EXPLORATION OF THE MICROBIOME OF NATIVE PLANTS FOR PLANT GROWTH-PROMOTING MICROBES TO MITIGATE EFFECTS OF ABIOTIC STRESSES IN CROP PLANTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1030994
• Grant No.: 2023-67014-40536
• Cumulative Award Amt.: $849,928.00
• Proposal No.: 2022-11070
• Project Start Date: Aug 1, 2023
• Project End Date: Jul 31, 2027
• Grant Year: 2023
• Program Code: [A1402]- Agricultural Microbiomes in Plant Systems and Natural Resources

Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322

Performing Department
Plants Soils & Climate

Non Technical Summary
Climate change induces extreme abiotic stresses such as drought and salinity, which are detrimental to agricultural production. Plants native to drought and salinity stress-affected regions can thrive in those conditions due to the interactions with their rhizosphere, a rich habitat of various microbes that directly or indirectly affect plant growth and development. The Intermountain West region in the US is rich in drought-tolerant native plants and recommended for low-water landscape plants. Native plants in this region have formed symbiotic relationships with native wildlife over thousands of years and offer the most sustainable habitat. Although many plant growth-promoting bacteria (PGPB) isolated from plant rhizospheres are used as successful biofertilizers, native plants have not been explored for their microbiome. One way to meet the increasing food demand due to the escalating world population is by exploring the microbiome of native plants that helps them establish and survive in their habitat. The proposed project will investigate the microbiome of two native plants,Ceanothus velutinus(Snowbrush),Cercocarpus ledifolius(Mountain Mahogany), of the Intermountain West Region of the US, and plants from the Great Salt Lake. The study will then isolate, purify, characterize the rhizosphere bacteria, and test them for plant growth promotion under abiotic stresses on the model plant Arabidopsis. Selected PGPB will be characterized further by whole genome sequencing and will be validated on selected grain (maize and wheat), vegetable (onion and watermelon), and forage (alfalfa) crops. The natural microbiome of native plants will help mitigate crop yield reductions caused by abiotic stresses.

Animal Health Component

70%

Research Effort Categories

Basic

15%

Applied

70%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
101	0199	1040	25%
111	1499	1010	25%
131	1510	1060	25%
605	4099	1103	25%

Knowledge Area
605 - Natural Resource and Environmental Economics; 111 - Conservation and Efficient Use of Water; 101 - Appraisal of Soil Resources; 131 - Alternative Uses of Land;

Subject Of Investigation
1499 - Vegetables, general/other; 4099 - Microorganisms, general/other; 1510 - Corn; 0199 - Soil and land, general;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1103 - Other microbiology; 1010 - Nutrition and metabolism;

Keywords

abiotic stress

ecosystem. crops

growth-promoting microbes

microbiome

native plants

Goals / Objectives
The proposed project will investigate the rhizosphere and endosphere microbiome of the native plants of the Intermountain West Region of the US. The study will isolate, purify, and characterize these microbes and test them for plant growth promotion under abiotic stresses on grain, vegetable, and forage crops. The final goal is to develop biofertilizers and biostimulants to promote the growth and development of various crops under drought and salt stress. Many plant growth-promoting bacteria (PGPB) have been characterized, and a few are successfully used as biofertilizers. The US. Intermountain West is rich in drought-tolerant native plants. Native plants have not been explored for their rhizosphere microbiome. Investigation of the rhizosphere microbiome of native plants may open a new chapter for using them as biofertilizers for sustainable agriculture under adverse conditions. In this study, we propose a complete metagenomic analysis of the rhizosphere and endosphere (microbes colonize inside the roots) microbiome of two native plants,Ceanothus velutinus(Snowbrush) andCercocarpus ledifolius(Mountain Mahogany), of the US. Intermountain West and halotolerant bacteria from the plants from the Great Salt Lake. The Great Salt Lake is rich in Halotolerant plants. We hypothesize that these plants have halotolerant bacteria in their rhizosphere and have the potential to mitigate the salinity stress in crop plants.Similarly, we hypothesize that above mentioned native plants harbor drought-tolerant bacteria that have the potential to impart drought tolerance to crop plants.?We propose to isolate, purify, and characterize microbes from these plants and plants from the Great Salt Lake. Investigate these microbiomes' ability to promote crop plants' growth and development under drought and salt stress in the greenhouse, and finally test them in fields of the grain crops wheat and maize, the vegetable crops onion and watermelon, and the forage crop alfalfa.Specific objectives:Establish the composition of microbial communities in the rhizosphere and endosphere of native plants -Ceanothus velutinus(Snowbrush) andCercocarpus ledifolius(Mountain Mahogany) and halotolerant bacteria from the plants of the Great Salt Lake by comparative metagenomics.Isolate, purify, and characterize rhizosphere bacteria of Snowbrush, Mountain Mahogany, and plants from the Great Salt Lake for plant growth-promoting activities.Establish and validate the plant growth-promoting ability of these bacterial isolates on Arabidopsis thaliana and conduct metatranscriptomics analysis to see the effect of bacteria on the plant's transcriptomics.Identify the plant growth-promoting bacteria by whole genome sequencing and evaluate the plant growth-promoting bacteria on maize, wheat, onion, watermelon, and alfalfa under normal, drought, and salt stress.An outline of the proposal with step-by-step objectives is depicted in Figure 1.

Project Methods
Methods:Establish the composition of microbial communities in the rhizosphere and endosphere of native plants -Ceanothus velutinus(Snowbrush),andCercocarpus ledifolius (Mountain Mahogany), and halotolerant bacteria from the plants of Great Salt Lake by comparative metagenomic.Rhizosphere soil and roots samples fromsnowbrushwill be collected from the Tony Grove Lake region near Logan, Utah. Rhizosphere soil and roots samples of Mountain Mahogany,will be collected from the area near the Wind Caves, Logan, Utah. The methodology described elsewhere will be used with slight modifications (McPherson et al., 2018). ?Microbial DNA will be isolated from the rhizosphere soil by Qiagen DNeasy PowerSoil Pro Kit and roots by DNeasy® Plant Mini Kit. The 16S rRNA gene will be amplified using the V4 variable region-specific primers 515F and 806Rand sequenced on the MiSeq using a 2×250 paired-end sequencing chip size (Illumina). .The sequence data will be analyzed using various packages available inRver 4.0(Team, 2021).Isolate, purify, and characterize rhizosphere bacteria of Snowbrush, Mountain Mahogany, and plants from the Great Salt Lake for plant growth-promoting activitiesThe rhizosphere pellets will be diluted by resuspending 10 grams of soil in 95 ml of sterilized water. This mixture will be serially diluted in 1:10 steps. Similarly, roots will be macerated and diluted to 1:10 (1g in 10ml water) and serially diluted in 1:10 steps. 100 µl of the 10-3, 10-4, and 10-5dilutions from rhizosphere soil will be spread onto agar plates with five different media compositions andplates will be incubated at 28oC for 3-5 days. Once growth appears, single colonies will be selected based on distinct visual characteristics such as color, texture, transparency, size, consistency, and any other distinct morphological trait, and selected colonies will be purified by the streak plate method(Woeste, 1996).The entire ~1,500 bp 16S rRNA region will be amplifiedfrom the isolated bacterial colonies using 27F and 1492Rprimers andsequenced. The sequences analyzed using BLAST (Basic Local Alignment Search Tool) against a 16S rRNA database on NCBI to identify the bacterial species.The bacterial colonies will be tested for their ability to produce siderophores, IAA, catalase, solubilizing phosphate, and to fix atmospheric nitrogen.Isolation of Halotolerant Bacteria from the plants in the Great Salt Lake:Roots samples will be collected aseptically in a sterile glass bottle from the plants in the Great Salt lake, brought into the lab in an ice cooler, and Rhizosphere soil will be separated from the roots as described above in section 1.1 and stored at 4oC.Samples will be enriched in liquid media or inoculated on different solid media with various NaCl concentrations. The objective is to obtain single colonies on the solid media to allow functional categorization and identification of halotolerant bacteria.Establish and validate the plant growth-promoting ability of these bacterial isolates onArabidopsis thalianaand conduct metatranscriptomics analysis to see the effect of bacteria on plant's transcriptomics.Testing PGPM onArabidopsis thaliana Col-0?One-week-old seedlings will be transferred to a 3.5-inch pot filled with LC1 soilless media (Sun Gro horticulture) and treated with 5ml of 1 OD600bacterial suspension in 0.9% NaCl solution. One seedling per pot and five replicates in the complete randomized block will be used per treatment. The bacterial treatment will be applied once again after one week. The biomass of plants will be measured after six weeks of treatment in a growth chamber.Under applied abiotic stresses, we will extract total RNA and conduct whole RNA comparative (before and after stresses) transcriptomics to study differential gene expressions.The selected PGPB based on plant growth promotion activities on Arabidopsis will be identified by whole genome sequencing(https://en.novogene.com/services/research-services/genome-sequencing/whole-genome-sequencing/#). TheGFPuv vector pDSK-GFPuv that contain GFPuv fragment and glow under UV light will be transformed in each bacterium to monitor the colonization of bacteria in the rhizosphere and in the roots(Wang et al., 2007). Identified PGPB containing pDSK-GFPuv will be screened on maize, wheat, onion, watermelon, and alfalfa under drought and salt stress in the greenhouse.Validation under drought stress:The seeds will be planted in the half-gallon pot filled with a soilless media (peat moss 75%, vermiculite 13%, and rice hulls 12%) and treated with 100ml of 1 OD600 bacterial suspension in 0.9% NaCl solution by pouring. Once the seed germinated, the individual seedlings will be transferred to individual half-gallon pots with soilless media and treated with 1 OD600bacterial suspension in 0.9% NaCl solution by pouring twice afterone week interval. The drought stress will be imposed slowly by withdrawing water for nine days or until the control plants start dying. After the drought stress, plants will be watered for one week. The plants will be observed for survival after the rewatering. Once the PGPB is screened for drought tolerance, the positive isolates will be retested for drought stress and various droughts stress related parameters such as electrolyte leakage, Relative Water content, Stomatal conductance, net photosynthetic rate, and gene expression of stress responsive genes.Validation under salinity stress:The testing for salt stress will be done in Dr. Devinder Sandhu's lab in Riverside, CA. Plants will be generated either from cuttings or seeds and submitted to target salinities, sufficient to trigger plant physiological, biochemical, and genetic responses to salinity in different plant species evaluated. Plants will be cultivated in pots filled with a sand/soil mixture and irrigated daily by dripping.Pots will be maintained in a greenhouse with controlled temperatures under natural illumination. Plants will be evaluated for biomass accumulation, and shoot and root ion compositions.In the final year of the project, the shortlisted PGPB ( without GFPuv plasmid) will be tested under field conditions for plant growth and development with and without abiotic stress (drought and salinity). Corn, wheat, and alfalfa testing will occur at two locations in central Utah, one in Uintah County and the other in Emery County. Both are farms associated with USU and the Utah Agricultural Experiment Station and have soils that are naturally high in salinity and irrigation systems that can be restricted to impose drought. Onion and watermelon will be tested at the USU Greenville Farm in Cache County where drought and salinity stress will be maintained artificially.Efforts and EvaluationThe results and outcomes will be presented at University, National, and international conferences, such asASA-CSSA-SSSA annual meeting,the ASPB,and ASHS meeting.The results will be published in the peer-reviewed journals such as Scientific reports, Frontiers of Plant Sciences, BMC- Microbiome, Crop Sciences, and Microbiology Spectrum.The evaluated PGPB in greenhouse and field conditions will be patented.We will also get data on how microbes interact with the plants and regulate plants' transcriptomics. Besides this, we will also obtain data about the microbial diversity in the rhizosphere and endosphere of two native plants and plants from GSL. All data will be deposited to the respective depositories at NCBI for public use.In the final year of the project, we will hold a field day at each of the three-field testing that are located in Cache, Emery, and Uintah Counties for farmers, crop consultants, and other clientele to learn about drought and salinity management and the potential for PGPB to improve crop growth under drought and salinity stress. Evaluation of each field day will occur by means of a retrospective assessment to measure the knowledge of participants before and after each educational event.

Progress 08/01/23 to 07/31/24

Outputs
Target Audience:The preliminary results were presented at field days organized by the Student Organic Farm at USU, Logan in August 2023. The target audience, which includes farmers, nursery producers, faculty, and students, is an integral part of this community. The audience was educated about the practical applications of the research findings, particularly the importance of native plants and their microbiome, plant-growth-promoting bacteria, and their use as biofertilizers in sustainable agriculture and nursery production. This knowledge empowers them to make informed decisions in their respective fields. Changes/Problems:We found drought-tolerant curl leaf and narrow-leaf mountain mahogany in the western desert of Utah. So, we decided to isolate PGPRs from these two varieties in the Western desert instead of the wind caves of Logan Canyon, where only the curl-leaf variety is available. Plants in this location have more potential to harbor drought-tolerant microbes. What opportunities for training and professional development has the project provided?One undergraduate student was trained and one Ph.D student was hired at the end of year. How have the results been disseminated to communities of interest? The results were disseminated to the scientific community, farmers, and stakeholders through conference presentations, field days, and manuscript publications. Filed days/ Fair Ty Wilson, Josthna Ganesh, Ananta Devkota, Katie Hewitt, and Amita Kaundal*, "Isolation of Native Plant's Microbiome for Plant Growth-Promoting Bacteria" CWEL Field Day, Greenville Research Farm, 2024. Ty Wilson, Josthna Ganesh, Ananta Devkota, Katie Hewitt, and Amita Kaundal*, "Isolation of Native Plant's Microbiome for Plant Growth-Promoting Bacteria" Student Organic Farm, Utah State University, 2024. Ty Wilson, Josthna Ganesh, Ananta Devkota, Katie Hewitt, and Amita Kaundal*, "Isolation of Native Plant's Microbiome for Plant Growth-Promoting Bacteria" Undergraduate Research Fair, Utah State University, 2024. Publications Ganesh, J., Hewitt, K., Devkota, A. R., Wilson, T., and Kaundal, A*. (2024). IAA-producing plant growth promoting rhizobacteria from Ceanothus velutinus enhance cutting propagation efficiency and Arabidopsis biomass. Frontiers in Plant Science, 15. :1374877. https://doi.org/10.3389/fpls.2024.1374877 Posters Devkota, A. R. and Kaundal, A. (2024). Plant growth-promoting rhizobacteria (PGPR) from the native plant Ceanothus velutinus promote growth in Tall Fescue. Food Security and Solutions Symposium, Utah State University, April 17, 2024 What do you plan to do during the next reporting period to accomplish the goals? Finish isolation and microbial diversity analysis of mountain mahogany. Finish isolation of halotolerant bacteria and microbial diversity analysis of plants from Great Salt Lake. Characterization and screening of drought tolerant PGPRs onArabidopsis thaliana. Testing of shortlisted isolates on maize, watermelon, and Wheat. Testing shortlisted isolates for salinity tolerance on maize, watermelon, and Wheat.

Impacts
What was accomplished under these goals? Climate-induced environmental stresses and overuse of chemical fertilizers are pressing issues that require a collaborative effort to address. To this end, we propose a study that will involve researchers and stakeholders from various fields. Our goal is to develop sustainable agriculture methods that can mitigate the effect of these environmental stresses on plants and improve soil health. As part of this approach, we are investigating the underground microbiome of two native plants of Utah and plants of Great Salt Lake, with the aim of developing them as biofertilizers to combat drought and salinity stress in plants and improve soil health. Accomplishment under Goal 1 We are pleased to announce the successful completion of a comparative analysis of the rhizosphere and endosphere microbiome ofCeanothus velutinusfrom three locations, marking a significant milestone in our research. Publication is in preparation for this work. Soil and root samples for two varieties ofCercocarpus ledifolius (Mountain Mahogany), curl-leaf mountain mahogany, and narrow-leaf mountain mahogany have been collected from the southwest desert of Utah. DNA has been extracted from the samples, and 16S rRNA sequencing isin progress. Accomplishment under Goal 2 Plant growth-promoting rhizobacteria have been isolated from the Ceanothus plants from their native location, greenhouse ceanothus plants treated with native soil, andcutting of ceanothus treated with native soil. Around 100 isolates were characterized for plant growth-promoting activities like the capability to produce Indole acetic acid (IAA), catalase, protease, ammonia, siderophore, ability to fix nitrogen, solubilize phosphate, oxidize sulfur, and the ability to use ACC as nitrogen source. Thirty isolates were selected for screening onArabidopsis thaliana for growth promotion.Two manuscripts are published from this study. The isolation of PGPRs from the rhizosphere of the curl leaf and narrow leaf is in progress. The isolation of halotolerant PGPRs from the plant of the Great Salt Lake is in progress. Accomplishment under Goal 3 Out of thirty isolates, seventeen were tested onA. thalianafor plant growth promotion. Eight isolates showed a significant increase in shoot biomass. Accomplishment under Goal 4 Eight isolates were tested on two wheat genotypes, and four were tested on two Tall fescue genotypes for growth promotion. Six isolates showed positive interactions with wheat and tall fescue. One manuscript from a tall fescue study is under review. Eight shortlisted isolates were tested for their ability to grow on 2-12% NaCl in LB media, and four of them were tested for salt tolerance on maize.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Ganesh, J., Hewitt, K., Devkota, A. R., Wilson, T., and Kaundal, A*. (2024). IAA-producing plant growth promoting rhizobacteria from Ceanothus velutinus enhance cutting propagation efficiency and Arabidopsis biomass. Frontiers in Plant Science, 15. :1374877. https://doi.org/10.3389/fpls.2024.1374877