferring resistance to combined abiotic stress'.
Although there was no significant effect of biota (B) inoculation on plant growth globally, there was a significant effect of biota treatment depending on the rootstock genetopye (G) used (GxB). Generally, grafted plants onto selected rootstocks performed better than the scion cultivar self-grafted control plants under the combined abiotic stresses applied. Therefore, the selected rootstocks for individual stresses could be used to alleviate the negative impact of the abiotic stress, which normally appears as a combination of two or more stresses. In particular, within the RIL population (Solanum lycopersicum x S. pimpinnelifolium), a couple of lines presented a good performance under all three combined conditions studied when used as rootstocks. Interestingly, other lines derived from a cross between Solanum lycopersicum and S. habrochaites, as some high vigour commercial rootstocks used as reference, exhibits better performance under drought and salinity stress. Similarly, an accession of Solanum lycopersicum var cerasiforme previously selected under individual stress conditions also showed a better performance under combined drought and salinity conditions. Both the near isogenic lines selected for the pre-existing QTL gFW9.1 and the functional lines, related with abscisic acid and cytokinin biosynthesis, behave better in any combined condition with salinity. Grafted plants onto some of the selected lines and NILs used as rootstocks increased nutrient and water use efficiency through maintaining growth and photosynthesis and reducing plant transpiration under combined Drought + Salinity and Low P + Salinity conditions. The higher performance of the selected RILs under individual stresses suggest that the selection process was successful in some cases, but the differences in rootstocks performance among the different experiments suggest an important genotype x environment interaction that requires further attention. However, the global analysis of the selected lines (RILs and non-RILs) suggests that genetic/physiological rootstock selection based on resistance for single stresses is adequate for stress tolerance under combined stress conditions.
• To link the generated genetic, physiological and agronomical knowledge through the modelling of genetic and environmental dependencies of root system architecture.
'A parameterized model of root architecture responses to low N with internal validation has been proposed in ROOTOPOWER'.
Using static root image datasets, a complete analysis pipeline to estimate automatically the main parameters of a dynamic model of root system architecture (RootTyp) for each line of the grafted population under control and Low N has been developed. At this stage, the whole experimental and analysis pipeline allows to reproduce a gross trend across the population and N levels, however, the limited value of the validation suggests that this information should not be trusted to predict details of the root architecture of individual genotypes. It can be anticipated that the quality of the simulations might be dramatically improved with an improved chain of image capture and analysis, which has been recently implemented in the aeroponics platform based on the results of the ROOTOPOWER experiments. Additionally, a non-destructive method for remote evaluation of plant physiological status has been developed. This method has proven to yield heritable indicators that are very sensitive to N-levels, although not linearly related to N-concentration. This method can facilitate the evaluation of plant status in N-limited environments and a non-invasive way, with costs that are essentially fixed.
The QTL analysis of root system architecture traits has been carried out on classical morphological indicators as well as on parameters of the RootTyp model. QTLs were detected at a rather low LOD threshold, indicating weak marker-trait associations. This result was somehow surprising in view of the reasonably large heritabilities and may be the consequence of the image capture and analysis chain. Under Low N, a reasonable good fit regarding the position and direction of allelic effects was found between manual and model-based estimates of root branching and of root system width, and an additional QTL was detected for the model-based estimate of root branching. However, low LOD thresholds have been encountered in several other QTL analyses of root traits in aeroponics, even with genetic material known to be diverse in field conditions, suggesting that the diversity of root architecture might be best expressed in constrained conditions. The results indicate that the genetic determinism of lateral root initiation is largely different at low and high N. This is an important result as lateral roots are known to respond to N supply and plant N status and contribute to most of the absorptive surface of the root system. Surprisingly, the heritability of the response of inter-lateral distance to N supply was negligible, indicating that the genetic determinism of N response is complex. Low LOD scores in aeroponics have been commonly observed in maize, barley, wheat and rice. This suggests that, in the absence of mechanical, gas and nutrient constraints, large collections of genotypes within a species tend to achieve a narrow range of phenotypes. In view of very large QTL effects reported in the literature in soil or other conditions, this would indicate that the scope for root architecture improvement lays more in the root responses to environmental constraints than on constitutive traits. If this was to be the case, the scientific community should probably reconsider much of the actual root architecture phenotyping practices.
• To develop a predictive model of root-mediated plant response to the above described abiotic stresses.
'Another conceptual model of root architectural responses to low P, low K, salinity, drought and mechanical impedance and validation with combined experiments has been approached in ROOTOPOWER'.
For all the individual and combined stresses evaluated in the project, the QTL results have been exploited, in particular (i) the collocation between root weight QTLs, shoot weight QTLs and hormone QTLs and (ii) the conditions in which these QTLs were detected. Based on this information, an integrated conceptual framework has been proposed where the effect of individual stresses on specific root growth and development processes are overlaid. Although several QTLs were detected in each experiment, no single co-location event could be detected across the different experiments. This support the proposition that QTLs for root traits should be seeked for in GxE designs instead of in G designs. This set of data would lead to recommend to address combined stresses in genetic analysis using especially dedicated experimental designs with in depth characterisation of the plant environment and which take into account the non-additive action of QTLs. Hovewer, further experiments will be necessary to validate this model and to evaluate to what extent these stresses operate in an additive manner on each growth/development processes.
'Stability of rootstock- and biotic- effects on fruit yield and quality'.
The proof of concept carried in Northern and Southern European conditions demonstrated that the rootstock genotype was the second important factor affecting plant growth, physiology, yield and quality of indeterminate tomato grown under combined abiotic stresses (Low N availability + Salinity or Low N + Drought). In the determinate tomato, mostly stress but also rootstock genotype affected majority of the parameters analysed in two different European locations. In the industry tomato, all the rootstocks evaluated increased crop yield with respect to the self-grafted scion plants in Spain (Low N + Salinity), while an accession from Solanum lycopersicum cv. cerasiforme, selected for drought tolerance, was the only genotype which performed better than the self-grafted scion in Turkey under Low N + Drought conditions. Moreover, the rootstock has the potential to improve water use efficiency, increasing photosynthesis of a commercially relevant scion under both optimum and combined stress conditions.
Indeed, commercial F1 rootstocks (inter-species hybrids) have an advantage in terms of tomato fruit yield (increased up to 50%) under different environmental stress conditions because of vigour. However, grafting onto some non-hybrid rootstocks also improves tomato fruit yield (up to 30%) under different environmental stress conditions through more specific (non-vigour related) mechanisms. Those rootstock-mediated yield improvements under stress conditions were not in detriment of fruit quality in different growing conditions. Although there is considerable overlapping of best performing rootstock genotypes in the experiments from all locations, the differences observed in rootstock performance between different locations suggest an important genotype x environment interaction that requires further attention. However, some RILs and other experimental lines tested in the ROOTOPOWER are promising genotypes for breeding purposes since they performed as better as the commercial F1 rootstocks across different locations and combined stress conditions.
Although there was no significant effect of biota (AMF + PGPR) inoculation on plant growth generally, there was a significant effect of biota treatment depending on the rootstock used (GxB) and/or stress applied on a few plant parameters, but this effect could be positive or negative. Overall, combined stresses reduced the indeterminate tomato fruit yield about 23% and 33% under with and without biota conditions, respectively.
'Biotic inoculant persistence and effects'.
Without surprise, the plant genotype plays a strong role in microbe–plant interactions. It influences the initial colonization, but also the functioning of the interactions. Data obtained in ROOTOPOWER indicate that cultivars harbour various susceptibilities to biota application, which can result in beneficial, neutral, or even detrimental on plant responses. However, although these observations are regularly reported, physiological determinisms that could elucidate these phenomena in the optic to predict and select positive symbiotic associations (in term of root colonization and/or benefit on plant performances) remains to be unravelled.
In that sense, studying the mycorrhizal behaviour throughout the exploitation of grafting technology is interesting, since it may highlight potential mobile factors and/or genotypic responses and/or metabolic contexts crucial to deepen knowledge in mycorrhizal physiology.
In general, the biota (PGPR and AMF) inoculation generated higher colonization in plant root when plants are inoculated in situ and/or grown in controlled greenhouse conditions (in sandy substrates or perlite) than under crop conditions. Although the various stresses affected more or less severely the non-inoculated plants (growth or quality), their impact on biota (colonization and plant response) did not allow to draw obvious conclusions among trials. The lack of correlation between laboratory, greenhouse and field results translates major complexities to study interactions in plant-microbe systems in various growth conditions, which is not new. In particular, data are often difficult to interpret among trial sites, probably owing of heterogeneity in trial implementation as expected from different geographical locations (plant inoculation time and harvesting, plant/rootstock genotype, culture conditions, cultural itinerary). As there exists multiple factors, often undetermined, which can influence root colonization in the soil, it is therefore difficult to design methods that will predict given characteristics under in vitro conditions. In addition, independently from the way that in planta screening is carried out, the outcome depends also on plant physiological state, known to vary for example by culture age, growing medium or temperature.
Responses of biota on plant under normal conditions were highly variable, according to genotype, graft combination and trials. Under P starvation or drought compared to normal conditions, mycorrhizal plants were less impacted than non-inoculated plants. Under salt stress, or when several stress conditions were combined, plant responses are more mitigated, mostly plant genotype/graft combination specific dependent. Under commercial conditions in which plant were pre-germinated under peat, no significant effect of biota was observed, even when the plants were inoculated in situ (melon and watermelon). This conclusion is consistent with the state of art on inoculation trials under field conditions.
Despite the low colonization detected in some experiments, the capacity of some rootstocks to improve the colonization ability and plant responsiveness to biota leads to interesting perspectives for developing this already well implemented commercial practice. ROOTOPOWER provides therefore some new insights in the rootstock x biota x environment genetic, physiological and agronomical interactions, opening challenging opportunities to deepen the understanding of plant-biota interactions and to master the potential benefits of biotic interactions in various culture and environmental conditions.
'Approach validation in other species'.
For most crops evaluated (tomato, eggplant, pepper, melon, watermelon, cucumber) grafting onto commercial rootstocks improved growth and yield-related parameters under combined abiotic stress. Biota treatment had a positive effect in some parameters evaluated in the grafted plants, but its effect was more evident in interaction with the stress treatment. Therefore, the results obtained demonstrate that grafting onto specific rootstocks improved plant performance and yield in other Solanaceae and Cucurbitaceae crops under abiotic stress, thus validating the approach used for tomato as a model crop species.
• To understand the genetic controls of different types of root-to-shoot signalling (hydraulic, ionomic and hormonal) that positively affect shoot performance under the above described abiotic stress conditions.
• To build and deliver a xylem profiling model (hormones + nutrients) that will provide physiological markers to assist root-targeted breeding for resistance to individual and combined stresses.
'Root signalling and soil biophysical properties'.
A huge amount of xylomic data including iononome and hormonome have been collected in 144 different rootstocks under 6 individual stress conditions and their respective controls and in eight different phenotyping platforms in five European countries. This unique set of information is being processed and it is expected that the elucidation of the specific physiological and genetic components of the rootstock-mediated effects on plant performance under those stressful conditions will be completed in a near future. The main results obtained so far can be summarized as follow:
Potassium (K) deficiency: Across all graft combinations, xylem K concentration of K-deprived plants was significantly lower than control plants, as expected. However, xylem Ca concentration of K-deprived plants was also decreased by a similar magnitude. Interestingly, there were also three micronutrients (B, Mn and Zn) that showed significant increases in K-deprived plants, compared to control plants. Potassium deprivation significantly decreased the xylem concentrations of multiple phytohormones and their precursors: abscisic acid, ethylene, gibberellins, auxin and cytokinins. Interestingly, potassium deprivation significantly decreased the bioactive cytokinin free-base zeatin concentration and increased its riboside form. Although there was no rootstock-mediated variation in xylem phytohormone concentrations under normal K conditions, both ethylene precursor ACC and trans-zeatin showed significant rootstock-mediated variation under K-deprivation. Within the K-deprived treatment, a principal component analysis showed that rootstock-mediated variation in plant response was not well correlated with either ionic or phytohormonal composition of the xylem sap. However, those hormonal and ionomic changes are helping to explain the behavior of contrasting rootstocks under normal and low-K nutrition.
Nitrogen (N) deficiency: Only K showed a significant decrease in xylem concentration under N deprivation. The remaining ions all showed significantly increased concentrations in N-deprived plants. The similar responses of many different ions suggest either a co-ordinated response to N deprivation, or that decreases in xylem sap flow rate (caused by N deprivation) concentrated the ions within the xylem sap. Under N-deprivation, there was significant rootstock-mediated variation in xylem B, Cu, Na and Zn concentrations. Nitrogen deprivation significantly increased xylem concentrations of abscisic, jasmonic and salicylic acids, and trans-zeatin riboside. In contrast, N deprivation almost halved xylem trans-zeatin concentration. Across all plants, the rootstock significantly altered xylem abscisic, jasmonic and salicylic acid concentrations under Low-N conditions.
Phosphorous (P) deficiency: Across all graft combinations, xylem P concentration of P-deprived plants was significantly lower (by 45%) than control plants, as expected. Xylem K concentration of P-deprived plants was decreased by 10%. In contrast, P deprivation significantly increased xylem concentrations of Ca, Mn and Mg. Interestingly, phosphorous deprivation significantly increased xylem concentrations of all gibberellins detected and free-base cytokinins. In contrast, P deprivation significantly decreased xylem the ethylene precursor ACC and jasmonic acid concentration. The rootstock significantly altered xylem jasmonic and salicylic acids, and trans-zeatin concentrations.
Soil drying: Compared to well-watered plants, soil drying significantly increased xylem concentrations of Fe, Mg, S and Zn. Across all plants, all ions analysed (B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Zn) showed significant rootstock effects. Xylem concentrations of many phytohormones were significantly altered by soil drying, with increases in gibberellin GA4 and jasmonic acid. In contrast, xylem concentrations of ethylene precursor ACC, gibberellin GA1, and cytokinins decreased. Surprisingly, soil drying did not significantly increase xylem ABA concentration in the tested conditions. Across all plants, there were significant rootstock-mediated effects on almost all the hormones analyzed in the xylem.
Mechanical impedance: Compared to other sites/stresses, xylem macronutrient (Ca, K, Mg, P and S) concentrations were higher in plants exposed to mechanical impedance. Only xylem Na concentration showed significant rootstock-mediated effects, while no significant rootstock-mediated effects were detected on xylem hormone concentrations.
Salinity: There was significant rootstock-mediated variation in xylem sap Fe and S concentrations. Of the phytohormones measured, only xylem the ethylene precursor ACC concentration showed significant rootstock mediated variation. Principal component analysis showed that shoot fresh weight was clustered with some xylem phytohormone concentrations (abscisic acid, ethylene precursor, gibberellins and auxin), while xylem trans-zeatin and Na concentrations were opposed to vegetative vigour.
In conclusion, significant rootstock-mediated variation in xylem sap composition (both ions and phytohormones) was detected in response to a range of different below-ground abiotic stresses, and in some cases rootstock-mediated effects on xylem sap composition were correlated with rootstock vigour. However, a more comprehensive and in deep analysis to elucidate the signaling components of plant response and adaption to different stresses in ongoing.
'Plant microbe interactions: root exudation and plant hormone status'.
Biota inoculation (AMF Rhizophagus irregularis + PGPR Variovorax paradoxus) sometimes had important effects on xylem macronutrient concentrations (eg. K, Mg, P). Especially consistent was an increase in xylem Mn concentration of between 15 and 30%, which was detected at 4 site/treatment combinations. In relation to the hormonal factors, trans-zeatin and salicylic acid were especially affected by biota inoculation under combined drought and salt stress. Principal component analysis revealed that most mineral nutrients varied in opposite direction to the shoot growth under combined Low P and salinity, while trans-zeatin and jasmonic and salicylic acids, and, especially, abscisic acid were the most important factors explaining the variability of shoot fresh weight in the population .
'Predicting signalling responses to combined abiotic stresses'.
Large differences in absolute hormone concentrations between experiments testing individual and combined stresses caused considerable inaccuracies in the modelled data. Thus predicting signalling (xylem sap composition) in response to combined abiotic stress remains problematic, and undertaking single and multiple abiotic stress experiments (eg. control, salt, Low P deprivation, salt and Low P combination) at the same time in the same experimental material seems necessary to make progress in this area, such as it has been done for drought and salt stress, where xylem ABA concentration in response to combined was entirely explained on the basis of its responses to individual drought stress during vegetative growth.
'Field variation in rootstock dependent root signaling'.
Frequency of successful root xylem sap collection varied substantially between species (and was especially low when relying on spontaneous exudation, rather than pressure-induced flow). Although collecting leaf xylem sap provided a viable alternative to guarantee sample availability, in this case xylem phytohormone concentrations in leaf xylem sap were poorly correlated with root xylem phytohormone concentrations, likely since root xylem sap was entirely apoplastic while leaf xylem sap will comprise both apoplastic and symplastic sources.
Biota inoculation generally had limited effects on root xylem hormone concentrations, significant effects only occurring within some species grown at some sites for certain phytohormones. Although a suitable growing substrate was optimized for biota colonization, poor colonisation of the root system by the inoculants in the field may have been responsible for this effect, especially since previous studies have shown that the PGPR can alter xylem abscisic acid and ethylene precursor ACC concentrations. Nevertheless, inoculated pepper plants showed a decreased xylem ACC concentration in response to Low N + salinity combined stress.
The stress treatment did not only clearly alter xylem concentrations of the traditional “stress hormones” ABA and ACC, but also of other phytohormones. However, the direction of change depended on the species/site combination. For indeterminate tomato, grown both in Southern and Northern European areas, root xylem ACC concentrations showed a consistent increase under stress conditions.
There were clear rootstock-mediated differences in root xylem phytohormone concentrations in determinate and indeterminate tomato when grown under semi-commercial conditions. These responses were not always consistent across sites and species, suggesting strong rootstock x environment interactions. Importantly, xylem ACC concentrations increased under Low N + salinity combine stress while trans-zeatin concentrations decreased. Furthermore, when consistent rootstock-mediated differences were detected, often it was the commercial rootstocks that showed lower concentrations of certain phytohormones (tras-zeatin, salicylic acid, and especially ethylene precursor ACC) under both optimal and stress conditions. Interestingly, self-grafted control plants showed amongst the lowest ACC concentrations under optimal conditions, while the highest under stress conditions. It is tempting to speculate that the commercial success of these widely adapted rootstocks is due to altered phytohormone profiles in the plant across different environments.
Taken together, all 3 factors controlled in these studies (rootstock, biota and stress) altered xylem phytohormone concentrations in plants grown under semi-commercial conditions. Of these, xylem ACC concentration (hitherto primarily considered as a signal of flooded soil) seems to be an important root-derived signal implicated in the response of the grafted plants to combined stress conditions.
In addition to hormonal components, by screening microarray data obtained from different plant species and under different stresses, a number of conserved stress-responsive genes whose expression was differentially regulated in tomato roots in response to one or several stresses have been identified. Ten of these genes have been validated as reliable biomarkers whose expression levels are related to different signalling pathways involved in adaptive stress responses. The genes identified could be used could be used to evaluate, at the molecular level, the stress responses of tomato cultivars that differ in stress tolerance. Due to conservation of genes and their downstream responses, they could also be used to evaluate the stress responses in different species where full transcriptomic information is not yet available.
Potential Impact:
According to the expected impact listed in the work programme, ROOTOPOWER can potentially contribute in the following ways:
“Knowledge and tools generated will help to (i) better understand root development and below-ground interactions under abiotic stress in field conditions”
ROOTOPOWER has generated valuable information and tools on how the different individual abiotic stresses (salinity, drought, soil compaction and nutrient –N,P,K- deficiencies) affect root and shoot development by analysing root system architecture, biomass and soil distribution by using different phenotyping platforms. A reliable method for remote evaluation of plant physiological status has been developed. This methods yields heritable indicators and is very sensitive to N-levels, although not linearly related to N-concentration. This method can facilitate the evaluation of plant status in N-limited environments in a non-invasive and low-cost way, and could be extended to other suboptimal environments.
The project has produced a huge amount of physiological information about how the rootstocks and/or the association with AMF and PGPR affect shoot performance under individual and combined stresses in terms of resource capture (complete ionome) and gas-exchange.
ROOTOPOWER has generated a huge amount of physiological information about how hormonal signalling is involved in (i) root-perception of stress, (ii) root-interactions with rhizosphere microorganisms; and (iii) root-to-shoot communication and its influence of shoot physiology (growth, water use, carbon assimilation, leaf senescence and fruit yield).
By analysing contrasting and functional lines, ROOTOPOWER has produced new information about how (i) stress, (ii) soil microorganisms and (iii) shoot genotype influence root development in tomato through analysing the candidate genes involved in auxin transport, cytokinin sensitivity and ABA biosynthesis, and other hormonal-related processes.
ROOTOPOWER has identified a list of QTLs and chromosomic regions as indicators of resistance to the above mentioned individual abiotic stresses. A set of QTLs have been assayed for validation under individual and multiple combined stresses in field conditions. A set of near isogenic lines have been generated for a particularly rich chromosomic region harbouring important QTLs, which have been used to generate new transcriptomic information about candidate genes involved in rootstock-mediated stress tolerance. Those lines are now available to the scientific community.
ROOTOPOWER has generated a predictive conceptual model to integrate genetic, physiological and agronomical data that will serve as a basis for future modelling studies. More specifically, a tomato version of the RootTyp model has been obtained and implemented a whole image analysis pipeline that is able to predict the main model parameters from images captured when plants are 3-week old (after transplantation). This model allows simulating realistic root systems in 4D (spatial and temporal dynamics) and can be used to different purposes. It can be used by breeders to test in silico the value of novel genotypes, considering different aspects of resource capture and soil profile exploration. It can also be used as a communication tool to illustrate the dynamic development of tomato root systems of different lines (including grafted and non-grafted plants). Using QTL data, a conceptual model of root architecture response to individual stresses have been designed. This model will be useful to design further plant physiology research on root responses to various abiotic stresses. Work is ongoing to extend this model to three scenarios combining two multiple stress conditions.
Upon a comprehensive analysis of the data, ROOTOPOWER will contribute to improve plant resistance to fluctuating environmental stress, by providing quantitative data useful to develop new mechanistic strategies of plants adaptation to their environment. The project will identify interactions among dominant physiological functions involved in the plant response submitted to several combined stress factors and has established some quantitative stress dose versus plant response relationships.
ROOTOPOWER results provide tools and knowledge to anticipate different stress combinations (ie. salinity and Low N, salinity and low P, drought and low P, drought and low N, salinity and drought) and thus contributing to adapt agriculture to specific time- and space- specific environmental conditions. The information generated will also serve to support the design of ideotypes, i.e. rootstock genotypes best adapted to a given set of environmental conditions while maintaining or optimizing yield and quality traits.
ROOTOPOWER will contribute to fill the gap in the understanding of plant responses to some combinations of abiotic stress factors. In particular ROOTOPOWER has identified regulatory networks at the gene and at the process levels in tomato which will help elucidate complex interactions in the plant response to combined stress factors, particularly drought and low P.
The information delivered in ROOTOPOWER will contribute to increase stress resistance of other species. Indeed the QTLs and key-candidate genes that will be identified underlying below-ground abiotic stress resistance are likely conserved within tomato, other Solanaceae and other crops. These traits can be expected to be found in other dicotyledonous crop species independent of their grafting susceptibility. Therefore ROOTOPOWER will provide the scientific community with new perspectives for knowledge transfer from the model and crop plant tomato to other crop species.
“Knowledge and tools generated will help to (ii) support the development of root-targeted breeding strategies to generate varieties with increased resource use efficiency and resistance to abiotic stresses”
ROOTOPOWER is releasing genetic markers for root-targeted traits under six different abiotic stress conditions and some of their natural co-occurring combinations, which will be used in marker assisted selection. Thus, ROOTOPOWER will help overcome current limitations of breeding strategies for increasing crop plant resistance to environmental challenges, by identifying QTLs and providing a list of candidate genes for breeding strategies to restrict negative side effects on growth and yield.
More specifically, for the S. lycopersicum var cerasiforme L5 x S. pimpinellifolium E9 recombinant inbred population that was used to report rootstock mediated QTL, the majority of SNP and InDel polymorphisms present have been identified by genomic resequencing. This will allow polymorphic candidate genes to be identified in the region of any finely mapped QTL from this population. The possible candidate genes would allow marker assisted selection of novel rootstock genotypes.
Near isogenic lines have been produced from the ancestors of the P-RILs representing the L5 and E9 DNA on chromosome 9. Different lines when used as rootstocks showed contrasting root growth and also scion growth under drought conditions. This forms the basis of further genetic analysis, and such rootstocks might be useful in their own right.
Moreover, tomato rootstocks overexpressing LeNCED1 gene (key enzyme for abscisic acid biosynthesis) improved the yield of fruit produced from the grafted scions when the plants were grown under mild salinity. Such rootstocks or other developed by approaching similar traits could improve tomato production in salt-affected areas (e.g. in Mediterranean region).
ROOTOPOWER results will contribute to develop new root-targeted breeding strategies based on the signalling processes existing between soil microorganisms, roots and shoots leading to increased resource acquisition and resource use efficiency and resistance to multiple and combined abiotic stresses which naturally co-occur.
ROOTOPOWER is generating genetic and biochemical markers (QTLs, candidate genes and signalling molecules) for root capacity to associate AMF and PGPR in search of synergistic plant-biota interactions to increase resource acquisition and resistance to abiotic stresses. Three sets of transcriptomic data were produced from tomato rootstocks and candidate genes were identified that might be responsible for QTL described, or be involved in rootstock responses to multistress conditions and colonization with AMF/PGPRs. This provides a resource to help in understanding the genetic basis of rootstock function which could eventually help find markers for marker assisted breeding.
ROOTOPOWER has identified recombinant inbred lines (RIL) and other genetic materials publically available that when used as roostocks provide contrasting degrees of resistance (assayed as shoot performance) to several abiotic stresses (moderate salinity, moderate water deficit and moderate soil impedance, decreased fertiliser input: N, P, K) both individually and in some real-world combinations in the field. ROOTOPOWER has also identified RILs with contrasting capacity to associate with AMF and PGPR.
More specifically, inbred tomato rootstocks improve vegetative growth and nutrient use efficiency under single (between 11% and 67% for the self-grafted variety) and combined (up to 100% for the self-grafted variety) abiotic stress conditions (salinity/drought and NPK nutrient deficiencies). While commercial F1 rootstocks (inter-specific hybrids) have an advantage in terms of tomato fruit yield (increased up to 50%) under different environmental stress conditions because of vigour, grafting onto some non-hybrid rootstocks evaluated in ROOTOPOWER also improves tomato fruit yield (up to 30%) under different environmental stress conditions through more specific (non-vigour related) mechanisms.
ROOTOPOWER will help breeders to interpret complex below-ground abiotic stress interactions and provide them with relevant physiological indicators to evaluate plant response to different stress combinations, discerning between vigour and non-vigour related mechanisms. This will build capacity to screen genetic diversity, and complemented existing approaches based more on phenotypic traits (vigour) than on underlying physiology and genetic (specific mechanisms).
Similarly, ROOTOPOWER will support the development of sustainable innovative production strategies by unravelling the synergistic or antagonistic actions of combined stresses and their dependency on different stress factors and some natural stress combinations. These data will allow the development and optimisation of cultural practices to predict whether resistance to a given stress factor can be extrapolated or not to another factor (ie. drought vs salinity).
ROOTOPOWER has the potential to rapidly benefit breeders since interesting lines have been selected under both controlled and real field conditions. Use of rootstocks carrying promising characteristics for abiotic stress resistance allows their almost- immediate exploitation with existing elite scion material, to increase resource use efficiency and enhance resistance to abiotic stresses. This represents a considerable acceleration in developing and adapting new breeding strategies to increase the resistance and yields of crop species in the future climatic context, compared with non-graftable species (such as cereals) where the time between selection and introduction to existing elite material may be 10 years or more.
The combination of approaches developed by ROOTOPOWER provides the possibility of translating results:
• From species like tomato via the modelling tools developed to other species with are amenable to grafting and thus to the use of rootstocks. This will allow taking advantage of genomic knowledge that is being continuously generated in tomato and other species like eggplant, pepper, watermelon, cucumber and melon.
• From phenotyping platforms to the field. The phenotyping methods developed both in the field and in platforms (through extending laboratory experiments to the field, and through directly testing these physiological hypotheses in the field) provide the possibility of increasing accuracy in future characterisation of the genotype performances in the field.
• From a few field experiments to a large range of stress scenarios in the field within the diversity of EU climates, and extended to forecasted climatic scenarios.
“Overall, results obtained will contribute to sustaining yields under more adverse climatic conditions”
• By providing knowledge to elucidate complex interactions in the plant response to different below-ground stress factors, and by setting new breeding strategies for improving crop resistance to below-ground abiotic stress combinations, ROOTOPOWER results will contribute to adapt European and world agriculture to forecasted climatic challenges and adverse environmental factors, of which drought, salinity and low nutrient availability represent the most severe constraint to agriculture
• Global warming is also predicted to affect most severely developing countries, where agricultural systems are most vulnerable to climatic conditions. Additionally, at global level, soil degradation and abandonment by factors as salinisation is equivalent every year to the total irrigated area. The development of crop varieties with increased resistance to below-ground abiotic stress factors is highly necessary in developing world countries which face nutrient shortages that limit productivity.
• Farmers have a major interest in extending the productive cropping period during the year and to recover and maintain the soil as a natural substrate. It is essential to help crop producers and breeders to face climate change and the forecasted higher intensity and frequency of extreme climatic events in a substantial part of Europe, including increased salinity, drought and other below-ground abiotic stresses. ROOTOPOWER results can certainly contribute to this challenge by improving our understanding of the root-targeted breeding and biotechnology -a field yet relatively unexplored.
• Establishing an association with non-profit organisations such as The World Vegetable Center (AVRDC) would allow promising rootstocks to be disseminated to some of the world’s poorest countries, since tomato and other graftable vegetables can be important to subsistence farmers (especially as cash crops). Some joint initiatives between some ROOTOPOWER partners AVRDC and are currently in process.
“Project results should clearly be of interest and potential benefit to SMEs”
• SME’s participating in ROOTOPOWER can benefit from yield-enhancing and environmentally friendly tomato rootstocks and from innovation in breeding programmes to ensure the competitiveness, sustainability and independence of European agriculture. Varieties released and used in modern agriculture have been developed over hundreds of years by farmers and plant breeders. Unfortunately, for main crops, the relative yield increase has been declining during the second half of the 20th century in most producing countries, including Europe. Thus, there is a crucial need to develop a sustainable agricultural production system by increasing and stabilizing the production with cultivars adapted to climate challenges, which require less input, and, at the same time, provide enhanced nutritional composition for consumers’ health and well-being and deliver consistent output traits to processors.
• The rootstocks and QTLs selected and identified in the project for their involvement in the resistance different abiotic stresses will be available for breeders upon its adequate publication. A whole population of RILs, used as rootstocks, has been screened, new alleles have been identified and negative side effects on agronomical traits have been checked during the project; these QTLs and genes can be introduced more rapidly and earlier in breeding programs for the development of new tomato cultivars and rootstocks that are more resistant to abiotic stresses.
• To test the resistance/resistance of plants to abiotic stresses (which are often complex traits,) breeders usually need large trials in controlled environments Moreover they usually test one environmental factor at a time to limit the trial size, and breeding for resistance to abiotic stress factors has been only recently initiated. The identification of single genes playing key roles in stress resistance to abiotic factors, will greatly contribute to breeding effectiveness since are more easily manipulated in breeding programs. Breeders can apply Marker Assisted Selection to significantly shorten the breeding process.
• Moreover lines selected by tilling in cultivated populations could be directly used in breeding programs. With knowledge gained through ROOTOPOWER, it is expected that SMEs could bring to the market new rootstocks within 5 to 8 years which is about half the time required with more traditional breeding strategies. These new rootstocks will present a broad spectrum of resistance to biotic and abiotic stress factors and thus will be adapted to contrasted climatic regions. Thus ROOTOPOWER will impact on the efficiency of breeding programs, and promote the creation of new tomato rootstocks and varieties with high yield and good fruit/grain quality, adapted to long cultivation in different parts of Europe.
• The project will support the development of new rootstocks of tomato adapted to multiple stress environments which is expected to have large economic impacts for producers in Europe (mostly SMEs). Tomato is the most important vegetable production in the world (129 millions of tons), the most consumed vegetable in Europe (after potato) and an important source of healthy compounds such as antioxidants. By increasing the plant resistance to abiotic stresses, ROOTOPOWER will contribute to decrease the dependence on chemical fertilisers, water resources and technological solutions (eg desalinisation plants; alternative substrates, climatic control, etc.) to face climatic challenges. Thus, the project will have a major quantitative impact in terms of intermediate inputs use and consequently a qualitative impact on food safety and environment preservation. It will therefore contribute to increase the sustainability of agriculture in Europe, a highly SME-intensive sector.
• The involvement of a SME in quality control of inoculant production has provided a unique opportunity for this SME to appraise the value of different biota (one of which, Variovorax paradoxus, has yet to be used commercially) to increase resistance to abiotic stress. Positive results have contributed to commercial development of single or combined inoculants that could be available for the European market in a near future.
• In intensive agricultural systems, biotechnological innovations (especially new inbred varieties) are a seminal factor of the production system that influences three basic dimensions:
(i) production structure by defining its integrating productive cycles, processes, methods and practises, including work organisation.
(ii) input consumption (water, fertilisers, etc) that has a strong effect on regulating plant production.
(iii) the technology matrix that integrates different types of engineering and biological technologies.
• One of the major problems of these technologies is their high development costs and the high degree of exclusivity and control by the multinational companies. Farmers (as users of these technologies) have relatively little input into their development. In the EU-27, about 90% of the farms comprise less than 20 ha and 95% less than 50 ha. Consequently, public support of the technological development of those companies is necessary to maintain agricultural competitiveness and sustainability.
• The overall impact of ROOTOPOWER deliverables is especially relevant to SMEs involved in the European horticultural sector. Micro, small and medium-sized enterprises (SMEs) with a high research focus are the economic powerhouse behind scientific and technological developments in agriculture, playing a pivotal role in the success of a European Knowledge-Based Bio-Economy. Although specialised horticulture accounts on average for about 8% of the total agricultural production in Europe, this percentage increases to 13% in Southern European countries such as Spain, Portugal and Italy, and to 40% in highly specialised regions such as Murcia, Andalucía and The Netherlands. The most recent available data in Eurostat does not include Agriculture, but most of the companies in this sector are SMEs and account for the largest employment generation (eg. 36% of the sector in Holland, year 2005).
• The inclusion within ROOTOPOWER of several SMEs with specialised skills (biotic inoculants, rootstock breeding and tomato growers) (i) ensures that the knowledge generated within this proposal is suitable for application in agricultural systems that the SMEs have extensive knowledge of, thus decreasing the time from scientific innovation to general application, and (ii) contributes to their strategic development into the economic sector.
‘’In relation to natural resource (water and nutrients) management’’
While natural resource management (water and nutrients) is a global issue, the results of the ROOTOPOWER project can have a greater impact in Mediterranean countries, where degradation of soil and water resources are major constraints to economic growth, lowering standards of living among rural Mediterranean inhabitants. About 70% of water resources are dedicated to irrigation in Mediterranean Countries, implying that any rootstock-mediated increase in crop water use efficiency will save a significant amount of water. This would enhance compliance with the EU water policy, especially in areas with high level of water stress on the aquifers, where it is compulsory to reduce and eliminate the gap between uptake and recharge. Development of more salt-tolerant rootstocks will allow aquifers that are currently unused (due to their high salt loads) to become a viable resource (thus decreasing pressure on those with high quality water in existing, likely unsustainable use). Changes in water use patterns could potentially make additional water available for other agricultural, industrial and domestic uses.
The transfer of nitrogen (N) and phosphorus (P) from land to water (termed “diffuse pollution”) presents a problem for most agricultural systems, with resulting eutrophic downstream impacts on rivers, lakes and even estuaries. Key processes involved include the accumulation of nutrient sources in the soil-crop system, mobilisation through solubilisation and subsequent leaching and runoff, mobilisation through physical (particle/colloid) detachment and subsequent runoff, mobilisation through incidental transfers of recently applied fertilizer (that is not yet equilibrated with the soil-plant system) and finally issues of subsequent transport through the landscape, with the combination of these processes across a scale known as the ‘transfer continuum’. Recent work has estimated the potential impact of climate change on mobilisation of diffuse nutrients as a result of changes in temperature. Phosphorus emissions from agriculture must be restricted if we are to address potentially catastrophic future P-limitations to agriculture. Climate change predictions of wetter winters and drier summers in northern Europe may increase soil water storage (and runoff or drainage below the root system) during the winter months, but cause crop water stress during summer. Crop rooting patterns (deep roots) buffered the impact of rainfall amount on crop yield. The discovery of rootstocks with increase nutrient use efficiency through a more adequate root system morphology and/or nutrient uptake capacity within the project may provide a way of mitigating diffuse pollution effects.
According to the Strategic Research Agenda produced in 2007 by the European Technology Platform 'Plants for the Future' which identified five challenges for Europe’s society and economy to which the plant sector generally can contribute, the ROOTOPOWER project results could potentially impact the following aspects:
Healthy, safe and sufficient food and feed: Agriculture faces new challenges such as climate change and society’s expectations of reducing the environmental footprint of agriculture, while at the same time providing sufficient food to a burgeoning world population. ROOTOPOWER has embraced these aspirations and can make a telling contribution to crop resource efficiency by identifying rootstocks and root-specific traits contributing to “produce more with less” thus allowing the sustainable intensification of agriculture.
Sustainable agriculture, forestry and landscape: the identification of genes conferring resource use efficiency within ROOTOPOWER represents a key output to enhance sustainability: both by diminishing nutrient losses from agriculture and fertiliser inputs (which are expensive to produce in the case of N, and increasingly scarce in the case of P and K).
‘’Translating the scientific discoveries to market ‘’
Agricultural productivity must increase by 60% to feed the expected population of 9.6 billion people in 2050, while climate change may also reduce crop productivity. Pressure for cheaper, high quality and enhanced nutrient content tomato and other horticultural crops is increasing worldwide. Moreover, EU farmers must manage expensive costs and environmental restrictions unlike producers in emerging countries. The only way to produce high quality products while minimising economic costs is to produce intensive and resource-efficient high-yielding crops. Plant breeding and the use of horticultural techniques including grafting and the use of microbial inoculants will play a key role in developing more profitable horticulture to address these challenges.
The use of rootstocks has enhanced productivity of woody perennial crops for centuries. Grafting of vegetable crops has developed very quickly in the last 50 years, mainly to induce shoot vigor and to overcome soil-borne diseases in susceptible crops. Rootstocks can optimize vegetable crop productivity by (i) increasing the yield potential; (ii) maintaining yield in the face of biotic (ie. fungi, bacteria, virus, nematodes) and abiotic (ie. salinity, drought, low nutrients, extreme temperatures, soil compaction) stresses; (iii) decreasing the use of environmentally damaging pesticides and fertilizers; and (iv) increasing the efficiency of use of natural resources of water and soil. The socioeconomic and environmental potential impact of grafting is enormous. However, both the development of new rootstocks and new applications in agriculture are hampered by limited knowledge of the physiological and genetic mechanisms underlying rootstock x scion (shoot) x environment interactions.
Overall, the results of ROOTOPOWER show that use of novel rootstocks can improve tomato yield under stress conditions. This paves the way for their use in plant breeding programs. Furthermore, the new genes that we identified could be markers for breeding in related species of Solanaceae (pepper and eggplant) and Cucurbitaceae (cucumber, melon and watermelon) botanical families.
Main dissemination activities and exploitation of results
The dissemination and exploitation of project results was addressed in WP7 “Dissemination and IPR”, whose general objectives were to make the largest possible use of project outcomes and maximize the benefits of advances in other initiatives.
In accordance with the strategy defined in Annex I, the dissemination of knowledge deriving from ROOTOPOWER has been made through the following activities:
• Participation in scientific events in which ROOTOPOWER partners have had the opportunity to present information and results on the project, for instance, the VI SEST-ISHS Symposium, the XVIII FESPB-EPSO Plant Biology Congress, the Environment Workshop 2013, the 10th Solanaceae Conference, the 1st ISHS International Symposium on Vegetable Grafting, the XVIIIth EUCARPIA Meeting on Genetics and Breeding of Tomato, 11th Solanaceae Conference (SOL2014), the SEB Annual Meeting in Manchester (SEB2014) and Prague (SEB2015), the 7th International Symposium on Root Development (2014), the 12th Solanaceae Conference (SOL2015), and the Plant and Animal Genome XXIV Conference (PAG2016), among other relevant conferences.
• Publication of scientific results in peer-reviewed journals and books: 8 articles have been published so far in Acta Horticulturae, G3-Genes Genomes Genetics, Theoretical and Applied Genetics, Journal of Experimental Botany, Functional Plant Biology and Molecular Breeding, while some others are under preparation. Given the high amount of data and the need to arrange it so as to draw up sound publications, most publications will be ready during the course of 2016.
• Key activities targeted to the general public has led to a better understanding of root-targeted strategies to minimize abiotic stress impacts on horticultural crops, and to increase the education of the general public and schoolchildren on the field. Activities specifically focused on the general public include publications in the general press, a TV interview, handing out brochures, publication of general information in the Twitter account of the project and participation in science fairs. As per schoolchildren, ROOTOPOWER took part in the XII Week of Science (Murcia, Spain) and the London International Youth Science Forum (Cranfield, UK). Furthermore, an educational workshop entitled “Vegetables do not grow in the fridge: scientists for one day” was organised for students attending a primary school in Murcia (Spain). Specific tailored material was prepared for this specific workshop.
• Website: the project website consists of a public and a restricted private area. The publicly accessible pages have provided information on this project throughout its lifetime. For instance, a calendar of conferences in which ROOTOPOWER participation was foreseen, a list of media activities, general project brochures and a summary of communications presented during scientific events were made available in the public area of the webpage. The private area was restricted to project partners in order to share and disseminate research related knowledge and data. The website has supported a close co-operation between the Consortium partners and has been useful for raising public awareness.
• Preparation of a project newsletter targeted at the academic and scientific community. This newsletter has been issued once per year and has been duly disseminated through the project webpage and other potential stakeholders interested in the progress of the project (e.g. ISHS, EPSO, etc.).
• Organization of scientific workshops: two international workshops have been organized with invited speakers and lectures specifically oriented to the academic and scientific community, namely the 1st Rootopower workshop entitled “Phenotyping, Physiology and Genetics of Rootstock-Mediated Tolerance to Soil Abiotic Stresses in Tomato” and held within the framework of the I COST Action FA1204 Annual Conference (Murcia, Spain) and the 2nd Rootopower workshop under the title “Understanding the Power of Root Traits for Producing More with Less”, jointly organized with the XXIX International Horticultural Congress (Brisbane, Australia). In order to ensure economies of scale and provide value for money, they were organized within already scheduled high-level international events in collaboration with major international organisations.
• Organization of technical workshops: a series of local workshops have been organized in different countries of participating partners, namely Spain, Turkey and the Netherlands. The goal of these informative workshops was to disseminate project results in its technological dimension and they were aimed towards industry stakeholders. They were mainly conducted during the last quarter of the project implementation in collaboration with national farmer, consumer organisations and governmental institutions. In other countries (Belgium and United Kingdom), contact has been constantly maintained with companies that may be interested in the project results and their potential application.
Two aspects worth highlighting are: 1) following Article 45 RfP – Article II.30.4 of the GA, the statement of financial support from the CE has been included in the dissemination material generated within the framework of the project, and 2) all publications, press releases, and other outward communications from the project have been sanctioned by the IP Committee of the project to avoid difficulties in the protection of potential foreground production.
List of Websites:
http://www.rootopower.eu/
Dr. Francisco Pérez-Alfocea (coordinator)
CEBAS-CSIC
Campus Universitario de Espinardo, s/n. Aptdo. 164
30100 - Murcia
SPAIN
'A paradigm shift to root system engineering'.
After more than fifty years of crop improvement principally selecting for above ground traits, scientists now perceive root system engineering as an opportunity to integrate new approaches to maintain sustainable crop production under changing environmental conditions while minimizing the demand for new resources. Root-specific traits such as root system architecture, sensing of edaphic stress and root-to-shoot communication can be exploited to improve resource capture and plant development under adverse conditions.
'ROOTOPOWER: understanding the power of root traits'.
ROOTOPOWER aims to develop a multidisciplinary suite of new tools targeted to the root system to enhance agronomical stability and sustainability of dicotyledonous crops under multiple and combined stress conditions. Central to our approach is the use of tomato as a model species since it can be very easily grafted, (and indeed is usually grafted in commercial protected cropping). This surgical technique attaches genetically different shoot and root systems, allowing precise assessment of the effect of altering root traits on crop performance independently of shoot traits, since the scion (shoot) is constant. Many physiological studies have employed grafting between different genotypes of dicotyledonous species (or even between species) to improve our understanding of root-to-shoot signalling or to alter shoot phenotype. This project has been analyzing and exploiting the natural genetic variability existing in wild-relative tomato species (used as rootstocks) and their beneficial interactions with natural soil microorganisms (arbuscular mycorrhizal fungi AMF, and plant growth promoting rhizobacteria, PGPR). This project has obtained genetic information and physiological understanding of mechanisms vital for high-performing root systems. The key research challenges approached by ROOTOPOWER have been therefore:
• To identify stress-resistant root systems and rhizosphere microorganisms (and their synergisms) for enhanced resistance to individual and combined abiotic stresses.
• To understand the underlying genetic and physiological mechanisms, which are potentially fundamental to all crops, and readily exploited in dicotyledonous crops.
This project has firstly identified genetic variation and quantitative trait loci (QTL) that allow tomato roots to confer crop resistance to a range of abiotic stresses, alone or in association with arbuscular mycorrhizal fungi (AMF) and/or plant growth promoting rhizobacteria (PGPR). This approach is establishing the physiological mechanisms and signalling processes conferred by key QTLs, and identifying candidates for the causative genes by obtaining and evaluating near isogenic lines (NILs) for selected QTLs. Primarily, ROOTOPOWER evaluated rootstocks of a unique recombinant inbred line population (RIL) from a cross between Solanum lycopersicum var. cerasiforme and S. pimpinellifolium for their performance under multiple abiotic stresses and for their interaction with AMF and PGPR. This project conducted detailed analysis of the underlying rootstock-derived molecular, physiological and morphological mechanisms that influence plant growth, fruit yield and quality under suboptimal conditions. Several combinations of below-ground abiotic stresses have been addressed in the project: salinity, water stress, soil compaction and low fertilizer (N, P, K) input.
Project Context and Objectives:
The strategic aim of ROOTOPOWER is to help crop producers and breeders deal with the predicted impacts of climate change and to overcome the consequences of unsustainable agricultural practices that are causing soil degradation and depleting natural resources. ROOTOPOWER seeks to improve crop stress resistance and develop more resource-efficient crops, allowing more efficient use of dwindling water and phosphorus resources, and to reduce excessive use of nitrogen fertilizers (which have a high carbon footprint). Without this research there will be a trend for below-ground abiotic stresses to decrease plant growth and development throughout Europe leading to serious crop yield losses.
'Taking advantage of a multidisciplinary approach'.
Since ROOTOPOWER aims to understand crop responses to multiple below-ground stresses (including root stress perception and communication to influence shoot growth and development), it has been analysing the relationships between root genotype and shoot performance. Beyond root and rootstock-influenced shoot phenotyping, the consortium has identified QTL and candidate genes (fine mapping, transcriptomics and RT-PCR) for rootstock-mediated abiotic stress resistances and interactions with beneficial rhizosphere fungi and bacteria that will be directly suitable for breeding programs. High throughput comprehensive hormonal and ionome analyses have been performed in root and leaf xylem sap to identify and quantify the signals involved in root perception and response to individual and combined stresses and selected biological interactions. Regulatory effects of root genotype and rhizosphere microbiology on shoot photosynthesis, organ growth and development, carbon, nitrogen, phosphorous, potassium accumulation and water fluxes have been identified. Data from analyses of the genome, transcriptome, hormone, ionome and phenome are being processed through advanced statistical and modelling approaches to define the mechanistic links between genes and trait responses to stress. Such understanding will enable delivery of specific combinations of root and biota-traits for use in root-targeted breeding strategies.
'Analysis of abiotic factors'.
The project has been focused on specific abiotic stress factors in particular salinity and water stress, soil compaction and low fertilizer (N, P, K) input, and indirectly apply extreme temperatures (by evaluating crops grown in different locations and growing seasons in commercial-scale experiments). This choice is fully justified since these factors are of great relevance for determining worldwide agricultural productivity, including horticultural crops such as tomato. It is widely accepted that climate changes occurring in the years ahead will be characterized by an unpredictable increase of extreme temperatures and frequency and intensity of precipitation and intensity of drought periods. This uneven distribution of water and temperature in space and time, in addition to unsustainable agricultural practises resulting in soil degradation and water contamination (eg. excessive irrigation causing nutrient leaching from crops, or irrigation with poor quality water causing salinity) will also contribute to the overall scarcity of suitable agricultural land.
'Analysis of rhizosphere biotic interactions'.
Major groups of rhizosphere organisms known to improve crop resource capture and enhance stress resistance include rhizobia that fix nitrogen in the legume nodule; mycorrhizal fungi that enhance water and nutrient uptake, and plant growth promoting rhizobacteria that have multiple beneficial impacts including changing root system architecture and root hormone status. Although not a legume, tomato is a suitable model species to investigate the genetics, genomics and signalling processes involved in the root associations with AMF and PGPR and the subsequent effect on root and shoot development under abiotic stress conditions. ROOTOPOWER has considered both how these organisms affect the root system (and thereby shoot processes) but also how root exudation to the rhizosphere affects colonisation of rhizosphere organisms.
The scientific objectives of the project are:
• To identify new QTLs, candidate genes and novel signalling processes involved in root specific responses of tomato to six individual abiotic stresses including 3 major nutrient deficiencies (e.g. moderate salinity, restricted soil water availability, moderate soil impedance, decreased nutrient supply: N, P, K), and some naturally occurring stress combinations
• To generate new genetic, genomic and physiological information, on the basis of the genetic variability of the P-RIL population, regarding the capacity or root colonisation by beneficial rhizosphere microorganisms (AMF and PGPR).
• To link the generated genetic, physiological and agronomical knowledge through the modelling of genetic and environmental dependencies of root system architecture.
• To evaluate selected and combined genetic plant material and microorganisms in controlled multi-stress environments and real Northern and Southern European cropping conditions.
• To understand biological interactions between roots and rhizosphere microorganisms (AMF and PGPR), and to identify at least one root genetic marker for each microorganism and the associated root-signalling mechanisms.
• To understand the genetic controls of different types of root-to-shoot signalling (hydraulic, ionomic and hormonal) that positively affect shoot performance under the above described abiotic stress conditions.
At the technological level, project objectives are:
• To deliver to the scientific community at least two contrasting tomato lines used as rootstocks for their induced differential shoot response to i) moderate salinity, (ii) decreased soil water availability (iii) moderate soil impedance, (iv) decreased nutrient supply (N, P, K) and their combinations, and (v) for their capacity to associate beneficial fungi and bacteria.
• To deliver to the plant breeding community between 8 and 20 tomato lines (P-RILs) harbouring dominant and additive traits for different individual and combined abiotic stresses, some of them with direct commercial interest.
• To deliver strategies for root-targeted, marker-assisted selection for resistance to individual and combined stresses by identifying DNA-based genetic markers and an understanding of the additive effects and epistatic interactions of multiple QTLs.
• To deliver to the scientific community at least 2 pairs of near isogenic lines (NILs) for at least 4 selected QTLs conferring resistance rootstock-mediated resistance to below-ground abiotic stresses.
• To build and deliver a xylem profiling model (hormones + nutrients) that will provide physiological markers to assist root-targeted breeding for resistance to individual and combined stresses.
• To develop a predictive model of root-mediated plant response to the above described abiotic stresses.
• To develop innovative methods for testing biotic interactions (eg. root vs PGPR) and for root phenotyping (e.g. changing root system architecture in response to different stimuli).
• To propose new targets for the development of root-targeted breeding strategies in crop-species for abiotic stress resistance.
• To propose new sustainable strategies of crop management on the basis of the availability of more resource efficient root systems and associated microorganisms.
• To develop a commercial product for abiotic stress alleviation based on AMF/PGPR.
Dissemination and technology transfer objectives include:
• Reporting our results via publications (at least 10) in high impact scientific journals.
• Disseminate novel scientific advances via PI attendance at scientific and trade, national and international conferences (eg. International Society for Horticultural Science meetings) and our interactions with the advisory board and the international Solanaceae community.
• Disseminate science at a general public level through school activities, and general public targeted information in order to convey to the public opinion clear notions on abiotic stress impacts on horticultural crops.
• To demonstrate rootstock- and biota-mediated benefits of a sustainable modern agriculture (via our field-scale trials at grower sites) to a wide community of horticultural farmers within (at least) The Netherlands, Turkey and Spain.
• To train a PhD student or postdoctoral researcher in rootstock breeding, providing a career opportunity of a further contract with the SME breeding company.
• To assist the SME breeding company to start a rootstock breeding programme for some selected abiotic stress conditions on the basis of the knowledge and tools issued from ROOTOPOWER.
• To organise a workshop on phenotyping at the outset of the project to standardise data collection across consortium partners.
Relevance to the addressed topic
Elucidating root perception and response to below-ground abiotic stresses comprises phytohormone, hydraulic signalling, complete ionome, transcriptome and candidate genes analyses at local (within the root), long-distance (root-shoot communication assayed via transport fluids) and systemic (shoot) levels in the whole RIL population and in selected contrasting lines. Analysis of relationships between signalling and soil biophysical variables under controlled uniform conditions (eg. soil matric potential, soil strength, and nutrient status) has been used to determine whether these relationships can predict signalling responses to heterogeneity and combinations of abiotic stresses. The predictive value of relationships developed under root-constrained conditions, and their moderation by developmental responses of unconstrained root systems, has been tested by upscaling experiments to the field. Root interactions with the biotic environment has been measured via rhizosphere colonization by introduced biotic inoculants (AMF and PGPR), the analysis of root exudates (defined as chemicals released by roots into the rhizosphere) and root xylem sap in response to inoculation, and root system architecture. The project relies on interdisciplinary approaches integrating different expertise within the consortium composition.
Project Results:
Although a better statement of the main results of the project will be possible upon a more comprehensive analysis of the huge amount of data obtained, below there is a description of the main scientific and technological results of the ROOTOPOWER project that have been highlighted so far in relation to the foreseen objectives.
• To identify new QTLs, candidate genes and novel signalling processes involved in root specific responses of tomato to six individual abiotic stresses including 3 major nutrient deficiencies (e.g. moderate salinity, restricted soil water availability, moderate soil impedance, decreased nutrient supply: N, P, K), and some naturally occurring stress combinations
'QTL detection for rootstock effects under individual and combined stresses'.
The EU ROOTOPOWER project (Grant # 289365) is exploiting the natural genetic variability existing in wild-relative tomato species (used as rootstocks) to enhance agronomic stability of tomato by maximising water and nutrient capture and sensing and adjusting plant growth and shoot physiology to six abiotic stresses (drought, salinity, high impedance and nutrient - NPK - deficits). For this purpose, a commercial tomato cultivar was grafted onto 144 different rootstocks: six accessions from S. lycopersicum and S. pimpinellifolium, selected for drought tolerance (sourced from AVRDC); nine introgression lines from S. lycopersicum x S. pennellii and x S. habrochaites, selected for high root/shoot ratio, salinity and drought tolerances (sourced from TGRC); and a population of 129 recombinant inbred lines derived from S. lycopersicum x S. pimpinellifolium (sourced from IVIA). Since grafting allows precise assessment of the effect of altering root traits on general crop performance, the production of a huge amount of uniform grafted plants to delivery in eight phenotyping sites across Europe has been a key milestone of the project. More than 150.000 datapoints have been obtained below-, between- and above-ground to unravel rootstock-specific QTLs, physiological mechanisms and candidate genes for high-performing root systems. Although the results reveal a rootstock-induced (1.5-5.2-fold) variability in shoot vegetative biomass across different environments, the lack of correlation between them suggests important genotypic x environment interactions. However, stronger correlations in selected lines for different abiotic stresses suggest that common trait(s) might be responsible for rootstock performance under different environments, as supported by genetic and physiological studies.
The phenotyping of those populations under individual stresses has allowed the identification not only of contrasting rootstock lines than can increase or decrease the plant performance respect to the ungrafted scion variety, but also of lines showing a better performance under low P and K nutrition compared to normal fertilization, which is of high interest for further studies and rootstock development for a more sustainable agriculture.
To integrate the tolerance response of the grafted tomato plants to the different stress factors tested separately and the composition of the extracted root xylem sap in each experiment, the phenotype data collected from 8 experiments in five different countries across Europe (Spain, Turkey, Netherlands, Belgium and United Kingdom) were used for genetic analysis. A total of 170 significant QTLs were identified for evaluated traits, with five particularly QTL-rich linkage groups.
Under moderate salinity (75 mM NaCl), only two fruit yield QTLs were detected and in different regions that the previous fruit yield QTLs detected under high salinity (125 mM NaCl). This result suggests that the detection of rootstock-dependent fruit yield QTLs highly depends on the level of salinity. In fact, the heritability estimates at both levels were different at high and moderate salinity levels, respectively, suggesting that the higher the salinity level is, the higher the heritability of the rootstock-dependent fruit yield seems to be. However, two salt tolerance QTLs at the vegetative stage did not co-locate with fruit yield QTLs at the same salinity level, suggesting that both vegetative and reproductive growth under salinity can be differentially affected by the rootstock. Interestingly, the majority of QTLs related to Na+ concentration of the leaf and xylem sap (under control, salinity, drought, low P) were detected on the same chromosome where HKT HKT1 (codifying for a Na+ transporter) genes are located.
Most relevant QTLs related to tolerance to P deprivation were linked to tolerance QTLs to low N. Moreover, most QTLs related to drought tolerance under field conditions were linked to a QTL for number of penetrating roots through geotex T1000 and a low N tolerance QTL. A low K tolerance QTL was detected at the same region of chromosome 9 (chr9) than a QTL for leaf area under low N and a drought tolerance QTL.
A set of linked QTLs controlling the concentrations of numerous cations in the xylem sap under drought, was detected on chr8, linked to no drought tolerance QTL but to two salt tolerance QTLs. Several QTLs controlling jasmonic acid concentration under both low P and low N were detected on chr9. Up to 14 QTLs controlling the cytokinin trans-zeatin concentration were detected on different linkage groups, suggesting an important role for this hormone in the rootstock-mediated responses under stress conditions. Other QTLs were detected for other hormones such as abscisic and salicylic acids
As far as genetic studies are concerned most of them are preliminary and need to be complemented with further data and analyses. There were RILs that, as rootstocks, increased fruit yield and quality of the commercial variety used as scion. QTL analysis of rootstock-mediated scion nutrition is a powerful forward genetic approach to identify wild genes for rootstock breeding. These results could have an impact on the characterization and utilization of germplasm in rootstock breeding programs of horticultural and fruit crops towards the sustainability of agriculture using marginal water.
• To deliver to the scientific community at least two contrasting tomato lines used as rootstocks for their induced differential shoot response to i) moderate salinity, (ii) decreased soil water availability (iii) moderate soil impedance, (iv) decreased nutrient supply (N, P, K) and their combinations, and (v) for their capacity to associate beneficial fungi and bacteria.
Under field conditions, 42% of the experimental rootstocks performed better (increased shoot fresh weight) under drought conditions. Similarly, under drought in glasshouse conditions, 45% of the lines showed increased biomass compared to self-grafted controls. Under salt stress in glasshouse experiments, 20% of the lines had a higher shoot fresh weight, with some lines reaching a 24% increase. Moreover inbred tomato rootstocks had improved vegetative growth and nutrient use efficiency under single (between 11% and 67% for the self-grafted variety) and combined (up to 100% for the self-grafted variety) abiotic stress conditions (salinity/drought and NPK nutrient deficiencies). Interestingly, some rootstocks improved shoot growth more under low nutrient supply than under conventional nutrition, which is of great interest for increasing sustainability. Finally, improved rootstock association with beneficial rhizosphere microorganisms detected in some lines (an order of magnitude increase in colonisation) will contribute to additional positive effects on alleviating stress penalties on yield.
• To deliver to the plant breeding community between 8 and 20 tomato lines (P-RILs) harbouring dominant and additive traits for different individual and combined abiotic stresses, some of them with direct commercial interest.
• To deliver strategies for root-targeted, marker-assisted selection for resistance to individual and combined stresses by identifying DNA-based genetic markers and an understanding of the additive effects and epistatic interactions of multiple QTLs.
• To propose new targets for the development of root-targeted breeding strategies in crop-species for abiotic stress resistance.
'QTL validation, fine mapping and analysis of candidate genes'.
A fruit yield QTL gFW9.1 reported before this project was chosen to be validated by the production and phenotyping of lines that differ by a single chromosomal region (near-isogenic lines, NILs) at the genomic region where it was detected in the tomato RIL population. For this purpose, a set of NILs around this QTL were obtained and then a high salt tolerance experiment had to be carried out to find out the physical interval (defined by the significant differences among the NILs) where gFW9.1 fine-maps. Rootstock QTL gFW9.1 was previously reported on chr9, and many additional rootstock QTL were identified on chr9. In order to identify candidate genes on chr9 that might underlie the QTL gFW9.1 and other QTL on the same region, a pair of NILs derived from the Solanum lycopersicum var cerasiforme (E9) x S. pimpinellifolium (L5) recombinant inbred line population that differ in a large section of chr9 was evaluated. For the transcriptome analysis, grafted NILs were grown in a control treatment and a Low Phosphorus + Drought combined stress treatment.
A total of 80 genes that were significantly differentially expressed in roots (when comparing NILs) and that also located to chr9 were identified; these all represent candidate genes that could be causally related to chr9 QTL effects. Within this group of genes, two genes can be highlighted: (1) a gene that is predicted to have a non-functional allele in L5, annotated as a transmembrane protein of unknown function; and (2) a UBX domain-containing protein, whose expression level shows a significant interaction between genotype and treatment. It belongs to the large family of proteins which are involved in the substrate recruitment for directed protein degradation that could have a role in abiotic stress resistance.
For the remaining genes within the group of 80, specific genes can be highlighted based on relevant annotation, extent of up- or down-regulation, or statistical robustness, but, to further refine the list of candidate genes, additional validation and fine-mapping of QTL is required to narrow the locus of interest.
These results provide a list of candidate genes on chr9 that could be causative genes underlying the QTL gFW9.1 or indeed any QTL found within the region of chr9 that differs between NILs. Since the gFW9.1 QTL was not detected in the experiments designed to fine map it, further work is needed to validate and to map it to a smaller interval, and then to narrow the list of candidate genes.
'Combined stress-induced genes'.
Data from root transcriptome study also highlights genes that are induced by the Low P + Drought combined stress treatment, irrespective of the genotype, and this may provide information about how tomato roots respond to multistress treatments. Many genes related to phosphate and nitrogen metabolism and to drought and root-related processes have been highlighted. Additional systematic analysis of gene expression patterns in relation to functional annotation will be undertaken to understand how the plants have responded to the Low P + Drought combined stress.
The most prominent genes upregulated by the Low P + Drought combined stress treatment (irrespective of genotype) were the four copies of the proteinase inhibitor genes on chr9. Homologous protease inhibitor genes in the related potato (Solanum tuberosum) were previously reported to have high expression under cold stress. Also of note are several pyridoxal phosphate phosphatase genes whose homologous genes are known to be induced by phosphate starvation in Arabidopsis. The most strongly downregulated genes include genes related to nitrate transport and a nitrate reductase, and also nodulin-like genes which are believed to be involved in amino acid transport. This, combined with the upregulation of proteinase inhibitor genes, support a shift in N metabolism. A more detailed analysis of the functional annotation of these differentially expressed genes may be helpful to understand how tomato roots respond to this common combined stress in the field.
• To deliver to the scientific community at least 2 pairs of near isogenic lines (NILs) for at least 4 selected QTLs conferring resistance rootstock-mediated resistance to below-ground abiotic stresses.
'Validation of new QTLs and analysis of candidate genes in ‘high ABA’ rootstocks'.
A cluster of eleven new QTL detected on chr9 that influence rootstock performance were investigated using a collection of eleven near-isogenic lines (NILs) designed for validation and mapping of QTL. The NILs were grown in root penetration assays in tubes, in drought experiments in polytunnels, and under low nitrogen conditions and data was collected on growth, xylem sap hormones and gas-exchange.
When used as rootstocks in these experiments, the different NILs often differentially influenced scion phenotypes in statistically significant ways that are dependent on their genotypes. For example, differences due to rootstock NIL genotype gave over 50% difference in shoot dry weights under drought conditions, double the number of penetrating roots, or 25% increase in fruit yield. However, there were no systematic differences between NILs related to their genotypic differences on chr9 that allowed the validation of any QTL in a simple single QTL model. More complex models are required to explain the results.
There are many explanations for the inability to validate QTL, for example: the QTLs were too weak to be detected with the level of replication available; they have too strong a genotype x environmental interaction; they are artefactual due to random variation in the data; they rely on epistatic interactions that are missing in the NIL lines; additional unknown residual heterozygous regions were segregating in NILs that confounded the chr9 effects. Further work is required to understand the genetic basis for the differential effects of the NILs.
Since there were no validated QTL that could be investigated by transcriptomics, the project microarray resources were used to investigate transgenic rootstocks that increased fruit yield under mild salinity conditions. The transgenic rootstocks overexpress a tomato gene encoding a rate-limiting enzyme in abscisic acid biosynthesis and are previously known to exhibit increased accumulation of abscisic acid in xylem sap; the rootstocks were shown to increase fruit yield of grafted scions in conditions of mild salinity stress. Transcriptomic data highlighted a number of classes of genes that are differentially expressed due to the presence of the LeNCED1 transgene; notably several specific aquaporin genes are either up or down regulated, ethylene signalling related genes are upregulated, and many pathogenesis related genes are downregulated. This data set can be used to understand how abscisic acid influences rootstock impact on scion performance and to identify a list of candidate genes involved.
• To generate new genetic, genomic and physiological information, on the basis of the genetic variability of the P-RIL population, regarding the capacity or root colonisation by beneficial rhizosphere microorganisms (AMF and PGPR).
• To understand biological interactions between roots and rhizosphere microorganisms (AMF and PGPR), and to identify at least one root genetic marker for each microorganism and the associated root-signalling mechanisms.
'Rootstock variation in AMF/PGPR dependency and plant growth promotion'.
The results obtained clearly demonstrate a variation in the colonisation performance of the different tomato RILs under in vivo and in vitro culture conditions, as well as a differential effect of the microbial inoculants (AMF Rhizophagus intrarradices or PGPR Variovorax paradoxus) on plant performance, depending on the RIL involved. Genetic analysis determined heritability for root capacity to be colonized by AMF and by PGPR, but no QTL was detected above significant threshold with the linkage map (158 loci) used as background in the project. Since root colonization by AMF appears as a heritable trait, further analysis will be required to locate putative genes involved. A comprehensive metabolomic analysis of signalling molecules involved in AMF colonisation revealed significant differences between contrasting lines. Interestingly, many of the molecules belong to the xenobiotics and tyrosine metabolism. Similarly, transcriptomic analysis revealed important differences in the gene expression profile between contrasting lines used as rootstocks, helping to explain their differential response to drought stress in presence or in absence of the biota AMF and PGPR. Protein abundance and phosphorylation status was analysed for aquaporins PIP1 and PIP2, although no clear relation was found with rootstock-mediated increase in root hydraulic conductivity and plant performance. However, many other differentially expressed genes were related to KEGG pathways involved in several metabolic processes that could be considered as candidate genes underlying the rootstock x biota mediated plant performance under water stress: amino acid biosynthesis, amino sugar and nucleotide metabolism, purine and pyrimidine metabolism, glycerophospholipid metabolism and carbon fixation in photosynthetic organisms, phosphatidylinositol signaling system or phenylpropanoid biosynthesis, among others. The information generated in ROOTOPOWER regarding genetics, signalling molecules and candidate genes involved in plant microbe interactions and resistance could be considered at the cutting edge of this scientific field and could serve to develop more efficient rootstocks with improved capacity to interact with soil biota and to increase plant performance under abiotic stresses.
• To develop a commercial product for abiotic stress alleviation based on AMF/PGPR.
'Validation of AMF production process in crop field conditions and technology transfer on commercial AMF strain'.
The production of mycorrhizal (AMF) inoculum has been optimize and validated at the field scale in ROOTOPOWER. The first production was done from in vitro starter inoculum and then enriched gradually to generate a sufficient volume of concentrated AMF inoculum. Several parameters were tested, such as the ability of propagules to germinate in different substrates (sand or vermiculite) or the influence of the host plant. Finally, a new AMF product based on pure mycorrhizal root powder was developed at one of the specialized SME partner, showing a significantly increase of the mycorrhizal development compared to indigenous AMF strains. The data set obtained under the frame of the project has allowed development of a concentrated mycorrhizal inoculum, able to generate mycorrhiza under greenhouse and field conditions while decreasing the amount of inoculum compared to classic crude inoculum use. Based on the best results obtained all along the ROOTOPOWER project with the R. irregularis MUCL41833 strain, a mass production was set up with a commercial strain of the same species, whose propagule richness was increased by 305 fold under vermiculite substrate (root powder prepared and mixed back in the remaining substrate) compared to initial capacity. In the next years, it is expected that an ultra-rich mycorrhizal inoculum (able to inoculate crop fields with several billions propagules per hectare) will be brought to market, which constitutes a significant step forward regarding the current available offers.
• To evaluate selected and combined genetic plant material and microorganisms in controlled multi-stress environments and real Northern and Southern European cropping conditions.
'Physiological and genetic interactions conferring resistance to combined abiotic stress'.
Although there was no significant effect of biota (B) inoculation on plant growth globally, there was a significant effect of biota treatment depending on the rootstock genetopye (G) used (GxB). Generally, grafted plants onto selected rootstocks performed better than the scion cultivar self-grafted control plants under the combined abiotic stresses applied. Therefore, the selected rootstocks for individual stresses could be used to alleviate the negative impact of the abiotic stress, which normally appears as a combination of two or more stresses. In particular, within the RIL population (Solanum lycopersicum x S. pimpinnelifolium), a couple of lines presented a good performance under all three combined conditions studied when used as rootstocks. Interestingly, other lines derived from a cross between Solanum lycopersicum and S. habrochaites, as some high vigour commercial rootstocks used as reference, exhibits better performance under drought and salinity stress. Similarly, an accession of Solanum lycopersicum var cerasiforme previously selected under individual stress conditions also showed a better performance under combined drought and salinity conditions. Both the near isogenic lines selected for the pre-existing QTL gFW9.1 and the functional lines, related with abscisic acid and cytokinin biosynthesis, behave better in any combined condition with salinity. Grafted plants onto some of the selected lines and NILs used as rootstocks increased nutrient and water use efficiency through maintaining growth and photosynthesis and reducing plant transpiration under combined Drought + Salinity and Low P + Salinity conditions. The higher performance of the selected RILs under individual stresses suggest that the selection process was successful in some cases, but the differences in rootstocks performance among the different experiments suggest an important genotype x environment interaction that requires further attention. However, the global analysis of the selected lines (RILs and non-RILs) suggests that genetic/physiological rootstock selection based on resistance for single stresses is adequate for stress tolerance under combined stress conditions.
• To link the generated genetic, physiological and agronomical knowledge through the modelling of genetic and environmental dependencies of root system architecture.
'A parameterized model of root architecture responses to low N with internal validation has been proposed in ROOTOPOWER'.
Using static root image datasets, a complete analysis pipeline to estimate automatically the main parameters of a dynamic model of root system architecture (RootTyp) for each line of the grafted population under control and Low N has been developed. At this stage, the whole experimental and analysis pipeline allows to reproduce a gross trend across the population and N levels, however, the limited value of the validation suggests that this information should not be trusted to predict details of the root architecture of individual genotypes. It can be anticipated that the quality of the simulations might be dramatically improved with an improved chain of image capture and analysis, which has been recently implemented in the aeroponics platform based on the results of the ROOTOPOWER experiments. Additionally, a non-destructive method for remote evaluation of plant physiological status has been developed. This method has proven to yield heritable indicators that are very sensitive to N-levels, although not linearly related to N-concentration. This method can facilitate the evaluation of plant status in N-limited environments and a non-invasive way, with costs that are essentially fixed.
The QTL analysis of root system architecture traits has been carried out on classical morphological indicators as well as on parameters of the RootTyp model. QTLs were detected at a rather low LOD threshold, indicating weak marker-trait associations. This result was somehow surprising in view of the reasonably large heritabilities and may be the consequence of the image capture and analysis chain. Under Low N, a reasonable good fit regarding the position and direction of allelic effects was found between manual and model-based estimates of root branching and of root system width, and an additional QTL was detected for the model-based estimate of root branching. However, low LOD thresholds have been encountered in several other QTL analyses of root traits in aeroponics, even with genetic material known to be diverse in field conditions, suggesting that the diversity of root architecture might be best expressed in constrained conditions. The results indicate that the genetic determinism of lateral root initiation is largely different at low and high N. This is an important result as lateral roots are known to respond to N supply and plant N status and contribute to most of the absorptive surface of the root system. Surprisingly, the heritability of the response of inter-lateral distance to N supply was negligible, indicating that the genetic determinism of N response is complex. Low LOD scores in aeroponics have been commonly observed in maize, barley, wheat and rice. This suggests that, in the absence of mechanical, gas and nutrient constraints, large collections of genotypes within a species tend to achieve a narrow range of phenotypes. In view of very large QTL effects reported in the literature in soil or other conditions, this would indicate that the scope for root architecture improvement lays more in the root responses to environmental constraints than on constitutive traits. If this was to be the case, the scientific community should probably reconsider much of the actual root architecture phenotyping practices.
• To develop a predictive model of root-mediated plant response to the above described abiotic stresses.
'Another conceptual model of root architectural responses to low P, low K, salinity, drought and mechanical impedance and validation with combined experiments has been approached in ROOTOPOWER'.
For all the individual and combined stresses evaluated in the project, the QTL results have been exploited, in particular (i) the collocation between root weight QTLs, shoot weight QTLs and hormone QTLs and (ii) the conditions in which these QTLs were detected. Based on this information, an integrated conceptual framework has been proposed where the effect of individual stresses on specific root growth and development processes are overlaid. Although several QTLs were detected in each experiment, no single co-location event could be detected across the different experiments. This support the proposition that QTLs for root traits should be seeked for in GxE designs instead of in G designs. This set of data would lead to recommend to address combined stresses in genetic analysis using especially dedicated experimental designs with in depth characterisation of the plant environment and which take into account the non-additive action of QTLs. Hovewer, further experiments will be necessary to validate this model and to evaluate to what extent these stresses operate in an additive manner on each growth/development processes.
'Stability of rootstock- and biotic- effects on fruit yield and quality'.
The proof of concept carried in Northern and Southern European conditions demonstrated that the rootstock genotype was the second important factor affecting plant growth, physiology, yield and quality of indeterminate tomato grown under combined abiotic stresses (Low N availability + Salinity or Low N + Drought). In the determinate tomato, mostly stress but also rootstock genotype affected majority of the parameters analysed in two different European locations. In the industry tomato, all the rootstocks evaluated increased crop yield with respect to the self-grafted scion plants in Spain (Low N + Salinity), while an accession from Solanum lycopersicum cv. cerasiforme, selected for drought tolerance, was the only genotype which performed better than the self-grafted scion in Turkey under Low N + Drought conditions. Moreover, the rootstock has the potential to improve water use efficiency, increasing photosynthesis of a commercially relevant scion under both optimum and combined stress conditions.
Indeed, commercial F1 rootstocks (inter-species hybrids) have an advantage in terms of tomato fruit yield (increased up to 50%) under different environmental stress conditions because of vigour. However, grafting onto some non-hybrid rootstocks also improves tomato fruit yield (up to 30%) under different environmental stress conditions through more specific (non-vigour related) mechanisms. Those rootstock-mediated yield improvements under stress conditions were not in detriment of fruit quality in different growing conditions. Although there is considerable overlapping of best performing rootstock genotypes in the experiments from all locations, the differences observed in rootstock performance between different locations suggest an important genotype x environment interaction that requires further attention. However, some RILs and other experimental lines tested in the ROOTOPOWER are promising genotypes for breeding purposes since they performed as better as the commercial F1 rootstocks across different locations and combined stress conditions.
Although there was no significant effect of biota (AMF + PGPR) inoculation on plant growth generally, there was a significant effect of biota treatment depending on the rootstock used (GxB) and/or stress applied on a few plant parameters, but this effect could be positive or negative. Overall, combined stresses reduced the indeterminate tomato fruit yield about 23% and 33% under with and without biota conditions, respectively.
'Biotic inoculant persistence and effects'.
Without surprise, the plant genotype plays a strong role in microbe–plant interactions. It influences the initial colonization, but also the functioning of the interactions. Data obtained in ROOTOPOWER indicate that cultivars harbour various susceptibilities to biota application, which can result in beneficial, neutral, or even detrimental on plant responses. However, although these observations are regularly reported, physiological determinisms that could elucidate these phenomena in the optic to predict and select positive symbiotic associations (in term of root colonization and/or benefit on plant performances) remains to be unravelled.
In that sense, studying the mycorrhizal behaviour throughout the exploitation of grafting technology is interesting, since it may highlight potential mobile factors and/or genotypic responses and/or metabolic contexts crucial to deepen knowledge in mycorrhizal physiology.
In general, the biota (PGPR and AMF) inoculation generated higher colonization in plant root when plants are inoculated in situ and/or grown in controlled greenhouse conditions (in sandy substrates or perlite) than under crop conditions. Although the various stresses affected more or less severely the non-inoculated plants (growth or quality), their impact on biota (colonization and plant response) did not allow to draw obvious conclusions among trials. The lack of correlation between laboratory, greenhouse and field results translates major complexities to study interactions in plant-microbe systems in various growth conditions, which is not new. In particular, data are often difficult to interpret among trial sites, probably owing of heterogeneity in trial implementation as expected from different geographical locations (plant inoculation time and harvesting, plant/rootstock genotype, culture conditions, cultural itinerary). As there exists multiple factors, often undetermined, which can influence root colonization in the soil, it is therefore difficult to design methods that will predict given characteristics under in vitro conditions. In addition, independently from the way that in planta screening is carried out, the outcome depends also on plant physiological state, known to vary for example by culture age, growing medium or temperature.
Responses of biota on plant under normal conditions were highly variable, according to genotype, graft combination and trials. Under P starvation or drought compared to normal conditions, mycorrhizal plants were less impacted than non-inoculated plants. Under salt stress, or when several stress conditions were combined, plant responses are more mitigated, mostly plant genotype/graft combination specific dependent. Under commercial conditions in which plant were pre-germinated under peat, no significant effect of biota was observed, even when the plants were inoculated in situ (melon and watermelon). This conclusion is consistent with the state of art on inoculation trials under field conditions.
Despite the low colonization detected in some experiments, the capacity of some rootstocks to improve the colonization ability and plant responsiveness to biota leads to interesting perspectives for developing this already well implemented commercial practice. ROOTOPOWER provides therefore some new insights in the rootstock x biota x environment genetic, physiological and agronomical interactions, opening challenging opportunities to deepen the understanding of plant-biota interactions and to master the potential benefits of biotic interactions in various culture and environmental conditions.
'Approach validation in other species'.
For most crops evaluated (tomato, eggplant, pepper, melon, watermelon, cucumber) grafting onto commercial rootstocks improved growth and yield-related parameters under combined abiotic stress. Biota treatment had a positive effect in some parameters evaluated in the grafted plants, but its effect was more evident in interaction with the stress treatment. Therefore, the results obtained demonstrate that grafting onto specific rootstocks improved plant performance and yield in other Solanaceae and Cucurbitaceae crops under abiotic stress, thus validating the approach used for tomato as a model crop species.
• To understand the genetic controls of different types of root-to-shoot signalling (hydraulic, ionomic and hormonal) that positively affect shoot performance under the above described abiotic stress conditions.
• To build and deliver a xylem profiling model (hormones + nutrients) that will provide physiological markers to assist root-targeted breeding for resistance to individual and combined stresses.
'Root signalling and soil biophysical properties'.
A huge amount of xylomic data including iononome and hormonome have been collected in 144 different rootstocks under 6 individual stress conditions and their respective controls and in eight different phenotyping platforms in five European countries. This unique set of information is being processed and it is expected that the elucidation of the specific physiological and genetic components of the rootstock-mediated effects on plant performance under those stressful conditions will be completed in a near future. The main results obtained so far can be summarized as follow:
Potassium (K) deficiency: Across all graft combinations, xylem K concentration of K-deprived plants was significantly lower than control plants, as expected. However, xylem Ca concentration of K-deprived plants was also decreased by a similar magnitude. Interestingly, there were also three micronutrients (B, Mn and Zn) that showed significant increases in K-deprived plants, compared to control plants. Potassium deprivation significantly decreased the xylem concentrations of multiple phytohormones and their precursors: abscisic acid, ethylene, gibberellins, auxin and cytokinins. Interestingly, potassium deprivation significantly decreased the bioactive cytokinin free-base zeatin concentration and increased its riboside form. Although there was no rootstock-mediated variation in xylem phytohormone concentrations under normal K conditions, both ethylene precursor ACC and trans-zeatin showed significant rootstock-mediated variation under K-deprivation. Within the K-deprived treatment, a principal component analysis showed that rootstock-mediated variation in plant response was not well correlated with either ionic or phytohormonal composition of the xylem sap. However, those hormonal and ionomic changes are helping to explain the behavior of contrasting rootstocks under normal and low-K nutrition.
Nitrogen (N) deficiency: Only K showed a significant decrease in xylem concentration under N deprivation. The remaining ions all showed significantly increased concentrations in N-deprived plants. The similar responses of many different ions suggest either a co-ordinated response to N deprivation, or that decreases in xylem sap flow rate (caused by N deprivation) concentrated the ions within the xylem sap. Under N-deprivation, there was significant rootstock-mediated variation in xylem B, Cu, Na and Zn concentrations. Nitrogen deprivation significantly increased xylem concentrations of abscisic, jasmonic and salicylic acids, and trans-zeatin riboside. In contrast, N deprivation almost halved xylem trans-zeatin concentration. Across all plants, the rootstock significantly altered xylem abscisic, jasmonic and salicylic acid concentrations under Low-N conditions.
Phosphorous (P) deficiency: Across all graft combinations, xylem P concentration of P-deprived plants was significantly lower (by 45%) than control plants, as expected. Xylem K concentration of P-deprived plants was decreased by 10%. In contrast, P deprivation significantly increased xylem concentrations of Ca, Mn and Mg. Interestingly, phosphorous deprivation significantly increased xylem concentrations of all gibberellins detected and free-base cytokinins. In contrast, P deprivation significantly decreased xylem the ethylene precursor ACC and jasmonic acid concentration. The rootstock significantly altered xylem jasmonic and salicylic acids, and trans-zeatin concentrations.
Soil drying: Compared to well-watered plants, soil drying significantly increased xylem concentrations of Fe, Mg, S and Zn. Across all plants, all ions analysed (B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Zn) showed significant rootstock effects. Xylem concentrations of many phytohormones were significantly altered by soil drying, with increases in gibberellin GA4 and jasmonic acid. In contrast, xylem concentrations of ethylene precursor ACC, gibberellin GA1, and cytokinins decreased. Surprisingly, soil drying did not significantly increase xylem ABA concentration in the tested conditions. Across all plants, there were significant rootstock-mediated effects on almost all the hormones analyzed in the xylem.
Mechanical impedance: Compared to other sites/stresses, xylem macronutrient (Ca, K, Mg, P and S) concentrations were higher in plants exposed to mechanical impedance. Only xylem Na concentration showed significant rootstock-mediated effects, while no significant rootstock-mediated effects were detected on xylem hormone concentrations.
Salinity: There was significant rootstock-mediated variation in xylem sap Fe and S concentrations. Of the phytohormones measured, only xylem the ethylene precursor ACC concentration showed significant rootstock mediated variation. Principal component analysis showed that shoot fresh weight was clustered with some xylem phytohormone concentrations (abscisic acid, ethylene precursor, gibberellins and auxin), while xylem trans-zeatin and Na concentrations were opposed to vegetative vigour.
In conclusion, significant rootstock-mediated variation in xylem sap composition (both ions and phytohormones) was detected in response to a range of different below-ground abiotic stresses, and in some cases rootstock-mediated effects on xylem sap composition were correlated with rootstock vigour. However, a more comprehensive and in deep analysis to elucidate the signaling components of plant response and adaption to different stresses in ongoing.
'Plant microbe interactions: root exudation and plant hormone status'.
Biota inoculation (AMF Rhizophagus irregularis + PGPR Variovorax paradoxus) sometimes had important effects on xylem macronutrient concentrations (eg. K, Mg, P). Especially consistent was an increase in xylem Mn concentration of between 15 and 30%, which was detected at 4 site/treatment combinations. In relation to the hormonal factors, trans-zeatin and salicylic acid were especially affected by biota inoculation under combined drought and salt stress. Principal component analysis revealed that most mineral nutrients varied in opposite direction to the shoot growth under combined Low P and salinity, while trans-zeatin and jasmonic and salicylic acids, and, especially, abscisic acid were the most important factors explaining the variability of shoot fresh weight in the population .
'Predicting signalling responses to combined abiotic stresses'.
Large differences in absolute hormone concentrations between experiments testing individual and combined stresses caused considerable inaccuracies in the modelled data. Thus predicting signalling (xylem sap composition) in response to combined abiotic stress remains problematic, and undertaking single and multiple abiotic stress experiments (eg. control, salt, Low P deprivation, salt and Low P combination) at the same time in the same experimental material seems necessary to make progress in this area, such as it has been done for drought and salt stress, where xylem ABA concentration in response to combined was entirely explained on the basis of its responses to individual drought stress during vegetative growth.
'Field variation in rootstock dependent root signaling'.
Frequency of successful root xylem sap collection varied substantially between species (and was especially low when relying on spontaneous exudation, rather than pressure-induced flow). Although collecting leaf xylem sap provided a viable alternative to guarantee sample availability, in this case xylem phytohormone concentrations in leaf xylem sap were poorly correlated with root xylem phytohormone concentrations, likely since root xylem sap was entirely apoplastic while leaf xylem sap will comprise both apoplastic and symplastic sources.
Biota inoculation generally had limited effects on root xylem hormone concentrations, significant effects only occurring within some species grown at some sites for certain phytohormones. Although a suitable growing substrate was optimized for biota colonization, poor colonisation of the root system by the inoculants in the field may have been responsible for this effect, especially since previous studies have shown that the PGPR can alter xylem abscisic acid and ethylene precursor ACC concentrations. Nevertheless, inoculated pepper plants showed a decreased xylem ACC concentration in response to Low N + salinity combined stress.
The stress treatment did not only clearly alter xylem concentrations of the traditional “stress hormones” ABA and ACC, but also of other phytohormones. However, the direction of change depended on the species/site combination. For indeterminate tomato, grown both in Southern and Northern European areas, root xylem ACC concentrations showed a consistent increase under stress conditions.
There were clear rootstock-mediated differences in root xylem phytohormone concentrations in determinate and indeterminate tomato when grown under semi-commercial conditions. These responses were not always consistent across sites and species, suggesting strong rootstock x environment interactions. Importantly, xylem ACC concentrations increased under Low N + salinity combine stress while trans-zeatin concentrations decreased. Furthermore, when consistent rootstock-mediated differences were detected, often it was the commercial rootstocks that showed lower concentrations of certain phytohormones (tras-zeatin, salicylic acid, and especially ethylene precursor ACC) under both optimal and stress conditions. Interestingly, self-grafted control plants showed amongst the lowest ACC concentrations under optimal conditions, while the highest under stress conditions. It is tempting to speculate that the commercial success of these widely adapted rootstocks is due to altered phytohormone profiles in the plant across different environments.
Taken together, all 3 factors controlled in these studies (rootstock, biota and stress) altered xylem phytohormone concentrations in plants grown under semi-commercial conditions. Of these, xylem ACC concentration (hitherto primarily considered as a signal of flooded soil) seems to be an important root-derived signal implicated in the response of the grafted plants to combined stress conditions.
In addition to hormonal components, by screening microarray data obtained from different plant species and under different stresses, a number of conserved stress-responsive genes whose expression was differentially regulated in tomato roots in response to one or several stresses have been identified. Ten of these genes have been validated as reliable biomarkers whose expression levels are related to different signalling pathways involved in adaptive stress responses. The genes identified could be used could be used to evaluate, at the molecular level, the stress responses of tomato cultivars that differ in stress tolerance. Due to conservation of genes and their downstream responses, they could also be used to evaluate the stress responses in different species where full transcriptomic information is not yet available.
Potential Impact:
According to the expected impact listed in the work programme, ROOTOPOWER can potentially contribute in the following ways:
“Knowledge and tools generated will help to (i) better understand root development and below-ground interactions under abiotic stress in field conditions”
ROOTOPOWER has generated valuable information and tools on how the different individual abiotic stresses (salinity, drought, soil compaction and nutrient –N,P,K- deficiencies) affect root and shoot development by analysing root system architecture, biomass and soil distribution by using different phenotyping platforms. A reliable method for remote evaluation of plant physiological status has been developed. This methods yields heritable indicators and is very sensitive to N-levels, although not linearly related to N-concentration. This method can facilitate the evaluation of plant status in N-limited environments in a non-invasive and low-cost way, and could be extended to other suboptimal environments.
The project has produced a huge amount of physiological information about how the rootstocks and/or the association with AMF and PGPR affect shoot performance under individual and combined stresses in terms of resource capture (complete ionome) and gas-exchange.
ROOTOPOWER has generated a huge amount of physiological information about how hormonal signalling is involved in (i) root-perception of stress, (ii) root-interactions with rhizosphere microorganisms; and (iii) root-to-shoot communication and its influence of shoot physiology (growth, water use, carbon assimilation, leaf senescence and fruit yield).
By analysing contrasting and functional lines, ROOTOPOWER has produced new information about how (i) stress, (ii) soil microorganisms and (iii) shoot genotype influence root development in tomato through analysing the candidate genes involved in auxin transport, cytokinin sensitivity and ABA biosynthesis, and other hormonal-related processes.
ROOTOPOWER has identified a list of QTLs and chromosomic regions as indicators of resistance to the above mentioned individual abiotic stresses. A set of QTLs have been assayed for validation under individual and multiple combined stresses in field conditions. A set of near isogenic lines have been generated for a particularly rich chromosomic region harbouring important QTLs, which have been used to generate new transcriptomic information about candidate genes involved in rootstock-mediated stress tolerance. Those lines are now available to the scientific community.
ROOTOPOWER has generated a predictive conceptual model to integrate genetic, physiological and agronomical data that will serve as a basis for future modelling studies. More specifically, a tomato version of the RootTyp model has been obtained and implemented a whole image analysis pipeline that is able to predict the main model parameters from images captured when plants are 3-week old (after transplantation). This model allows simulating realistic root systems in 4D (spatial and temporal dynamics) and can be used to different purposes. It can be used by breeders to test in silico the value of novel genotypes, considering different aspects of resource capture and soil profile exploration. It can also be used as a communication tool to illustrate the dynamic development of tomato root systems of different lines (including grafted and non-grafted plants). Using QTL data, a conceptual model of root architecture response to individual stresses have been designed. This model will be useful to design further plant physiology research on root responses to various abiotic stresses. Work is ongoing to extend this model to three scenarios combining two multiple stress conditions.
Upon a comprehensive analysis of the data, ROOTOPOWER will contribute to improve plant resistance to fluctuating environmental stress, by providing quantitative data useful to develop new mechanistic strategies of plants adaptation to their environment. The project will identify interactions among dominant physiological functions involved in the plant response submitted to several combined stress factors and has established some quantitative stress dose versus plant response relationships.
ROOTOPOWER results provide tools and knowledge to anticipate different stress combinations (ie. salinity and Low N, salinity and low P, drought and low P, drought and low N, salinity and drought) and thus contributing to adapt agriculture to specific time- and space- specific environmental conditions. The information generated will also serve to support the design of ideotypes, i.e. rootstock genotypes best adapted to a given set of environmental conditions while maintaining or optimizing yield and quality traits.
ROOTOPOWER will contribute to fill the gap in the understanding of plant responses to some combinations of abiotic stress factors. In particular ROOTOPOWER has identified regulatory networks at the gene and at the process levels in tomato which will help elucidate complex interactions in the plant response to combined stress factors, particularly drought and low P.
The information delivered in ROOTOPOWER will contribute to increase stress resistance of other species. Indeed the QTLs and key-candidate genes that will be identified underlying below-ground abiotic stress resistance are likely conserved within tomato, other Solanaceae and other crops. These traits can be expected to be found in other dicotyledonous crop species independent of their grafting susceptibility. Therefore ROOTOPOWER will provide the scientific community with new perspectives for knowledge transfer from the model and crop plant tomato to other crop species.
“Knowledge and tools generated will help to (ii) support the development of root-targeted breeding strategies to generate varieties with increased resource use efficiency and resistance to abiotic stresses”
ROOTOPOWER is releasing genetic markers for root-targeted traits under six different abiotic stress conditions and some of their natural co-occurring combinations, which will be used in marker assisted selection. Thus, ROOTOPOWER will help overcome current limitations of breeding strategies for increasing crop plant resistance to environmental challenges, by identifying QTLs and providing a list of candidate genes for breeding strategies to restrict negative side effects on growth and yield.
More specifically, for the S. lycopersicum var cerasiforme L5 x S. pimpinellifolium E9 recombinant inbred population that was used to report rootstock mediated QTL, the majority of SNP and InDel polymorphisms present have been identified by genomic resequencing. This will allow polymorphic candidate genes to be identified in the region of any finely mapped QTL from this population. The possible candidate genes would allow marker assisted selection of novel rootstock genotypes.
Near isogenic lines have been produced from the ancestors of the P-RILs representing the L5 and E9 DNA on chromosome 9. Different lines when used as rootstocks showed contrasting root growth and also scion growth under drought conditions. This forms the basis of further genetic analysis, and such rootstocks might be useful in their own right.
Moreover, tomato rootstocks overexpressing LeNCED1 gene (key enzyme for abscisic acid biosynthesis) improved the yield of fruit produced from the grafted scions when the plants were grown under mild salinity. Such rootstocks or other developed by approaching similar traits could improve tomato production in salt-affected areas (e.g. in Mediterranean region).
ROOTOPOWER results will contribute to develop new root-targeted breeding strategies based on the signalling processes existing between soil microorganisms, roots and shoots leading to increased resource acquisition and resource use efficiency and resistance to multiple and combined abiotic stresses which naturally co-occur.
ROOTOPOWER is generating genetic and biochemical markers (QTLs, candidate genes and signalling molecules) for root capacity to associate AMF and PGPR in search of synergistic plant-biota interactions to increase resource acquisition and resistance to abiotic stresses. Three sets of transcriptomic data were produced from tomato rootstocks and candidate genes were identified that might be responsible for QTL described, or be involved in rootstock responses to multistress conditions and colonization with AMF/PGPRs. This provides a resource to help in understanding the genetic basis of rootstock function which could eventually help find markers for marker assisted breeding.
ROOTOPOWER has identified recombinant inbred lines (RIL) and other genetic materials publically available that when used as roostocks provide contrasting degrees of resistance (assayed as shoot performance) to several abiotic stresses (moderate salinity, moderate water deficit and moderate soil impedance, decreased fertiliser input: N, P, K) both individually and in some real-world combinations in the field. ROOTOPOWER has also identified RILs with contrasting capacity to associate with AMF and PGPR.
More specifically, inbred tomato rootstocks improve vegetative growth and nutrient use efficiency under single (between 11% and 67% for the self-grafted variety) and combined (up to 100% for the self-grafted variety) abiotic stress conditions (salinity/drought and NPK nutrient deficiencies). While commercial F1 rootstocks (inter-specific hybrids) have an advantage in terms of tomato fruit yield (increased up to 50%) under different environmental stress conditions because of vigour, grafting onto some non-hybrid rootstocks evaluated in ROOTOPOWER also improves tomato fruit yield (up to 30%) under different environmental stress conditions through more specific (non-vigour related) mechanisms.
ROOTOPOWER will help breeders to interpret complex below-ground abiotic stress interactions and provide them with relevant physiological indicators to evaluate plant response to different stress combinations, discerning between vigour and non-vigour related mechanisms. This will build capacity to screen genetic diversity, and complemented existing approaches based more on phenotypic traits (vigour) than on underlying physiology and genetic (specific mechanisms).
Similarly, ROOTOPOWER will support the development of sustainable innovative production strategies by unravelling the synergistic or antagonistic actions of combined stresses and their dependency on different stress factors and some natural stress combinations. These data will allow the development and optimisation of cultural practices to predict whether resistance to a given stress factor can be extrapolated or not to another factor (ie. drought vs salinity).
ROOTOPOWER has the potential to rapidly benefit breeders since interesting lines have been selected under both controlled and real field conditions. Use of rootstocks carrying promising characteristics for abiotic stress resistance allows their almost- immediate exploitation with existing elite scion material, to increase resource use efficiency and enhance resistance to abiotic stresses. This represents a considerable acceleration in developing and adapting new breeding strategies to increase the resistance and yields of crop species in the future climatic context, compared with non-graftable species (such as cereals) where the time between selection and introduction to existing elite material may be 10 years or more.
The combination of approaches developed by ROOTOPOWER provides the possibility of translating results:
• From species like tomato via the modelling tools developed to other species with are amenable to grafting and thus to the use of rootstocks. This will allow taking advantage of genomic knowledge that is being continuously generated in tomato and other species like eggplant, pepper, watermelon, cucumber and melon.
• From phenotyping platforms to the field. The phenotyping methods developed both in the field and in platforms (through extending laboratory experiments to the field, and through directly testing these physiological hypotheses in the field) provide the possibility of increasing accuracy in future characterisation of the genotype performances in the field.
• From a few field experiments to a large range of stress scenarios in the field within the diversity of EU climates, and extended to forecasted climatic scenarios.
“Overall, results obtained will contribute to sustaining yields under more adverse climatic conditions”
• By providing knowledge to elucidate complex interactions in the plant response to different below-ground stress factors, and by setting new breeding strategies for improving crop resistance to below-ground abiotic stress combinations, ROOTOPOWER results will contribute to adapt European and world agriculture to forecasted climatic challenges and adverse environmental factors, of which drought, salinity and low nutrient availability represent the most severe constraint to agriculture
• Global warming is also predicted to affect most severely developing countries, where agricultural systems are most vulnerable to climatic conditions. Additionally, at global level, soil degradation and abandonment by factors as salinisation is equivalent every year to the total irrigated area. The development of crop varieties with increased resistance to below-ground abiotic stress factors is highly necessary in developing world countries which face nutrient shortages that limit productivity.
• Farmers have a major interest in extending the productive cropping period during the year and to recover and maintain the soil as a natural substrate. It is essential to help crop producers and breeders to face climate change and the forecasted higher intensity and frequency of extreme climatic events in a substantial part of Europe, including increased salinity, drought and other below-ground abiotic stresses. ROOTOPOWER results can certainly contribute to this challenge by improving our understanding of the root-targeted breeding and biotechnology -a field yet relatively unexplored.
• Establishing an association with non-profit organisations such as The World Vegetable Center (AVRDC) would allow promising rootstocks to be disseminated to some of the world’s poorest countries, since tomato and other graftable vegetables can be important to subsistence farmers (especially as cash crops). Some joint initiatives between some ROOTOPOWER partners AVRDC and are currently in process.
“Project results should clearly be of interest and potential benefit to SMEs”
• SME’s participating in ROOTOPOWER can benefit from yield-enhancing and environmentally friendly tomato rootstocks and from innovation in breeding programmes to ensure the competitiveness, sustainability and independence of European agriculture. Varieties released and used in modern agriculture have been developed over hundreds of years by farmers and plant breeders. Unfortunately, for main crops, the relative yield increase has been declining during the second half of the 20th century in most producing countries, including Europe. Thus, there is a crucial need to develop a sustainable agricultural production system by increasing and stabilizing the production with cultivars adapted to climate challenges, which require less input, and, at the same time, provide enhanced nutritional composition for consumers’ health and well-being and deliver consistent output traits to processors.
• The rootstocks and QTLs selected and identified in the project for their involvement in the resistance different abiotic stresses will be available for breeders upon its adequate publication. A whole population of RILs, used as rootstocks, has been screened, new alleles have been identified and negative side effects on agronomical traits have been checked during the project; these QTLs and genes can be introduced more rapidly and earlier in breeding programs for the development of new tomato cultivars and rootstocks that are more resistant to abiotic stresses.
• To test the resistance/resistance of plants to abiotic stresses (which are often complex traits,) breeders usually need large trials in controlled environments Moreover they usually test one environmental factor at a time to limit the trial size, and breeding for resistance to abiotic stress factors has been only recently initiated. The identification of single genes playing key roles in stress resistance to abiotic factors, will greatly contribute to breeding effectiveness since are more easily manipulated in breeding programs. Breeders can apply Marker Assisted Selection to significantly shorten the breeding process.
• Moreover lines selected by tilling in cultivated populations could be directly used in breeding programs. With knowledge gained through ROOTOPOWER, it is expected that SMEs could bring to the market new rootstocks within 5 to 8 years which is about half the time required with more traditional breeding strategies. These new rootstocks will present a broad spectrum of resistance to biotic and abiotic stress factors and thus will be adapted to contrasted climatic regions. Thus ROOTOPOWER will impact on the efficiency of breeding programs, and promote the creation of new tomato rootstocks and varieties with high yield and good fruit/grain quality, adapted to long cultivation in different parts of Europe.
• The project will support the development of new rootstocks of tomato adapted to multiple stress environments which is expected to have large economic impacts for producers in Europe (mostly SMEs). Tomato is the most important vegetable production in the world (129 millions of tons), the most consumed vegetable in Europe (after potato) and an important source of healthy compounds such as antioxidants. By increasing the plant resistance to abiotic stresses, ROOTOPOWER will contribute to decrease the dependence on chemical fertilisers, water resources and technological solutions (eg desalinisation plants; alternative substrates, climatic control, etc.) to face climatic challenges. Thus, the project will have a major quantitative impact in terms of intermediate inputs use and consequently a qualitative impact on food safety and environment preservation. It will therefore contribute to increase the sustainability of agriculture in Europe, a highly SME-intensive sector.
• The involvement of a SME in quality control of inoculant production has provided a unique opportunity for this SME to appraise the value of different biota (one of which, Variovorax paradoxus, has yet to be used commercially) to increase resistance to abiotic stress. Positive results have contributed to commercial development of single or combined inoculants that could be available for the European market in a near future.
• In intensive agricultural systems, biotechnological innovations (especially new inbred varieties) are a seminal factor of the production system that influences three basic dimensions:
(i) production structure by defining its integrating productive cycles, processes, methods and practises, including work organisation.
(ii) input consumption (water, fertilisers, etc) that has a strong effect on regulating plant production.
(iii) the technology matrix that integrates different types of engineering and biological technologies.
• One of the major problems of these technologies is their high development costs and the high degree of exclusivity and control by the multinational companies. Farmers (as users of these technologies) have relatively little input into their development. In the EU-27, about 90% of the farms comprise less than 20 ha and 95% less than 50 ha. Consequently, public support of the technological development of those companies is necessary to maintain agricultural competitiveness and sustainability.
• The overall impact of ROOTOPOWER deliverables is especially relevant to SMEs involved in the European horticultural sector. Micro, small and medium-sized enterprises (SMEs) with a high research focus are the economic powerhouse behind scientific and technological developments in agriculture, playing a pivotal role in the success of a European Knowledge-Based Bio-Economy. Although specialised horticulture accounts on average for about 8% of the total agricultural production in Europe, this percentage increases to 13% in Southern European countries such as Spain, Portugal and Italy, and to 40% in highly specialised regions such as Murcia, Andalucía and The Netherlands. The most recent available data in Eurostat does not include Agriculture, but most of the companies in this sector are SMEs and account for the largest employment generation (eg. 36% of the sector in Holland, year 2005).
• The inclusion within ROOTOPOWER of several SMEs with specialised skills (biotic inoculants, rootstock breeding and tomato growers) (i) ensures that the knowledge generated within this proposal is suitable for application in agricultural systems that the SMEs have extensive knowledge of, thus decreasing the time from scientific innovation to general application, and (ii) contributes to their strategic development into the economic sector.
‘’In relation to natural resource (water and nutrients) management’’
While natural resource management (water and nutrients) is a global issue, the results of the ROOTOPOWER project can have a greater impact in Mediterranean countries, where degradation of soil and water resources are major constraints to economic growth, lowering standards of living among rural Mediterranean inhabitants. About 70% of water resources are dedicated to irrigation in Mediterranean Countries, implying that any rootstock-mediated increase in crop water use efficiency will save a significant amount of water. This would enhance compliance with the EU water policy, especially in areas with high level of water stress on the aquifers, where it is compulsory to reduce and eliminate the gap between uptake and recharge. Development of more salt-tolerant rootstocks will allow aquifers that are currently unused (due to their high salt loads) to become a viable resource (thus decreasing pressure on those with high quality water in existing, likely unsustainable use). Changes in water use patterns could potentially make additional water available for other agricultural, industrial and domestic uses.
The transfer of nitrogen (N) and phosphorus (P) from land to water (termed “diffuse pollution”) presents a problem for most agricultural systems, with resulting eutrophic downstream impacts on rivers, lakes and even estuaries. Key processes involved include the accumulation of nutrient sources in the soil-crop system, mobilisation through solubilisation and subsequent leaching and runoff, mobilisation through physical (particle/colloid) detachment and subsequent runoff, mobilisation through incidental transfers of recently applied fertilizer (that is not yet equilibrated with the soil-plant system) and finally issues of subsequent transport through the landscape, with the combination of these processes across a scale known as the ‘transfer continuum’. Recent work has estimated the potential impact of climate change on mobilisation of diffuse nutrients as a result of changes in temperature. Phosphorus emissions from agriculture must be restricted if we are to address potentially catastrophic future P-limitations to agriculture. Climate change predictions of wetter winters and drier summers in northern Europe may increase soil water storage (and runoff or drainage below the root system) during the winter months, but cause crop water stress during summer. Crop rooting patterns (deep roots) buffered the impact of rainfall amount on crop yield. The discovery of rootstocks with increase nutrient use efficiency through a more adequate root system morphology and/or nutrient uptake capacity within the project may provide a way of mitigating diffuse pollution effects.
According to the Strategic Research Agenda produced in 2007 by the European Technology Platform 'Plants for the Future' which identified five challenges for Europe’s society and economy to which the plant sector generally can contribute, the ROOTOPOWER project results could potentially impact the following aspects:
Healthy, safe and sufficient food and feed: Agriculture faces new challenges such as climate change and society’s expectations of reducing the environmental footprint of agriculture, while at the same time providing sufficient food to a burgeoning world population. ROOTOPOWER has embraced these aspirations and can make a telling contribution to crop resource efficiency by identifying rootstocks and root-specific traits contributing to “produce more with less” thus allowing the sustainable intensification of agriculture.
Sustainable agriculture, forestry and landscape: the identification of genes conferring resource use efficiency within ROOTOPOWER represents a key output to enhance sustainability: both by diminishing nutrient losses from agriculture and fertiliser inputs (which are expensive to produce in the case of N, and increasingly scarce in the case of P and K).
‘’Translating the scientific discoveries to market ‘’
Agricultural productivity must increase by 60% to feed the expected population of 9.6 billion people in 2050, while climate change may also reduce crop productivity. Pressure for cheaper, high quality and enhanced nutrient content tomato and other horticultural crops is increasing worldwide. Moreover, EU farmers must manage expensive costs and environmental restrictions unlike producers in emerging countries. The only way to produce high quality products while minimising economic costs is to produce intensive and resource-efficient high-yielding crops. Plant breeding and the use of horticultural techniques including grafting and the use of microbial inoculants will play a key role in developing more profitable horticulture to address these challenges.
The use of rootstocks has enhanced productivity of woody perennial crops for centuries. Grafting of vegetable crops has developed very quickly in the last 50 years, mainly to induce shoot vigor and to overcome soil-borne diseases in susceptible crops. Rootstocks can optimize vegetable crop productivity by (i) increasing the yield potential; (ii) maintaining yield in the face of biotic (ie. fungi, bacteria, virus, nematodes) and abiotic (ie. salinity, drought, low nutrients, extreme temperatures, soil compaction) stresses; (iii) decreasing the use of environmentally damaging pesticides and fertilizers; and (iv) increasing the efficiency of use of natural resources of water and soil. The socioeconomic and environmental potential impact of grafting is enormous. However, both the development of new rootstocks and new applications in agriculture are hampered by limited knowledge of the physiological and genetic mechanisms underlying rootstock x scion (shoot) x environment interactions.
Overall, the results of ROOTOPOWER show that use of novel rootstocks can improve tomato yield under stress conditions. This paves the way for their use in plant breeding programs. Furthermore, the new genes that we identified could be markers for breeding in related species of Solanaceae (pepper and eggplant) and Cucurbitaceae (cucumber, melon and watermelon) botanical families.
Main dissemination activities and exploitation of results
The dissemination and exploitation of project results was addressed in WP7 “Dissemination and IPR”, whose general objectives were to make the largest possible use of project outcomes and maximize the benefits of advances in other initiatives.
In accordance with the strategy defined in Annex I, the dissemination of knowledge deriving from ROOTOPOWER has been made through the following activities:
• Participation in scientific events in which ROOTOPOWER partners have had the opportunity to present information and results on the project, for instance, the VI SEST-ISHS Symposium, the XVIII FESPB-EPSO Plant Biology Congress, the Environment Workshop 2013, the 10th Solanaceae Conference, the 1st ISHS International Symposium on Vegetable Grafting, the XVIIIth EUCARPIA Meeting on Genetics and Breeding of Tomato, 11th Solanaceae Conference (SOL2014), the SEB Annual Meeting in Manchester (SEB2014) and Prague (SEB2015), the 7th International Symposium on Root Development (2014), the 12th Solanaceae Conference (SOL2015), and the Plant and Animal Genome XXIV Conference (PAG2016), among other relevant conferences.
• Publication of scientific results in peer-reviewed journals and books: 8 articles have been published so far in Acta Horticulturae, G3-Genes Genomes Genetics, Theoretical and Applied Genetics, Journal of Experimental Botany, Functional Plant Biology and Molecular Breeding, while some others are under preparation. Given the high amount of data and the need to arrange it so as to draw up sound publications, most publications will be ready during the course of 2016.
• Key activities targeted to the general public has led to a better understanding of root-targeted strategies to minimize abiotic stress impacts on horticultural crops, and to increase the education of the general public and schoolchildren on the field. Activities specifically focused on the general public include publications in the general press, a TV interview, handing out brochures, publication of general information in the Twitter account of the project and participation in science fairs. As per schoolchildren, ROOTOPOWER took part in the XII Week of Science (Murcia, Spain) and the London International Youth Science Forum (Cranfield, UK). Furthermore, an educational workshop entitled “Vegetables do not grow in the fridge: scientists for one day” was organised for students attending a primary school in Murcia (Spain). Specific tailored material was prepared for this specific workshop.
• Website: the project website consists of a public and a restricted private area. The publicly accessible pages have provided information on this project throughout its lifetime. For instance, a calendar of conferences in which ROOTOPOWER participation was foreseen, a list of media activities, general project brochures and a summary of communications presented during scientific events were made available in the public area of the webpage. The private area was restricted to project partners in order to share and disseminate research related knowledge and data. The website has supported a close co-operation between the Consortium partners and has been useful for raising public awareness.
• Preparation of a project newsletter targeted at the academic and scientific community. This newsletter has been issued once per year and has been duly disseminated through the project webpage and other potential stakeholders interested in the progress of the project (e.g. ISHS, EPSO, etc.).
• Organization of scientific workshops: two international workshops have been organized with invited speakers and lectures specifically oriented to the academic and scientific community, namely the 1st Rootopower workshop entitled “Phenotyping, Physiology and Genetics of Rootstock-Mediated Tolerance to Soil Abiotic Stresses in Tomato” and held within the framework of the I COST Action FA1204 Annual Conference (Murcia, Spain) and the 2nd Rootopower workshop under the title “Understanding the Power of Root Traits for Producing More with Less”, jointly organized with the XXIX International Horticultural Congress (Brisbane, Australia). In order to ensure economies of scale and provide value for money, they were organized within already scheduled high-level international events in collaboration with major international organisations.
• Organization of technical workshops: a series of local workshops have been organized in different countries of participating partners, namely Spain, Turkey and the Netherlands. The goal of these informative workshops was to disseminate project results in its technological dimension and they were aimed towards industry stakeholders. They were mainly conducted during the last quarter of the project implementation in collaboration with national farmer, consumer organisations and governmental institutions. In other countries (Belgium and United Kingdom), contact has been constantly maintained with companies that may be interested in the project results and their potential application.
Two aspects worth highlighting are: 1) following Article 45 RfP – Article II.30.4 of the GA, the statement of financial support from the CE has been included in the dissemination material generated within the framework of the project, and 2) all publications, press releases, and other outward communications from the project have been sanctioned by the IP Committee of the project to avoid difficulties in the protection of potential foreground production.
List of Websites:
http://www.rootopower.eu/
Dr. Francisco Pérez-Alfocea (coordinator)
CEBAS-CSIC
Campus Universitario de Espinardo, s/n. Aptdo. 164
30100 - Murcia
SPAIN
