Research Project: Biology, Ecology, and Genomics of Pathogenic and Beneficial Microorganisms of Wheat, Barley, and Biofuel Brassicas

Location: Wheat Health, Genetics, and Quality Research

2020 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: Define the pathogen diversity, host range, and geographical distribution of fungal and nematode root pathogens, especially those causing emerging diseases in cereal-based cropping systems in the Pacific Northwest. Subobjective 1A: Using conventional and molecular techniques, determine the biogeographical distribution and risk of emerging and chronic pathogens and diseases. Subobjective 1B: Examine the genetic and pathogenic diversity of emerging and chronic pathogens. Subobjective 1C: Develop and evaluate agronomic, genetic and cultural methods of root disease management. Objective 2: Determine the soil microorganisms, microbial communities, and molecular mechanisms that promote or reduce plant health in wheat, barley and canola in the Pacific Northwest. Subobjective 2A: Determine how cultural practices and chemical inputs affect the plant and soil microbiomes in wheat cropping systems. Subobjective 2B: Characterize the rhizosphere microbiome of wheat in take-all decline soils. Subobjective 2C: Evaluate the effect of the wheat cultivar on the robustness of biological control by Pseudomonas spp. and in take-all decline soils. Objective 3: Identify and characterize molecular mechanisms of host-microbe interactions, including the action of host genes governing disease resistance and biological control against soilborne pathogens of wheat, barley and canola. Subobjective 3A: Identify host responses to soilborne pathogens, biocontrol bacteria and bacterial metabolites. Subobjective 3B: Identify and characterize germplasm with resistance to soilborne pathogens.

Approach
Biological control of soilborne fungal pathogens such as Gaeumannomyces, Rhizoctonia, Pythium, Fusarium and plant-parasitic nematodes by naturally-occurring and recombinant microorganisms will be developed and quantified in agricultural soils. Molecular approaches will be used to detect and quantify soilborne pathogens and their microbial antagonists, and next-generation sequencing will be used to characterize the microbiomes of conducive and suppressive soils and the rhizosphere of small grain crops. Genetic determinants and molecular mechanisms responsible for root colonization and pathogen suppression will be characterized with emphasis on the genetics and regulation of phenazine and phloroglucinol biosynthesis in vitro and in situ. The genetic and physiological diversity of populations of root pathogens and their microbial antagonists, and influence of cropping systems on pathogens and antagonists will be determined. Genomes of pathogens and antagonists will be sequenced and analyzed. New sources and mechanisms of host resistance will be identified. Practical disease control will be accomplished by maximizing the activity of natural biocontrol agents.

Progress Report
This is the fourth report for this project which began in March of 2017. Wheat, barley, and biofuel crops are infected by soilborne pathogens that reduce yields 10 to 30% annually. Diseased crops cannot take full advantage of fertilizers and irrigation water, and unused nitrates move into surface and ground water and pollute the environment. The overarching goal of this project is to develop biologically-based technology for controlling root diseases of wheat, barley and biofuel brassica crops; to identify the diversity, host range, and geographical distribution of root pathogens, especially those causing emerging diseases; to determine the soil microbes, microbial communities, and molecular mechanisms that promote or reduce plant health, and to characterize the host microbe interactions involved in disease resistance and biocontrol of root diseases. Progress was made on all three objectives and their sub-objectives. Under Sub-objective 1A, we continued to monitor for existing and emerging diseases in Washington State. This includes cereal cyst nematode (Heterodera avenae and H. filipjevi). We continue to survey for black leg of canola (Leptosphaeria maculans), making collections, extracting and sequencing DNA, and making identifications. Under Sub-objective 1B, we have transferred over 300 isolates to a collaborator at University of Idaho to determine races and avr genes. These isolates have now been analyzed and the student is reporting on them. Under Sub-objective 2A, we have determined the core rhizosphere microbiome of wheat across a range of precipitation zones and cropping systems. In collaboration with the Cook Farm, Long-Term Agriculture Research (LTAR) network, we have identified bacteria associated with soil health, yield and edaphic factors such as organic matter and pH. We have identified the effect of depth and time on bacterial communities. This is the first work to decipher the microbial black box of soil health, by using next-generation sequencing to identify specific components of the bacterial community from over 120 sampling sites on the LTAR. In collaboration with University of Oregon, we have completed a study on the effect of liming on microbial communities and submitted two papers. Under Sub-objective 2B, the eighth year of a field plot has continued at Lind, Washington, comparing the microbial communities in dryland vs irrigated plots, and quantifying the levels of DAPG (2,4-diacetylphloroglucinol) and phenazine. These are two antifungal antibiotics produced by the bacterium Pseudomonas on the roots of wheat that control root diseases. We have analyzed seven years of dynamics of the bacterial and fungal communities in the soil, rhizosphere, and root, both between years and within growing seasons. Also under Sub-objective 2B, we developed and environmentally-validated an approach to detect bacteria with genes for the synthesis and degradation of phenazine antibiotics in soil. We discovered that in addition to phenazine-producing Pseudomonas bacterial species, approximately the same number of Streptomyces bacterial species are carrying phenazine genes in the wheat rhizosphere, while bacteria degrading the antibiotic are rare. Under Sub-objective 2C, we identified amino acids and compatible solutes in exudates of the wheat cultivars Tara, Buchanan and Finley, which differentially support production on roots of the antifungal metabolite 2,4-diacetylphloroglucinol. Under Sub-objective 3A, using RNA sequencing (transcriptomics) and proteomics approaches, we have identified wild oat seed genes and proteins induced during infection by a seed-decaying isolate of the soilborne fungal pathogen Fusarium avenaceum. Bioinformatic analyses indicated that wild oat seed harbored several hundred defense-related proteins. In contrast, Fusarium harbored modest numbers of defense and pathogenicity proteins when associated with semi-dry host seed, but 12- to 15-fold more proteins were found in the aqueous environment Since infection of wild oat, an unwanted weed pest, is desired, breeding programs could benefit by selection of crop genotypes or lines with naturally elevated expression of specific defense proteins, especially during early seedling growth. Under Sub-objective 3B, 48-h and 72-h root phenotyping assays were developed to identify wheat genotypes with resistance to Rhizoctonia solani, a root rotting fungus on wheat. These rapid assays showed that very early differences in root growth rate and root biomass accumulation could distinguish strong resistance from strong susceptibility.

Accomplishments
1. Wheat plants have a core microbiome. Plant roots exude carbon, nitrogen, and other nutrients that support a microbial community on the root surface, much like the gut microbiome in humans. Is there a finite set of core microbes on wheat roots present across a wide range of environments? ARS scientists in Pullman, Washington, sampled wheat roots across a range of precipitation zones in eastern Washington. A core set of bacteria and fungi were found in over 95 percent of rhizosphere or bulk soil samples. These bacteria and fungi may play a critical role in plant health and provide an indicator of soil health for wheat growers.

2. Liming and pH shifts in microbial communities. Soil acidification is an increasing problem in dryland wheat production, because of long-term use of ammonium fertilizer and nitrification. Growers are looking at using lime to raise the pH, but what effect does this have on microbial communities in the soil? Oregon State University scientists at Pendleton, Oregon, and ARS scientists in Pullman, Washington, analyzed the soil microbiome in replicated field plots with the addition of different levels of lime. Liming significantly increased the relative abundance of some bacterial families, including Pseudomonadaceae, Opitutaceae, and Flavobacteriaceae, while decreasing others, such as the Bradyrhizobiaceae, though this effect was often seen only at the 0-3 inch depths. This information is important for growers because liming will reshape soil communities, primarily impacting bacteria that influence plant health.

3. Wheat microbiome shifts during the growing season. Most soil microbiome work involves samples taken only once in the spring, but little is known about the community dynamics over a full growing season. ARS scientists in Pullman, Washington, collected samples at the Cook Agronomy Farm Long-Term Agricultural Research project from November 2017 to November 2018 and compared an aspirational farm (no-till) to an adjacent business as usual (reduced tillage) farm. Bacterial communities in the spring were very similar, but then became quite different during the dry season. When the fall rains returned, the late fall communities resembled the spring communities. This information is important to growers to understand how the microbiome affects soil and plant health.

4. Phenazine-producing bacteria are enriched in plant microbiomes. Dryland wheat on the Columbia Plateau of the Pacific Northwest enrich for biofilm-forming populations of phenazine antibiotic-producing Pseudomonas spp. on the surface of the root. Phenazine antibiotics control root rotting fungi on the roots of wheat. These biofilms retain soil moisture and suppress a wide range of soilborne plant pathogens. Scientists at ARS in Pullman, Washington, in collaboration with the California Institute of Technology, developed a computational method to analyze bacterial metagenomes from across the world and showed that phenazine biosynthesis is a highly prevalent trait that is enriched in plant microbiomes relative to bulk soils. In contrast, phenazine biodegradation genes are rarely detected in these environments. These results highlight new and potentially important associations between phenazine producing bacterial clades and provide evidence for extending the application of phenazine-producing biocontrol agents to other major cereal crops grown in semiarid regions.

5. Molecular communication in the wheat rhizosphere. Plant roots secrete compounds in exudates that sustain and mediate communication with their rhizosphere microbiome, but the biochemical basis of these processes in cereals is poorly understood. ARS scientists, with collaborators at Southern Mississippi University, identified amino acids and compatible solutes in exudates of the wheat cultivar Louise, which supports increased production on roots of the antifungal metabolite phenazine-1-carboxylic acid. These exudate compounds, and the technology developed to recover and analyze them, are important because they can help to explain why cultivars of wheat such as Louise support root colonization and biofilm formation by phenazine-producing strains that protect wheat from fungal pathogens in the semiarid farming regions of the Pacific Northwest.

6. Wild oat seed proteome and transcriptome. Wild oat can be a major yield-limiting factor in dryland cereal production regions of the Pacific Northwest and other parts of the world, Fusarium avenaceum, a fungal plant pathogen, causes decay in dormant seeds of wild oat, and may be a potential biocontrol agent against the weed. In collaboration with Washington State University researchers, an ARS scientist at Pullman, Washington, used RNA (transcriptomics) and protein (proteomics) sequencing to identify genes and proteins expressed during the wild oat-F. avenaceum interaction. The findings indicated that both wild oat and the pathogen used reactive oxygen species for defense. Wild oat deployed several other defense pathways, whereas the pathogen produced enzymes for mycotoxin biosynthesis, detoxification and host cell wall degradation. This data provides insights into the relative importance of these biochemical processes in defense and invasion.

Review Publications
Carter, A.H., Allan, R.E., Balow, K., Burke, A., Chen, X., Engle, D.A., Garland Campbell, K.A., Hagemeyer, K., Morris, C.F., Murray, T., Paulitz, T.C., Shelton, G. 2020. How ‘Madsen’ has shaped Pacific Northwest wheat and beyond. Journal of Plant Registrations. 14(3):223-233. https://doi.org/10.1002/plr2.20049.
Zhang, J., Yang, M., Mavrodi, D.V., Kelton, J., Thomashow, L.S., Weller, D.M. 2020. Pseudomonas synxantha 2-79 transformed with pyrrolnitrin biosynthesis genes has improved biocontrol activity against soilborne diseases of wheat and canola. Phytopathology. 110: 1010-1017. https://doi.org/10.1094/PHYTO-09-19-0367-R.
Yin, C., McLaughlin, K., Paulitz, T.C., Kroese, D.R., Hagerty, C.H. 2020. Population dynamics of root pathogens of wheat under different tillage systems in NE Oregon. Plant Disease. https://doi.org/10.1094/PDIS-03-19-0621-RE.
Yuan, J., Wen, T., Zhang, H., Zhao, M., Penton, R., Thomashow, L.S., Shen, Q. 2020. Predicting disease occurrence with high accuracy based on soil macroecological patterns of Fusarium wilt. ISME Journal. https://doi.org/10.1038/s41396-020-0720-5.
Wang, X., Glawe, D.A., Weller, D.M., Okubara, P.A. 2019. Real-time PCR assays for the quantification of native yeast DNA in grape berry and fermentation extracts. Journal of Microbial Methods. 168:105794. https://doi.org/10.1016/j.mimet.2019.105794.
Lewis, R.W., Okubara, P.A., Fuerst, P.E., He, R., Gang, D., Sullivan, T. 2020. Chronic sublethal aluminum exposure and wild oat caryopsis decay influence gene expression of Fusarium avenaceum F.a.1. Frontiers in Microbiology. 11:51. https://doi.org/10.3389/fmicb.2020.00051.
Basaid, K., Cheblia, B., Mayad, E., Furze, J.N., Bouharroud, R., Krierd, F., Barakate, M., Paulitz, T.C. 2020. Biological activities of essential oils and lipopeptides applied to control plant pests and diseases: A review. International Journal of Pest Management. https://doi.org/10.1080/09670874.2019.1707327.
Schlatter, D.C., Baugher, C., Kahl, K., Johnson-Maynard, J.L., Huggins, D.R., Paulitz, T.C. 2019. Bacterial communities of soil and earthworm casts of native Palouse Prairie remnants and no-till wheat cropping systems. Soil Biology and Biochemistry. 139. https://doi.org/10.1016/j.soilbio.2019.107625.
Schlatter, D.C., Hansen, J.C., Schillinger, W.F., Sullivan, T.S., Paulitz, T.C. 2019. Common and unique rhizosphere microbial communities of wheat and canola in a semiarid Mediterranean environment. Soil Biology and Biochemistry. 144:170-181. https://doi.org/10.1016/j.apsoil.2019.07.010.
Yang, M., Mavrodi, D.V., Mavrodi, O.V., Thomashow, L.S., Weller, D.M. 2019. Exploring the phytotoxic effect of Pseudomonas brassicacearum Q8r1-96 on tomato. Plant Disease. 104(4):1026-1031. https://doi.org/10.1094/PDIS-09-19-1989-RE.
Salamone, A.L., Okubara, P.A. 2020. Real-time PCR quantification of a Rhizoctonia solani AG-3 variant of potato. Journal of Microbiological Methods. 172. https://doi.org/10.1016/j.mimet.2020.105914.
Ozer, G., Imren, M., Bayraktar, H., Paulitz, T.C., Muminjanov, H., Dababat, A.A. 2019. First report of Fusarium hostae causing crown rot on wheat in Azerbaijan. Plant Disease. 103(12):3278. https://doi.org/10.1094/pdis-05-19-1035-pdn.
Ozer, G., Alkan, M., Imren, M., Muminjanov, H., Paulitz, T.C., Dababat, A.A. 2020. Identity and pathogenicity of fungi associated with crown and root rot of dryland winter wheat in Azerbaijan. Plant Disease. 104(4):2149-2157. https://doi.org/10.1094/PDIS-08-19-1799-RE.
Schlatter, D.C., Yin, C., Hulbert, S., Paulitz, T.C. 2020. Core rhizosphere microbiomes of dryland wheat are influenced by location and land use history. Applied and Environmental Microbiology. 86(5):e02135-19. https://doi.org/10.1128/AEM.02135-19.
Dababat, A., Duman, N., Ozer, G., Mokrini, F., Imren, M., Paulitz, T.C. 2020. Genetic and pathogenic variation in Heterodera latipons populations from Turkey. Journal of Nematology. https://doi.org/10.1163/15685411-bja10029.