Periodic Reporting for period 1 - FUNVINE (Global assessment of grapevine microbiome function to promote sustainable vineyard management)
Período documentado: 2022-10-16 hasta 2025-01-15
Vineyards represent a large potential source of ecological impact as they cover a vast portion of land worldwide, and also represent an important source of income for millions of people. Given these ecological and sociological roles, it is important to identify new ways to promote vineyard sustainability, including through the investigation of ‘the unseen majority’ of microbiota that associate with grapevines. The plant microbiome, composed of bacteria, fungi, and micro-fauna, performs a suite of vital functions that promote the stress resistance, nutrition, productivity, and fitness of wild and cultivated species across the globe. Plant microbiomes play an outsized role in plant health and are notably important for nutrient cycling, resistance to abiotic and biotic stress, and can dramatically influence overall plant fitness through pathogenesis and symbiosis. The microbiome of grapevines (Vitis vinifera) is an integral component of the production system, from the vineyard to the winery. While the microbiome of vineyards is often studied at the local scale considering single grape varieties, the existence of a functional and taxonomic microbiome shared by grapevines across contrasting environments is undetermined. For an industry experiencing the immediate impacts of climate change, with a global market size of $417 billion USD in 2020, and a footprint of >7.4 million hectares, my research project elucidating the capacity of microbial functions, e.g. pathogen suppression or enhanced nutrient cycling, will provide important knowledge to support economic and ecological benefits that can help to promote vineyard sustainability worldwide.
Despite advances in exploration of the grapevine microbiome in recent years, there is an important knowledge gap regarding the integration of myriad environmental stressors, growing regions, and varieties in order to understand grapevine microbiome composition and functionality, particularly in response to a rapidly changing global climate. To overcome this knowledge gap, I will expand the goals of a global initiative identifying the grapevine microbiome composition (VINE MICROBIOME) by characterizing the functional profiles of the grapevine microbiome globally (FUNVINE). This knowledge is essential to provide a better understanding of the key relationships between functional microbiomes and crop health and sustainability, helping to support the EU viticulture industry and UN Sustainable Development Goals (SDG-2; 13; 15). We will identify the global patterns of functional genes associated with plant productivity and plant fitness across contrasting environmental conditions and grape varieties. Critical functions performed by the soil microbiomes have been shown to mitigate pathogen efficacy, increase resistance to environmental stressors such as drought or temperature increases, and notably alleviate nutrient limitations through mutualism. These functional capacities of microbiomes can alter the health, nutritional value, and productivity of crops worldwide. Given the vast ecological and socio-economic importance of the global wine industry, FUNVINE will deliver results that can expand knowledge of the grapevine microbiome to researchers and viticulturists. Additionally, FUNVINE aims to identify sustainable management scenarios that consider the impacts of global climate change stressors as well as the declining returns of expensive and deleterious chemical fertilizers, pesticides, or insecticides used in the industry relative to the declines in pathogenesis, stress-relief, and nutritional provisioning of region-specific, grape variety-specific microbiomes. FUNVINE will include observational and experimental work to investigate the functional microbiome of vineyards aiming to address critical knowledge gaps and alleviate the environmental change-driven challenges to vineyards.
The FUNVINE project had two major research objectives:
Objective 1: To characterize the leaf and root functional microbiome of grapevines across the globe.
Objective 2: Experimental management of the grapevine microbiome under global change stress.
Despite advances in exploration of the grapevine microbiome in recent years, there is an important knowledge gap regarding the integration of myriad environmental stressors, growing regions, and varieties in order to understand grapevine microbiome composition and functionality, particularly in response to a rapidly changing global climate. To overcome this knowledge gap, I will expand the goals of a global initiative identifying the grapevine microbiome composition (VINE MICROBIOME) by characterizing the functional profiles of the grapevine microbiome globally (FUNVINE). This knowledge is essential to provide a better understanding of the key relationships between functional microbiomes and crop health and sustainability, helping to support the EU viticulture industry and UN Sustainable Development Goals (SDG-2; 13; 15). We will identify the global patterns of functional genes associated with plant productivity and plant fitness across contrasting environmental conditions and grape varieties. Critical functions performed by the soil microbiomes have been shown to mitigate pathogen efficacy, increase resistance to environmental stressors such as drought or temperature increases, and notably alleviate nutrient limitations through mutualism. These functional capacities of microbiomes can alter the health, nutritional value, and productivity of crops worldwide. Given the vast ecological and socio-economic importance of the global wine industry, FUNVINE will deliver results that can expand knowledge of the grapevine microbiome to researchers and viticulturists. Additionally, FUNVINE aims to identify sustainable management scenarios that consider the impacts of global climate change stressors as well as the declining returns of expensive and deleterious chemical fertilizers, pesticides, or insecticides used in the industry relative to the declines in pathogenesis, stress-relief, and nutritional provisioning of region-specific, grape variety-specific microbiomes. FUNVINE will include observational and experimental work to investigate the functional microbiome of vineyards aiming to address critical knowledge gaps and alleviate the environmental change-driven challenges to vineyards.
The FUNVINE project had two major research objectives:
Objective 1: To characterize the leaf and root functional microbiome of grapevines across the globe.
Objective 2: Experimental management of the grapevine microbiome under global change stress.
Work was conducted through 8 work packages (WP) and 13 deliverables (D). At the end of the MSCA project period, the fellow has completed, in some form, WP1-7 and is currently preparing manuscripts to complete WP-8 (publication of two manuscripts), and delivered D1, D2, D3-6, and D11-13. Deliverables 7-11 are in preparation and include; organization of a sustainable viticulture research symposium in Andalucia, Spain (D7), simple public summary reports for objectives 1 & 2 with infographics (D8), a YouTube video detailing our findings (D9), and at minimum two peer-reviewed scientific journal articles (D10). These deliverables will be completed after the project reporting period as the accumulation of large datasets has taken some time; metagenomics data for Objective 1 is still being sequenced, and microbiome community and function (metagenomics) data for Objective 2 was recently received and still being processed. See below for more details.
FUNVINE Research Objective 1: Global grapevine microbiome functional profiling
Global vineyard sampling, processing, metadata and metagenomic analyses:
Globally, grapevine leaf and rhizosphere soils were collected from ~40 vineyards across 16 countries to perform total DNA extractions and metagenomic sequencing to identify functional profiles for the above and belowground compartments of grapevines. From each sampling site (n = 242), we determined environmental data using available databases, for example various climate metrics were extracted for each site using the WorldClim database (Mean annual temperature, Mean annual precipitation, Maximum temperature, Precipitation seasonality, etc.). Bulk soils were collected from all sites to identify biogeochemical metrics across the global survey. These bulk soil analyses included: soil pH, C (total and mineral organic), N (total and available), P (available) and micronutrients, bulk density, texture, aggregate fractionation as well as grapevine leaf C (total), N (total) and micronutrients. Microbial respiration was measured to determine microbial activity, and enzymatic activity was calculated to determine the metabolic potential in each soil (C cycle: (i) β-1,4-glucosidase, β-xylosidase, β-D-cellobiohydrolase and α-1,4-glucosidase; N cycle: β-1,4-N-acetylglucosaminidase, and leucine-aminopeptidase; P cycle: phosphatase; S cycle: arylsulfatase), and ultimately all of these analyses will be paired with data of microbiome composition (GLOBAL VINE) as well as functional gene expression data from metagenomic sequencing (FUNVINE).
Main Achievements: Global surveys are complex endeavors; collection and analysis of all samples is laborious, and therefore this portion of the FUNVINE project will take some months to complete final analysis and manuscript preparation. To date, DNA from all soils and leaves has been sent to the sequencing facility to begin metagenomic sequencing. Further, we have processed bulk soils from each site for all other biogeochemical analyses as well as microbial respiration and enzymatic activity analysis, which determines the potential of the microbiome to process soil organic matter, nitrogen, phosphorus, or sulfur.
FUNVINE Research Objective 2: Experimental management of the grapevine microbiome under global change stress
Experimental design, sampling, analyses:
A controlled experiment was carried out using two grape varieties (Merlot and Garnacha), under factorial combinations of drought (intense and prolonged 2 week drought versus no drought) and fertilization (moderately high and no fertilization), and also received either no soil inoculation or inoculation of whole soils sourced from 22 vineyards across 8 countries in the EU. This experiment began in March of 2023 and was completed in November of 2023, with rootstocks of grapevines (i.e. first year planting) growing for a total of ~6 months in 18L pots prior to harvest of aboveground biomass (new growth) and soil sampling for biogeochemical analyses as well as microbiome analyses of either a) microbiome composition (targeted amplicon sequencing using 16S and ITS regions for arachael/bacterial or fungal community analyses, respectively) or total DNA sequencing for b) microbiome functional profiling using metagenomics. Analyses for each targeted set of analyses are summarized below:
Main Achievements:
Grapevine microbiome management under global change stress - COMPOSITION
Soil samples (n = 200) were collected at the end of the 2-week drought period for all pots and taken to the lab for DNA extraction, as well as soil chemical and metabolic potential analyses. DNA was extracted using Qiagen Powersoil Pro kit following manufacturer protocol, DNA was quantified using Qubit, normalized concentration to 30 ng/uL, and shipped to sequencing facility for 2 x 150bp sequencing on Illumina MiSeq. Bioinformatics were performed using QIIME2 (99% similarity with Greengenes database for 16S (archaea/bacteria) or 97% similarity with the UNITE database for ITS (fungi). Statistical analyses were performed in R. For soil chemical analyses, we performed total C, total N analyses (combustion CHN analysis), as well as available N and available P using fluorometric analyses (Mehlic extractions). Total C and N was also determined for all grapevine leaves using CHN analysis.
Grapevine microbiome management under global change stress - FUNCTION
A subset of soil samples (n = 74) were used to perform metagenomic analyses. All 22 soil inocula were sequenced, as well as 2 homogenized mixes of original background soils. The experimental samples were from one variety of grapevine (Merlot), under unfertilized conditions, which experienced drought or no drought and all inoculations (n = 22) or 3 controls (only background soils) for both drought or no-drought treatments. Metagenomics protocol: Total genomic DNA was determined on 1% agarose gel, fragmented at 300bp with Covaris M220, and paired-end libraries were constructed using the TruSeq DNA Sample Prep Kit. Bridge PCR was performed using HiSeq 3000/4000 PE Cluster Kit, and sequencing was performed with HiSeq 3000/4000 SBS Kits. Bioinformatics is being performed using the SAMSA2 pipeline and QIIME2. Functional genes are being annotated using the KEGG database, and statistical analyses will be performed using R.
FUNVINE Research Objective 1: Global grapevine microbiome functional profiling
Global vineyard sampling, processing, metadata and metagenomic analyses:
Globally, grapevine leaf and rhizosphere soils were collected from ~40 vineyards across 16 countries to perform total DNA extractions and metagenomic sequencing to identify functional profiles for the above and belowground compartments of grapevines. From each sampling site (n = 242), we determined environmental data using available databases, for example various climate metrics were extracted for each site using the WorldClim database (Mean annual temperature, Mean annual precipitation, Maximum temperature, Precipitation seasonality, etc.). Bulk soils were collected from all sites to identify biogeochemical metrics across the global survey. These bulk soil analyses included: soil pH, C (total and mineral organic), N (total and available), P (available) and micronutrients, bulk density, texture, aggregate fractionation as well as grapevine leaf C (total), N (total) and micronutrients. Microbial respiration was measured to determine microbial activity, and enzymatic activity was calculated to determine the metabolic potential in each soil (C cycle: (i) β-1,4-glucosidase, β-xylosidase, β-D-cellobiohydrolase and α-1,4-glucosidase; N cycle: β-1,4-N-acetylglucosaminidase, and leucine-aminopeptidase; P cycle: phosphatase; S cycle: arylsulfatase), and ultimately all of these analyses will be paired with data of microbiome composition (GLOBAL VINE) as well as functional gene expression data from metagenomic sequencing (FUNVINE).
Main Achievements: Global surveys are complex endeavors; collection and analysis of all samples is laborious, and therefore this portion of the FUNVINE project will take some months to complete final analysis and manuscript preparation. To date, DNA from all soils and leaves has been sent to the sequencing facility to begin metagenomic sequencing. Further, we have processed bulk soils from each site for all other biogeochemical analyses as well as microbial respiration and enzymatic activity analysis, which determines the potential of the microbiome to process soil organic matter, nitrogen, phosphorus, or sulfur.
FUNVINE Research Objective 2: Experimental management of the grapevine microbiome under global change stress
Experimental design, sampling, analyses:
A controlled experiment was carried out using two grape varieties (Merlot and Garnacha), under factorial combinations of drought (intense and prolonged 2 week drought versus no drought) and fertilization (moderately high and no fertilization), and also received either no soil inoculation or inoculation of whole soils sourced from 22 vineyards across 8 countries in the EU. This experiment began in March of 2023 and was completed in November of 2023, with rootstocks of grapevines (i.e. first year planting) growing for a total of ~6 months in 18L pots prior to harvest of aboveground biomass (new growth) and soil sampling for biogeochemical analyses as well as microbiome analyses of either a) microbiome composition (targeted amplicon sequencing using 16S and ITS regions for arachael/bacterial or fungal community analyses, respectively) or total DNA sequencing for b) microbiome functional profiling using metagenomics. Analyses for each targeted set of analyses are summarized below:
Main Achievements:
Grapevine microbiome management under global change stress - COMPOSITION
Soil samples (n = 200) were collected at the end of the 2-week drought period for all pots and taken to the lab for DNA extraction, as well as soil chemical and metabolic potential analyses. DNA was extracted using Qiagen Powersoil Pro kit following manufacturer protocol, DNA was quantified using Qubit, normalized concentration to 30 ng/uL, and shipped to sequencing facility for 2 x 150bp sequencing on Illumina MiSeq. Bioinformatics were performed using QIIME2 (99% similarity with Greengenes database for 16S (archaea/bacteria) or 97% similarity with the UNITE database for ITS (fungi). Statistical analyses were performed in R. For soil chemical analyses, we performed total C, total N analyses (combustion CHN analysis), as well as available N and available P using fluorometric analyses (Mehlic extractions). Total C and N was also determined for all grapevine leaves using CHN analysis.
Grapevine microbiome management under global change stress - FUNCTION
A subset of soil samples (n = 74) were used to perform metagenomic analyses. All 22 soil inocula were sequenced, as well as 2 homogenized mixes of original background soils. The experimental samples were from one variety of grapevine (Merlot), under unfertilized conditions, which experienced drought or no drought and all inoculations (n = 22) or 3 controls (only background soils) for both drought or no-drought treatments. Metagenomics protocol: Total genomic DNA was determined on 1% agarose gel, fragmented at 300bp with Covaris M220, and paired-end libraries were constructed using the TruSeq DNA Sample Prep Kit. Bridge PCR was performed using HiSeq 3000/4000 PE Cluster Kit, and sequencing was performed with HiSeq 3000/4000 SBS Kits. Bioinformatics is being performed using the SAMSA2 pipeline and QIIME2. Functional genes are being annotated using the KEGG database, and statistical analyses will be performed using R.
FUNVINE Research Objective 1: Global grapevine microbiome functional profiling
- Due to logistical issues and delays in shipment and sequencing of global survey samples, this objective is still in the initial bioinformatic analysis phase and will most likely be completed within 6 months.
- We intend to identify common functional patterns on the extremes of environmental gradients across the global sampling effort, and predict that microbiome functional shifts associated with environmental gradients across scales will be dependent on plant factors including plant age, grape variety, and plant compartment.
- This will be the first global analysis of grapevine microbiome functional capacity, and have implications for vineyards around the world.
FUNVINE Research Objective 2: Experimental management of grapevine microbiome under global change stress
COMPOSITION:
- Inoculation with whole vineyard soil produced differential growth responses to drought for two important wine varieties. There were 14 of 22 inoculations which produced positive productivity responses after experiencing drought for Merlot, while there were only 8 for Garnacha.
- Merlot and Garnacha grapevines maintained significantly different fungal communities.
- Prokaryotic community composition was significantly different for both grapevine varieties under drought when compared to no-drought controls.
- Soil bacterial and fungal species richness was greater after drought under Merlot vines.
- Source-specific microbial taxa were identified that have positive relationships with vine productivity and leaf nutrition under drought conditions, suggesting important microbial functional roles in promoting grapevine tolerance to water stress.
FUNCTION:
Metagenomic analysis is in progress. We anticipate this part of the project will be completed within 6 months.
All results associated with the FUNVINE project will be uploaded to the FUNVINE project page on FigShare at: https://figshare.com/projects/FUNVINE_MSCA-PF-2021-101064192_/236507(se abrirá en una nueva ventana)
- Due to logistical issues and delays in shipment and sequencing of global survey samples, this objective is still in the initial bioinformatic analysis phase and will most likely be completed within 6 months.
- We intend to identify common functional patterns on the extremes of environmental gradients across the global sampling effort, and predict that microbiome functional shifts associated with environmental gradients across scales will be dependent on plant factors including plant age, grape variety, and plant compartment.
- This will be the first global analysis of grapevine microbiome functional capacity, and have implications for vineyards around the world.
FUNVINE Research Objective 2: Experimental management of grapevine microbiome under global change stress
COMPOSITION:
- Inoculation with whole vineyard soil produced differential growth responses to drought for two important wine varieties. There were 14 of 22 inoculations which produced positive productivity responses after experiencing drought for Merlot, while there were only 8 for Garnacha.
- Merlot and Garnacha grapevines maintained significantly different fungal communities.
- Prokaryotic community composition was significantly different for both grapevine varieties under drought when compared to no-drought controls.
- Soil bacterial and fungal species richness was greater after drought under Merlot vines.
- Source-specific microbial taxa were identified that have positive relationships with vine productivity and leaf nutrition under drought conditions, suggesting important microbial functional roles in promoting grapevine tolerance to water stress.
FUNCTION:
Metagenomic analysis is in progress. We anticipate this part of the project will be completed within 6 months.
All results associated with the FUNVINE project will be uploaded to the FUNVINE project page on FigShare at: https://figshare.com/projects/FUNVINE_MSCA-PF-2021-101064192_/236507(se abrirá en una nueva ventana)