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

Location: Wheat Health, Genetics, and Quality Research

2022 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
Wheat, barley and biofuel crops are infected by soilborne pathogens that reduce yields 10-30% 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. This summarizes the progress made over the last five years on all three objectives and their sub-objectives, all of which fall under NP 303 and encompass Component 1 Problem 1A, Component 2 Problem 2C or Component 3 Problem 3A or B. In support of Sub-objective 1A, extensive surveys of blackleg (Leptosphaeria maculans) were conducted in the Pacific Northwest (PNW) for three years. This was a new and emerging pathogen of canola and threatened the Brassica seed industry in northwest Washington. Our annual results were reported at grower meetings and in publications and we received additional funding from the National Plant Disease Recovery System for three years. We conducted 10 years of survey for cereal cyst nematode, developing molecular methods to distinguish Heterodera avenae from H. filipjevi. H. filipjevi was discovered for the first time in Washington State in 2010, and both species are almost indistinguishable morphologically. We assisted international collaborators with disease surveys, hosting Fulbright scholars and students. They visited our lab and learned these techniques. Progress on Sub-objective 1B was achieved in collaboration with a researcher at the University of Idaho. Over 200 isolates of Leptosphaeria maculans and L. biglobosa were collected. They were sequenced for species identification and returned to the University of Idaho to assist in the completion of an MSc project. She characterized 86 isolates from the Washington collection using a combination of plant host differentials and Avirulence (AVR) specific Polymerase chain reaction (PCR) primers. We have tested H. filipjevi on a set of differentials acquired from colleagues in Turkey, to determine the pathotypes or races of this nematode in the Pacific Northwest. This is important so we know which resistance genes to deploy. The results show that our Pacific Northwest races do not match any of the known races. Progress on Sub-objective 1C focused on screening varieties for resistance to cereal cyst nematode and Fusarium crown rot. This continues almost two decades of work, but several important discoveries were made. Research on the cereal cyst nematode inlcuded the development of greenhouse methods that have enabled us to screen over 1000 lines of winter and spring wheat varieties for the Washington State University and ARS breeding programs and in the process, identified a number of resistant varieties. Kompetitive allele specific PCR (KASP) markers were developed to screen for known Cre genes, major resistance genes. To identify new resistance genes in PNW material, we crossed known resistant varieties such as Chara and ARS Crescent and susceptible lines Alpowa, Seahawk, Bruehl, Ouyen, and Louise. We imported known differential varieties from Turkey and described the pathotypes of H. filipjevi. We looked for resistance in the synthetic wheat population based on the D-genome Nested Association Mapping (DNAM) population. The DNAM population is the result of eight different hybridizations of Ae. tauschii with a hexaploid hard white Kansas breeding line. A genome-wide association study (GWAS) was conducted on varieties in the Regional Nursery and variety testing programs. Marker-trait associations (MTA) were detected on chromosomes 2B, 5B, and 7B; and two putative quantitative trait loci (QTL) on chromosomes 3A and 6D. As for research on Fusarium crown rot, large collections of germplasm have continued to be screened in the greenhouse. Consistent, accurate, and reproducible methods of phenotyping this disease have been developed. This was done by manipulating temperature, restricting water at the end of the cycle, and developing more accurate quantification of inoculum. Several sources of novel germplasm have also been developed to look for resistance, including a large Spring Wheat CAP population, with over 3000 lines. We have a set of facultative synthetic wheat germplasm developed by the International Maize and Wheat Improvement Center (CIMMYT) in Turkey, populations developed by backcrossing Iranian landrace AUS28451 to Louise, doubled haploid population from Cara X Xerpha cross, and a DNAM recombinant inbred lines developed by direct crosses between a hard white winter wheat and the wheat wild relative, Aegilops tauschii. We have a synthetic population crossed to Louise about 10 years ago and developed for resistance to Rhizoctonia. We continued to evaluate cultural and chemical methods to control disease, including greenbridge management and seed treatment. In support of Sub-objective 2A, over the last five years, several landmark papers have been published on how cultural and chemical inputs affect the wheat microbiome. We were the first to document, with next-generation sequencing, that glyphosate applied at rates used by farmers, has minimal impact on both bacterial and fungal communities in the rhizosphere and bulk soil. We showed the succession of fungal colonization of dying roots killed with glyphosate and clethodim and identified a previously unknown Pythium spp. (P. volutum) that may play a role in the greenbridge. Biosolids from treated sewage are applied to fields in eastern Washington as a source of nitrogen. We showed that biosolids can shift both bacterial and fungal communities, even when the application was four years previously. We documented the bacterial and fungal microbiome of dust emitted from these biosolid treated fields. Many of the soils in eastern Washington are becoming acidified because of use of ammonia fertilizers over 60 years, and growers are adding lime to increase soil potential of hydrogen (pH). In a series of papers, we examined how liming affects bacterial and fungal microbiomes. Tillage can have an effect on microbial communities. In a series of papers comparing long-term no-till sites to conventionally tilled sites, we found that tillage had a stronger effect on fungal communities than bacterial communities. Earthworms play a major role in no-till systems, by moving carbon into deep soil profiles. We identified specific bacterial communities present in the drilosphere, lining the burrows of the earthworms, and unique communities present in the gut and casts. Soils in the hilly Palouse region of Eastern Washington are very deep wind deposited loess soils, with topsoil up to 10 feet deep. We showed very distinct communities at each depth in a no-till system. Finally, we defined the core microbiome of wheat grown across four locations in distinct precipitation zones of eastern Washington. In support of Sub-objective 2B, over a period of eight years, we have evaluated the microbiome of wheat in side by side, long-term, dryland and irrigated plots at the Washington State University Lind Dryland Research Station. We found that compared to the dryland plots, three seasons of irrigation influenced the overall diversity within the rhizosphere microbiome, leading to significant differences in the relative abundance of specific taxa with known plant growth-promoting activity. We showed a season periodicity of some bacteria over a season. Some bacterial communities maintained in the rhizosphere over the eight years in both dry and irrigated (Pseudomonas, Variovorax, and Chryseobacterium) while some persisted in dryland but not irrigated (Mucilaginbacter, Sphingomonas, Massilia, and Burkholderia). Actinobacteria such as Streptomyces declined over 8 years in both dry and wet conditions. This is the first long-term study of the microbiome of wheat. For Sub-objective 2C, we discovered that 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. in the P. fluorescens complex are primarily responsible for a natural suppression of take-all of wheat known as take-all decline (TAD) in fields in the United States. We determined how the wheat cultivar affects the level of take-all suppression when grown in a TAD soil, and how cultivars respond to colonization by P. brassicacearum. Our results indicate that wheat cultivars grown in a TAD soil modulate both the robustness of take-all suppression and the potential phytotoxicity of the antibiotic DAPG. We also developed a new method for generating sterile wheat root exudates for the analysis of major and minor exudate constituents that differ among cultivars, influencing both the rate of root colonization and the kinetics of DAPG production. For Sub-objective 3A, biochemical, transcriptomic, and proteomic approaches were used to identify wheat root genes that are induced or repressed during interactions with the cereal nematodes Pratylenchus thornei and P. neglectus. We identified wild oat seed genes and proteins that are regulated during interactions with the seed decay pathogen Fusarium avenaceum. For Sub-objective 3B, in order to examine the mechanisms of resistance to soilborne pathogens, reverse genetics or transgenic approaches were used to identify specific genes involved in resistance to soilborne fungal pathogens, primarily Rhizoctonia and Pythium, and synthetic hexaploid wheat genotypes were assessed for field resistance to R. solani AG8.

Accomplishments
1. Bacterial community abundance in the soil fluctuates over the growing season. Bacterial communities in the soil play a major role in wheat health, nutrient uptake, residue decomposition and tolerance to abiotic stress. Most previous studies look at one time point, but little is known about how populations change over the growing season. ARS scientists at Pullman, Washington, and the Cook Long Term Agroecosystems Research site sampled every two weeks during the growing season (March-November) over three years and examined bacterial communities with amplicon sequencing of the 16S gene. Some bacteria families declined during the hot and dry months (June-August), but others maintained their populations. Previous rotation crops also influenced populations. This will help growers determine soil health in their field and how management practices may be adapted to favor beneficial microbiomes and increase yield and sustainability.

2. Camelina has a core microbiome of bacteria around its roots. 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. But nothing is known about the microbial communities on the roots, and how this may influence crop performance and nutrient uptake. ARS researchers at Pullman, Washington, and Washington State University scientists, funded by a grant from Department of Energy, grew camelina in 33 soils and extracted DNA from the bulk soil, rhizosphere, and roots. The core microbiome present in all the locations included Sphingomonas, Rhizobium, Micrococcaceae, Gemmatimonadaceae, Phenylobacterium, and Streptomyces. We have made a culture collection to isolate and test the ability of these bacteria to increase camelina health and performance in the greenhouse and field. This research will help in the adaption of this crop to eastern Washington.

3. Plant root exudates feed bacteria on roots. Plant root exudates provide nutrients for soil microorganisms and modulate their affinity to host plants, but molecular details of this process are largely unresolved. ARS scientists at Pullman, Washington, and Mississippi State University researchers addressed this gap by characterizing the molecular dialog between eight well-characterized beneficial strains of the Pseudomonas fluorescens group and Brachypodium distachyon, a model for economically important grass family crops. RNA-seq profiling of the bacteria amended with root exudates revealed changes in the expression of genes and products that are important for the multiple benefits that Pseudomons fluorescens provides to the plant, including protection against plant disease. These results collectively reveal the diversity of cellular pathways and physiological responses underlying the establishment of mutualistic interactions between these beneficial rhizobacteria and their plant host.

Review Publications
Mokrini, F., Laasli, S., Benseddik, Y., Joutei, A.B., Blenzar, A., Lakhal, H., Sbaghi, M., Imren, M., Ozer, G., Paulitz, T.C., Lahlali, R., Dababat, A.A. 2021. Potential of Moroccan entomopathogenic nematodes for the control of the Mediterranean fruit fly Ceratitis capitata Wiedemann (Diptera: Tephritidae). Scientific Reports. 10. Article 19204. https://doi.org/10.1038/s41598-020-76170-7.
Lewis, R.W., Okubara, P.A., Sullivan, T.S., Madden, B.J., Johnson, K.L., Charlesworth, C.M., Fuerst, E.P. 2022. Proteome-wide response of dormant caryopses of the weed, Avena fatua, after colonization by a seed-decay isolate of Fusarium avenaceum. Phytopathology. 112(5):1103-1117. https://doi.org/10.1094/PHYTO-06-21-0234-R.
Gargouri, S., Bouatrous, A., Murray, T.D., Paulitz, T.C., Khemir, E., Souissi, A., Chekali, S., Burgess, L.W. 2022. Occurrence of eyespot of cereals in Tunisia and identification of Oculimacula species and mating types. Canadian Journal of Plant Pathology. 44(3):345-353. https://doi.org/10.1080/07060661.2021.1995501.
Hagerty, C., Gardner, S., Kroese, D.R., Yin, C., Paulitz, T.C., Pscheidt, J.W. 2022. Occurrence of mummy berry associated with huckleberry (Vaccinium membranaceum) caused by Monilinia spp. in Oregon. Plant Disease. 106(2):357-359. https://doi.org/10.1094/PDIS-04-21-0691-SC.
Ahmadi, M., Mirakhorli, N., Erginbas-Orakci, G., Ansari, O., Braun, H., Paulitz, T.C., Dababat, A. 2022. Interactions among cereal cyst nematode Heterodera filipjevi, dryland crown rot Fusarium culmorum, and drought on grain yield components and disease severity in bread wheat. Canadian Journal of Plant Pathology. 44(3):415-431. https://doi.org/10.1080/07060661.2021.2013947.
Alkan, M., Bayraktar, H., Imren, M., Ozdemir, F., Lahlali, R., Mokrini, F., Paulitz, T.C., Dababat, A.A., Ozer, G. 2022. Monitoring of host suitability and defense-related genes in wheat to Bipolaris sorokiniana. The Journal of Fungi. 8(2). Article 149. https://doi.org/10.3390/jof8020149.
Schlatter, D.C., Hansen, J.C., Carlson, B.R., Leslie, I.N., Huggins, D.R., Paulitz, T.C. 2022. Are microbial communities indicators of soil health in a dryland wheat cropping system? Applied Soil Ecology. 170. Article 104302. https://doi.org/10.1016/j.apsoil.2021.104302.
Kim, D., Jeon, C., Cho, G., Thomashow, L.S., Weller, D.M., Paik, M., Lee, Y., Kwak, Y. 2021. Glutamic acid reshapes the plant microbiota to protect plants against pathogens. Microbiome. 9. Article 244. https://doi.org/10.1186/s40168-021-01186-8.