Performing Department
(N/A)
Non Technical Summary
Bioinoculant products in agriculture represent one of the fastest-growing markets in the world, with an approximate 17% annual increase and an anticipated market value of $12 billion US by 2026. New regulatory constraints placed on the agrochemical industry are expected to reduce consumer demand for chemical inputs and position the microbial-based inoculants industry to absorb part of this current $250 billion market. The adoption of biological tools offers a promising pathway toward more resilient, efficient, and ecologically responsible agricultural systems, ultimately shaping the future of sustainable food production. However, the variable efficacy of microbial inoculants and our limited understanding of their effects on soil health and the microbiome have severely hampered the widespread adoption of microbial-based products. This proposed research will directly contribute to improving microbial inoculant success and advance our understanding of the interannual effects of biological amendments in soil. Aims 1 and 2 will leverage ecological theory to experimentally manipulate the components of propagule pressure (e.g., number of inoculations and number of cells per inoculation) to enhance inoculant establishment,improve plant performance, and understand the implications of microbial inoculations on soil health. This study focuses ontwo distinct organismal types (i.e., arbuscular mycorrhizal fungi - Rhizophagus irregularis, and bacteria - Bacillus licheniformi) associated with microbial-mediated phosphorus acquisition in plants.Our experimental approach will assess the effects of microbial amendments on plant performance, the soil microbiome, and a suite of soil health parameters. We will grow plants in a climate-controlled greenhouse over two consecutiveyears. At the end of each growing season, we will use high-throughput genomic tools (e.g., 16S and ITS amplicon sequencing) todescribe the soil microbiome following biological amendments applied at different rates. Additionally, we will employ a suite of biogeochemical measurementsand soilassays to understand the implications of microbialamendments on soil health and the associatedtemporal patterns over two growing seasons. In particular, the data generated from this experiment will directly contribute toimproving microbial inoculation methodsand aid in understanding microbial amendments' implications for increasing phosphorous acquisition. Together, this work willleadto a decreased dependence on synthetic agrochemicals in production agriculture systems and promote sustainablepractices in production agriculture.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
0%
Goals / Objectives
The overarching goals of this proposed research are (1) to improve the efficacy of commercially available microbial inoculants through the incorporation of ecologically informed recommendations; (2) to assess the effects of microbial inoculants on the soil microbiome's composition, function, and soil health parameters over growing seasons; and (3) to establish an inclusive and diverse K-12 outreach program with the participation of plant scientists and microbial ecologists working in agroecosystems. This project will capitalize on the convergence of low-cost sequencing and biogeochemical assays to study the effects of microbial inoculations in agricultural systems. This proposal directly relates to the AFRI Farm Bill Priority Area "Plant Health and Production and Plant Products" through (i) the potential applications of the work to plant growth and health in the face of agricultural intensification and global change, and (ii) the large economic potential of agricultural microbial amendments. In the long term, this project will enhance the sustainability of agricultural systems by improving the adoption of bioinoculants and decreasing dependence on synthetic agrochemicals.Objective1 - Improving AMF inoculations for enhanced phosphorous uptake by plants & Objective 2 - Improving bacterial inoculations for enhanced phosphorous uptake by plants:Similar methods will be applied across bacterial and fungal inoculations to improve the efficacy and establishment of these promising biological amendments. We will use both MCS and non-MCS wheat to understandthe importance of this root trait for the establishment of root-microbe symbioses.Goals: 1) produce data to show the components of propagule pressureaffect the establishment of biological inoculants by sequencing the soil microbiome and collecting various metrics of soil health, and 2) Publish results from this project in an open-access peer-reviewed journal focused.Objective3 - Outreach activity to increase the involvement of URMs in agroecology:Outreach efforts will promote URM involvement in STEM. Goals: 1) Create anactivity that will be available for use after the fellowship, 2)reach > 100 K-12 students through targeted outreach efforts at local K-12 schools.
Project Methods
The project will experimentally study the effect of MCS in roots and varying levels of propagule pressure of the biological amendments on the symbiosis establishment, plant growth benefit, and soil health parameters over two growing seasons. In these aims, I will apply either R. irregularis [36] (AGTIV® - Premier Tech Horticulture, Riviere-du-Loup, Quebec, Canada) or B. licheniformi (commercial product - SoluPhos® SP, JH Biotech, Ventura, CA, USA)- AMF and bacterial inoculant, respectively. Plants will be grown under controlled conditions and measurements of plant performance, soil health parameters, and the overall effect on the soil microbiome will be assessed across treatments varying in MSC/no-MCS and propagule pressure. Next, I will overwinter pots and replant them without additional inoculations to examine the extent to which varying levels of propagule pressure result in legacy effects on plant performance and soil properties.Soil collections and greenhouse growing conditions: Phosphorus-limited soils ('Low Phosphorous Field 300', soil P-levels 7-10 ppm) will be collected from the Russel E. Larson Agricultural Research Center located in the Pennsylvania Furnace, PA. This P-limited soil will increase the dependence of plants on its microbiome to assist with P acquisition. The experiment will be performed using wheat (Triticum spp.) as a model plant. Wheat is the second most widely grown cereal crop worldwide. This project will use the wheat genotype Triticum aestivum cv. RB07 (CV-1028, PI 652930 -- hard red spring wheat) - varying in the presentence of MCS. Pots will be set under an automated irrigation system to maintain moisture levels at 60% of water holding capacity over the entire growth cycle. Individual pots will be fertilized every 15 days with Hoagland nutrient solution depleted in P. Following the first collection at the end of the growing season, pots will be overwintered and replanted the following summer under the same conditions.Experimental design: The first growing season will include inoculations of commercially available microbial amendments that promote phosphorous uptake by plants. Plants will be grown for three months under greenhouse conditions. Harvests at the end of the first and second growing seasons will include the assessment of plant performance metrics (e.g., height, root and shoot biomass, leaf P- and chlorophyll content), soil physicochemical characteristics (e.g. P-availability, total C & N, organic matter content, wet aggregate stability), microbial community composition (e.g., 16S and ITS amplicon sequencing), and microbial function (e.g., extracellular enzyme assays). In the second growing season, plants will be grown under the same conditions, except there will be no microbial inoculations. A second growing season allows for an evaluation of the legacy effects of microbial inoculants on plant performance and soil health parameters.Microbial inoculations: In year one, inoculations will be performed at one of nine possible combinations of propagule size (e.g., the number of individual microbial cells introduced each time) and number (e.g., the number of independent introductions). Inoculations will range from one to three independent events in the first growing season (i.e., propagule number = 1, 2, or 3) and propagule sizes ranging from 0.33x to 3x manufacturer recommendations (e.g., the number of cells introduced at each event). Additionally, non-inoculated controls will be included to determine the baseline P acquisition and growth promotion from the native microbial community in the soils. This design will result in 10 treatment groups, with five replicates each and with two wheat root phenotypes. A total of 100 independent samples will be collected in each year of the experiment [2 root phenotypes x 10 treatments x 5 replicates = 100 samples] x 2 years = 200 total samples.Plant harvest, soil collection, AMF colonization, and plant performance measurements: At the end of each three-month growing season, soil samples will be collected for downstream analyses, and plant performance will be assessed. First, before harvest, individual plant height will be recorded. Next, plants will be carefully removed from pots, and excess soil adhering to the roots will be removed by shaking vigorously. Soil samples will be collected by combining four 5g subsamples to capture variation within each pot. These tubes will be frozen at -80 °C for DNA extraction and -20 °C for enzymatic analysis. Additionally, 50 g of soil will be collected from the bulk soil of each pot. This soil will be used for the physicochemical analyses. After soil collection, above- and belowground plant biomass will be assessed by clipping the plant at the crown and these compartments will be weighed separately. Plant roots will be subjected to AMF quantification via microscopy using a standard staining protocol based on 5% blue ink/vinegar. Additionally, measures of leaf P and chlorophyll content will be assessed via colorimetry, and a SPAD-502 chlorophyll meter (Minolta Camera Co., Tokyo, Japan), respectively. To statistically assess the effect of propagule pressure on plant performance and AMF colonization, I will use generalized linear models with propagule size, number, and MCS status as factors.Soil physicochemical and enzyme analyses: Soil physicochemical properties and enzyme assays will be measured on subsamples of bulk soils. All physicochemical analyses will be conducted by the Penn State Agricultural Analytical Services Lab. The measured parameters will include soil pH, extractable P, K, Ca, and Mg via the Mehlich 3 (ICP) method, total organic matter content via loss on ignition, total N and C via combustion, and wet aggregate stability. To statistically assess the effect of propagule pressure on soil health and enzyme activities, I will use the same modeling approaches as for plant performance.DNA extraction, amplicon sequencing, and analysis - Microbial community composition will be profiled by amplicon sequencing on Illumina's HiSeq platform with 2x250 paired-end reads, using the primer pair 515f and 806r, for bacteria, and the primer pair fITS7 and ITS4 for fungi. Sequencing will be performed at PSU's genomics core facility. Briefly, DNA will be extracted from 0.25 g of rhizosphere soil using a DNeasy PowerSoil Pro extraction kit (Qiagen). The DNA extracts will be used to prepare amplicon libraries. Sequence data will be processed in Rusing DADA2.Patterns of community composition will be analyzed using common microbiome statistical approaches to understand variation across treatments, growing season, and MCS status (e.g., PERMANOVA and PCoA of Bray-Curtis distances). Microbial count regression modelswill be used to identify taxa that are differentially abundant across treatments and MCS status.