Research Project: Ecology and Genomics of Soilborne Pathogens, Beneficial Microbes, and the Microbiome of Wheat, Barley, and Biofuel Brassicas

Location: Wheat Health, Genetics, and Quality Research

2024 Annual Report

Objectives
The long-term objective of this project is to develop biologically based technologies for controlling soilborne pathogens of wheat, barley and brassica crops grown as part of cereal-based production systems. Three specific objectives will be addressed over the next five years. Objective 1: Determine the components of plant and soil microbiomes that promote the health of wheat, barley, and biofuel brassica crops and are responsible for disease suppressive soils. Sub-objective 1A: Define components of the wheat microbiome correlated with soil health. Isolate, culture and identify specific bacterial and fungal taxa. Test in the greenhouse for ability to protect against biotic stress (soilborne pathogens) and abiotic stress. Sub-objective 1B: Define the microbiome of rotational crops in cereals (canola, pea, camelina) and how they drive the microbiomes of the following wheat crop and influence yield and disease. Objective 2: Determine the molecular and biochemical mechanisms of host-microbe interactions, including plant-pathogen, plant-beneficial microbe, and host genetics. Sub-objective 2A: Determine the effect of the wheat cultivar on root exudate composition and the growth, production of DAPG and phytotoxicity of P. brassicacearum Q8r1-96. Sub-objective 2B: Characterize the rhizosphere microbiome of the wheat cultivars Tara, Finley, Louise and Buchanan in take-all decline soils. Sub-objective 2C: Identify Streptomyces strains associated with the soil, rhizosphere, and endosphere of dryland wheat, assess their effect on fungal root pathogens and wheat health under drought conditions, and identify antifungal metabolites they produce. Objective 3: Integrate disease management strategies to control root diseases of wheat, barley, and biofuel brassica crops. Sub-objective 3A: Screen wheat (germplasm and varieties) for resistance to cereal cyst nematode and Fusarium crown rot. Sub-objective 3B: Identify the microbial communities (fungal pathogens and bacteria components) involved in the greenbridge, role of weeds, dynamics and succession of populations in roots, and antagonists that displace pathogens from dying roots.

Approach
Objective 1: Hypothesis: By using correlations of communities with field traits, we can target our culturing and select isolates that can be tested for causation in the greenhouse. Hypothesis: The microbiome of the rotation crop will affect the microbiome of the next wheat crop and yield by stimulating growth and inhibiting pathogens. Approach: Sites at the Cook LTAR farm will be sampled for bacterial and fungal microbiomes and the taxa correlated with yield, biomass, organic matter, and pH. Organisms will be isolated and tested for ability to protect against pathogens, drought, and acidity. The microbiome of legume and oilseed crops grown in rotation with wheat will be characterized. Contingencies: The culture collection may be skewed toward copiotrophs, but the use of low nutrient media will capture Streptomyces and oligotrophs. Several techniques will be used to ensure a wide sampling of the fungal community. Objective 2: Hypothesis: Differences in root exudates of cultivars drive the differential responses of wheat to P. brassicacearum, the bacteria responsible for take-all decline (TAD). Hypothesis: The microbiomes of wheat cultivars differ and contribute to their ability to support TAD. Hypothesis: Dryland wheat is colonized by Streptomyces that promotes growth under drought conditions and inhibits pathogens. Approach: Root exudates will be produced from cvs. Tara, Finley and Buchanan; their composition compared and their ability to support the growth and antibiotic production of P. brassicacearum determined. The root microbiomes of Tara, Finley and Buchanan in TAD and conducive soils in the presence and absence of Gaeumannomyces tritici will be characterized. Streptomyces spp. will be isolated on semi-selective media and identified using DNA sequencing. Isolates will be tested for ability to protect wheat against diseases and drought stress. Contingencies: Wheat root exudates are easily produced, and their chemical composition can be determined. If fungi or bacteria of interest are not isolated, Q-PCR primers will be used to detect and quantify specific taxa. Objective 3: Hypothesis: Genetic resistance to these two diseases exists in exotic germplasm sources and adapted varieties. Certain weeds act as reservoirs for the greenbridge, and a succession of primarily fungi displace pathogens from the dying roots over time. Approach: Wheat germplasm will be screened for resistance to cereal cyst nematode (CCN) and Fusarium crown rot. Greenbridge reduction is a cultural control measure, but the microbiology of this phenomenon is not understood. We will identify the microbial communities involved in the greenbridge, role of weeds, succession of microbes in roots, and antagonists that displace pathogens from dying roots. Contingencies: Resistance genes may be difficult to identify. Genes for resistance to CCN can be indentified, but phenotyping in the field is still needed. For Fusarium crown rot, germplasm from many sources is used to increase our chances of finding effective genes. Weather conditions may prevent plot establishment. but we can rely on other years’ trials for data.

Progress Report
This report documents FY 2024 progress for project 2090-22000-019-000D, "Ecology and Genomics of Soilborne Pathogens, Beneficial Microbes, and the Microbiome of Wheat, Barley, and Biofuel Brassicas", which began in May 2022. In support of Sub-objective 1A, ARS researchers in Pullman, Washington, completed the sequencing for a metagenomics study to look at the function of the microbiome of camelina under high and low N conditions. Ten camelina accessions were grown in the greenhouse in high and low N soil and sampled at three growth stages. DNA was extracted from the rhizosphere. Shotgun sequencing was performed by Joint Genome Institute (JGI) and annotated. Analysis is currently being performed. A metabolomics study was performed on 40 representative bacterial isolates from a collection of 3,000 from the rhizosphere of camelina. Isolates were grown in defined media mimicking the rhizosphere, and comparisons were made to the starting media and media in which the bacteria were grown. Only a few compounds including 4-aminobenzoic acid were frequently detected in higher levels in the spent media of the bacterial cultures. Initial untargeted profiling of the spent media found a dramatic range in the number of features (ions associated with one or more organic compounds) detected in spent media with a putative Pedobacter and Arthrobacter spp. among the most prolific. These included kynurenine and acetylated-amino acids as common putative identified hits. The root microbiome of camelina from 33 locations in eastern Washington was detailed in a manuscript submitted to the journal Phytobiomes. ARS researchers identified the core bacterial (several Actinobacteria including Aeromicrobium and Marmoricola, as well as the genera Rhizobium, Clostridium, and Sphingomonas) and fungal community (Pseudogymnoascus, Fusarium and Mortierella). ARS researchers also determined the environmental and soil drivers of microbial communities. Soil factors such as pH, Mg, and Ca were strong predictors of bacterial communities, whereas environmental factors such as precipitation were stronger predictors of fungal communities. Under Sub-objective 1A, soil samples containing weed seed were collected from 70 sites by a project collaborator from eastern Washington, northeastern Oregon, and western Idaho. Seeds will be extracted from the samples, identified, and utilized for seed microbiome analysis. A protocol for extracting DNA for seed microbiome research for wheat seeds was obtained, and it is being amended to for weed seeds that are generally smaller in size compared to wheat seeds. DNA extracted from these samples will be sent to the University of Minnesota for amplicon sequencing to determine bacterial taxa and fungal taxa present in the seeds. Once reliable DNA extraction protocols have been determined for weed species of interest, these protocols could be utilized for future research where the change of the weed seed microbiome in response to agricultural practices can be evaluated over time and compared to crop species. In support of Sub-objective 1B, ARS researchers in Pullman, Washington, are continuing to sample rotation experiments in Ritzville, Washington, in canola, pea, triticale, barley, and wheat. ARS researchers are optimizing primers to quantify arbuscular mycorrhizal fungi (AMF) with amplicon sequencing. AMF are beneficial to the rotation crops by providing increased uptake of phosphorus. However, canola is not a host for AMF fungi, and may affect populations in the following rotation crop. For Objective 2, fifteen pyroxsulam-resistant Italian ryegrass populations were identified and provided by a project collaborator. These populations displayed increased sensitivity after treatment with pyroxsulam and malathion, a cytochrome P450 (CYP)-inhibitor. The current hypothesis is pyroxsulam resistance is due to CYP-mediated detoxification, which is inhibited by malathion. Involvement of CYPs in Italian ryegrass pyroxsulam detoxification is further substantiated by similarly structured herbicides being detoxified by CYPs and the primary metabolite formed in the wheat detoxification pathway being a demethylated metabolite, which is likely the result of CYP-mediated dealkylation. However, additional research will be performed to detect enhanced detoxification via liquid chromatography–mass spectrometry. The following progress was achieved under Sub-objective 2B. Wheat and its associated microbiome are impacted by abiotic factors that shape their interactions over time, but long-term trends in microbial community dynamics are poorly characterized. ARS researchers determined seasonal and long-term bacterial population dynamics in monocropped dryland and irrigated wheat in central Washington State. Analyses revealed that some bacterial genera exhibit distinct seasonal periodicity whereas others maintain stable populations over time or decrease in abundance independent of irrigation. Taxa in the wheat endosphere declined in abundance regardless of irrigation, indicating that the maturing host, and not water, is the main driver of these populations. ARS researchers submitted a manuscript on this work to the journal Phytobiomes. This research is continuing with funding from a NIFA grant “Drought shapes the dryland root metabolome and microbiome to protect against biotic and abiotic stress” with the University of Southern Mississippi and ARS St. Paul, Minnesota. ARS researchers are sampling the Lind plots in 2024 to do a metagenomics study to look at the function of microbes in dryland and irrigated conditions. This will move our understanding beyond just taxonomic descriptions of the bacteria. In support of Sub-objective 2C, ARS researchers in Pullman, Washington, continue to develop a collection of Streptomyces species from the irrigated and dryland plots at Lind, Washington. ARS researchers have a total of 1,568 isolates, 738 from the endosphere, and 830 from the rhizosphere. They are also extracting DNA from the collection to identify with multilocus sequence analysis. The goal is to test these isolates for their ability to suppress fungal root pathogens and to ameliorate drought stress in crops grown under a variety of soil moisture conditions. Under Objective 3, ARS researchers in Pullman, Washington, are planning field trials to determine how weed management practices affect soil seedbank dynamics and the microbiome of the seed and soil. In the future, field trials will evaluate different methods of cultural, chemical, and mechanical weed control for their effects on the incidence of species that emerge, the incidence of seeds that enter the soil seedbank, emergence timing, seed germination, seed longevity, and soil and seed microbiome. In the summer of 2024, an experiment was conducted to compare plots that are subjected to surface tillage and plots that are treated with glyphosate (no-till) throughout different timings for differences in the incidence of weed species that emerge, emergence timings, root microbiome, soil microbiome and rhizosphere microbiome. DNA extraction protocols for root microbiome, soil microbiome, and rhizosphere microbiome were obtained and will be amended as needed to achieve sufficient quality and yield. A protocol is also being developed to estimate seed viability by evaluating the RNA integrity of RNA extracted from seed samples of specific weed species. This experiment will require harvesting seeds from multiple species with varying seed longevity (i.e., Russian thistle seed longevity is relatively short while common lambsquarters is relatively long), burying the seed, and retrieving the samples at multiple time points to verify that the RNA integrity correlates with seed viability and age. The goal is to have a protocol that could be utilized to estimate the viability and relative age of weed seeds obtained from soil samples, which could be influenced by weed management practices. In support of Sub-objective 3A, wheat varieties and lines being grown in greenhouses and close to release are continuously being screened. ARS researchers in Pullman, Washington, are also testing prebreeding lines to find additional sources of resistance to Fusarium crown rot, including crosses between CIMMYT (International Maize and Wheat Improvement Center) synthetic lines and Pacific Northwest varieties, double haploid lines between Cara (tolerant) and Xerpha (susceptible) and DNAM tauschii crosses (D-genome Nested Association) focusing on the D genome. The D genome of wheat may contain unexplored sources of resistance. Under Sub-objective 3B, greenbridge field trials at Pendleton, Oregon, have been set up with ARS's collaborator from Oregon State University, and the pathogens in the root systems are being analyzed using quantitative PCR (qPCR). Quizalofop herbicide was applied to a CoAXium winter wheat trial infested with feral rye (Secale cereale L.). Wheat and feral rye were sampled from the trial, and qPCR was used to quantify the pathogens in the crop and weed. Both Fusarium pseudograminearum and F. culmorum were present in high levels in feral rye. These preliminary results prompted a survey to understand the pathogen load present in grassy weeds common in the winter wheat production system. In 2023, downy brome (Bromus tectorum) was collected at three locations in northeast Oregon (Pendleton, Moro, and Sherman). Both Fusarium species were detected in downy brome. These studies indicate the role of grassy weeds as a reservoir for Fusarium to bridge to winter wheat.

Accomplishments
1. Nematode communities can be described by DNA techniques. Nematodes are the most numerous soil invertebrate and occupy all trophic levels in the food web, from fungal and bacterial feeders to herbivores to predators. At present, they can only be described by extracting live nematodes from the soil, and identified by morphological characters under the microscope, which few trained nematologists can do. ARS scientists in Pullman, Washington, and Corvallis, Oregon, and Washington State University scientists developed a database of 18s sequences for amplicon sequencing. This was validated with potato and wheat soils across eastern Washington and Oregon, including soils that have never been cropped. Nematodes were morphologically identified and sequenced with amplicon sequencing, with a high degree of correlation between the two methods. This research opens up the possibility of more extensive use of nematode communities as indicators for soil health, especially by those not skilled in nematology taxonomy.

2. Bacterial and fungal communities in rhizosphere of camelina are driven by soil and environmental factors. Camelina, a member of the Brassicaceae family, is a potential low-input bioenergy crop that can be grown in rotation with wheat in dryland areas. Microbial communities on the roots may influence crop performance and nutrient uptake. ARS researchers in Pullman, Washington, and Washington State University and Montana State University scientists, funded by a grant from the U.S. Department of Energy, described the microbiome of camelina from the roots of camelina grown in 33 different locations in eastern Washington and Montana. Bacterial communities were highly influenced by soil pH, Ca and Mg, while fungal communities were more affected by precipitation. Influences were stronger in the bulk soil but less in the rhizosphere and root, indicating that the plant selects the community from the soil. This is part of a larger project to identify key components of the camelina bacterial community that may play a role in increasing nutrient uptake, pathogen resistance and drought tolerance in this important biofuels crop.

3. Disease-suppressive soils have a broader role in plant defense. Disease-suppressive soils are examples of natural microbial-based defense of plant roots against soilborne pathogens. They are defined as soils in which, because of their microbial makeup and activity, a pathogen does not establish or persist, establishes but causes little or no disease, or establishes and causes disease at first but then the disease declines with successive cropping of a susceptible host even though the pathogen may still persist in the soil. Take-all decline (TAD) is a natural suppression of take-all disease of wheat (caused by Gaeumannomyces tritici) that develops during wheat monoculture and following a severe outbreak of the disease. ARS scientists in Pullman, Washington, in collaboration with colleagues at Utecht University, the Netherlands, determined the ability of a TAD soil from Washington state to induce systemic resistance (ISR) in Arabidopsis thaliana against Pseudomonas syringae pv. tomato (Pst) DC3000, causal agent of bacterial speck. Arabidopsis seedlings grown in an autoclaved soil/sand mixture amended with 10% (wt/wt) TAD soil demonstrated strong induced resistance against Pst, but a non-suppressive soil did not induce resistance in Arabidopsis. This is the first report of induction of systemic resistance by a suppressive soil and indicates that TAD can have a broader role in foliar disease suppression, beyond the control of its target disease, take-all.

4. Chromosome 5A in allohexaploid wheat contains genes encoding herbicide-detoxifying enzymes. Allohexaploid wheat (Triticum aestivum L.) achieves tolerance to many synthetic auxin herbicides, including halauxifen-acid (HA), through enhanced detoxification. While the detoxification pathway of HA in wheat has been characterized, the genes encoding the detoxification enzymes have yet to be identified, which would be useful for breeding for herbicide tolerance or developing in vitro assays for herbicides. ARS researchers in Pullman, Washington, in collaboration with scientists at the University of Illinois, measured the abundance of HA, over time in wheat alien substitution and nullisomic-tetrasomic lines via liquid chromatography-mass spectrometry, and they observed lines lacking chromosome 5A consistently accumulated significantly more HA. Chromosome 5A contains genes encoding HA-detoxifying enzymes and the lack of these genes results in higher accumulation of HA relative to lines that maintain 5A. This finding could facilitate future development of wheat resistant to auxin herbicides, which would provide growers another tool in managing weeds that have become resistant to other herbicide classes.

5. Detection of the early effects of plant disease. Phenomics imaging technologies as applied to phytopathology allow the acquisition of high-dimensional phenotypic data on morphological and physiological changes in infected plants during disease development without destructive sampling of plant parts. Spectral analysis of plants can detect rapid and subtle changes in tissues and organs before symptoms are visible. ARS scientists in Pullman, Washington, and Dutch scientists used the Helios phenomics system at The Netherlands Eco-phenotyping Centre (NEPC) to describe the early phase of infection of tomato seedlings by Pseudomonas syringae pv. tomato (Pst) DC3000, causal agent of bacterial speck of tomato. Twenty days after planting in an autoclaved potting soil/sand mixture, tomato seedlings were inoculated by dipping the foliage into a suspension of Pst DC3000. In less than 24 hours after inoculation, and prior to appearance of any symptoms of bacterial speck, Helios detected significant changes in the morphology and physiology of tomato plants, including a significant decline in the density of leaves and stems, efficiency of energy harvesting, and leaf area. This research demonstrates how phenomics technologies can be used for early detection of foliar diseases in field- or greenhouse-grown crops, allowing more rapid removal of infected plants and preventing pathogen spread to healthy plants.

Review Publications
Yin, C., Larson, M., Lahr, N.D., Paulitz, T.C. 2023. Wheat rhizosphere-derived bacteria protect soybean from soilborne diseases. Plant Disease. 108(6):1565-1576. https://doi.org/10.1094/PDIS-08-23-1713-RE.
Landau, O.A., Concepcion, J.C., Riechers, D.E. 2024. Metabolism of halauxifen acid is regulated by genes located on wheat chromosome 5A. Weed Science. https://doi.org/10.1017/wsc.2024.24.
Laasli, S., Mokrini, F., Iraqi, D., Shtaya, M., Amiri, S., Dabaat, A.A., Paulitz, T.C., Khfif, K., Lahali, R. 2023. Phytopathogenic nematode communities infesting Moroccan olive agroecosystems: Impact of agroecological patterns. Plant and Soil. https://doi.org/10.1007/s11104-023-06190-5.
Benjlil, H., Filali Alaoui, I., Ait Hamza, M., Braimi, A., Oubidari, T., Idhmida, A., Ihitassen, A., Tazi, H., El Kherrak, H., Paulitz, T.C., Fossati-Gaschignard, O., Ferji, Z., Cherifi, K., Mayad, E. 2023. Nematodes associated with saffron II: Bioindication for soil health assessment and impact of agricultural practices. Applied Soil Ecology. 193. Article 105111. https://doi.org/10.1016/j.apsoil.2023.105111.
Schillinger, W., Paulitz, T.C., Hansen, J.C. 2023. Canola rotation effects on soil water and subsequent wheat in the Pacific Northwest USA. Agronomy Journal. 115(1):314-324. https://doi.org/10.1002/agj2.21248.
Weller, D.M., van Pelt, J.A., Thomashow, L.S., Mavrodi, D.V., Mavrodi, O., Pieterse, C.M., Bakker, P.A. 2024. Disease-suppressive soils induce systemic resistance in Arabidopsis thaliana against Pseudomonas syringae pv. tomato. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-02-24-0012-R.
Chekali, S., Ayed, S., Khemir, E., Gharbi, M., Marzougui, S., Paulitz, T.C., Gargouri, S. 2024. Response of durum wheat vs. bread wheat to Fusarium foot and root rot under semi-arid conditions. Journal of Plant Pathology. https://doi.org/10.1007/s42161-024-01659-3.
Spooren, J., van Bentum, S., Thomashow, L.S., Pieterse, C.M., Weller, D.M., Berendsen, R.L. 2024. Plant-driven assembly of disease-suppressive soil microbiomes. Annual Review of Phytopathology. 62:11.1-11.30. https://doi.org/10.1146/annurev-phyto-021622-100127.