Location: Wheat, Sorghum and Forage Research
2020 Annual Report
Objectives
Objective 1: Identify and manipulate the genetic and biochemical mechanisms controlling lignin deposition to develop improved sorghum germplasm for bioenergy and forage uses.
Subobjective 1A: Characterize the effects six recently identified brown midrib mutants (bmr) on phenylpropanoid metabolism and lignin deposition.
Subobjective 1B: Evaluate ways to increase lignin deposition and alter phenolic composition of biomass through overexpression of monolignol genes.
Objective 2: Identify and manipulate the genetic and biochemical mechanisms controlling starch and phosphorus composition of grain to develop novel sorghum traits for food, biofuel and livestock production.
Subobjective 2A: Identify and characterize mutants that alter phosphorus composition and reduce phytate in grain.
Subobjective 2B: Develop germplasm with altered starch composition and content in grain.
Objective 3: Identify resistance to fungal pathogens in lignin modified sorghum germplasm for development of stalk rot-resistant grain, bioenergy, and forage sorghums.
Subobjective 3A: Determine the responses of sorghum lines with six recently identified bmr mutants to stalk pathogens.
Subobjective 3B: Assess impact of the stalk pathogen Fusarium thapsinum on sorghum with altered the monolignol synthesis.
Subobjective 3C: Determine the response of sorghum stalk moisture phenotypes on stalk rot pathogens.
Subobjective 3D: Determine whether beneficial microorganisms increase protection of bmr mutants against root and stalk pathogens.
Objective 4: Identify resistance interactions between sorghum grain with novel composition and fungal pathogens for food, fuel, and feed uses.
Subobjective 4A: Determine whether pericarp pigments provide protection against grain pathogens.
Subobjective 4B: Determine whether grain tannins prevent fungal infection.
Approach
Sorghum (Sorghum bicolor) is a climate resilient crop, which is capable of providing grain and forage (biomass) to both the existing agricultural markets and the emerging bioenergy markets in the United States. To compete in these markets, compositional improvements to both sorghum grain and forage are needed and an understanding how these changes affect the fungal pathogens of sorghum. The objectives of this project will focus on the genetic, biochemical, and physiological mechanisms affecting the composition of sorghum biomass and grain. Efforts will result in sorghum with altered lignin content and/or composition of biomass, and increased starch content and reduced phytate content of grain for improved bioenergy conversion, livestock utilization and human nutrition. The impacts of fungal pathogens on sorghum with compositionally modified biomass and grain will be determined. Sorghum germplasm with desirable traits enhancing sorghum biomass and grain utilization will be developed, fully characterized, released and deposited into USDA–ARS National Plant Germplasm System (NPGS) for use by public and private sector plant breeders for developing improved hybrids and cultivars. The project consists of three integrated components: germplasm development, molecular biology, and plant pathology. Molecular and conventional methodologies will be utilized, and the project scale will range from gene-level to field-level. The project also has extensive formal and informal collaborations enhancing our ability to conduct this research. Anticipated products include improved sorghum germplasm for the sorghum seed industry with value-added traits and biotic stress tolerance, and tools to assess these biological pathways and fungal pathogen responses of sorghum.
Progress Report
Lignin is a component of the plant cell wall and its presence affects the use of sorghum as livestock forage or bioenergy feedstock. Objective 1: Candidate genes for each brown midrib (bmr) loci 29, 30, 31 and 32 were identified in previous fiscal years. The candidate gene for bmr30 was confirmed through the identification of a second mutation in this gene with the brown midrib phenotype. The three alleles of bmr32 were confirmed to be alleles of the previously described bmr19 through DNA sequencing and DNA marker analyses. Experiments are being performed to verify that the mutations identified are responsible for the changes to lignin observed in bmr29 and 31 mutant plants. The identification of the bmr30 and 32 genes validate the use of the brown midrib phenotype to discover new genes that affect lignin synthesis. The newly developed bmr29 through 32 mutant lines in three sorghum varieties were grown in the field for the second year of a two-year trial at two Nebraska locations. Field traits were measured and biomass from these lines was collected for future analyses. The biomass samples from the bmr plants have been processed, and chemical compositional analyses are currently underway to determine the effects of these mutants on lignin synthesis. The discovery of the genes underlying these bmr mutants through this approach will pave the way for the development of rational strategies to combine bmr mutants to alter lignin content and composition for bioenergy and forage uses in sorghum and other grasses. Understanding the impact of each brown midrib mutant in different varieties will lay the foundation for the development of the next generation of brown midrib hybrids that will benefit the livestock and bioenergy industries. Lines overexpressing different genes in lignin synthesis were combined together and with bmr mutants to further elevate phenolic compounds related to lignin in sorghum biomass. The analyses of biomass from this strategy are currently underway. This approach may lead to new resources for renewable chemical applications, because the elevated phenolic compounds in the biomass may not affect plant growth and may be valuable precursors for green chemistry.
Phytate is a major phosphorus storage compound in seeds, but it is also an antinutrient for both animals and humans, and its presence in animal waste leads to phosphorus management problems for surface water. Objective 2: Several sorghum mutants with reduced levels of phytate were identified in previous fiscal years. Further testing narrowed the focus to two mutants, which are in two separate genes. Reduced phytate levels in grain were not observed in the subsequent generation for these two mutants, hence seeds from the previous generation were grown, and the seed produced are being reassessed for the low phytate trait this summer. The ability to develop reduced phytate sorghum would increase the use of sorghum in animal feed.
Starch is a major component of sorghum grain, and it is also the starting material for ethanol production and provides nutritional energy for both humans and livestock. Objective 2: Sorghum lines with increased starch content in the grain were identified and crossed into elite sorghum lines in previous fiscal years. The resulting lines were self-pollinated for six generations. These lines were planted in the field this spring, and starch content will be analyzed in fiscal year 2021. Increasing the starch concentration of grain will open new opportunities for sorghum as a livestock feed and ethanol-based biofuels.
Stalk rot pathogens are destructive to sorghum, which impacts both grain and biomass yield. Plant resistance and biocontrols are the main strategies to protect grain and forage sorghums from these fungal pathogens. The cell wall polymer lignin has been implicated as a defense against pathogens including stalk rots and altering its synthesis and composition may improve sorghum for forage and bioenergy uses. Objective 3: we tested several novel bmr mutants decreased in lignin content for susceptibility to three stalk pathogens and determined that these lines were not more susceptible to the pathogen than normal lines. One line was even more resistant to charcoal rot than its normal counterpart. Likewise, lines developed for altered lignin synthesis through combining bmr lines with transgenic ones were screened for resistance to stalk rot pathogens. These lines were as resistant as normal lines to these pathogens, or had slightly increased resistance to the pathogen as compared to normal lines. Together these data demonstrate that altering lignin synthesis does not increase susceptibility to these potentially devastating pathogens of sorghum, which is important information for forage and bioenergy sorghum efforts. The D locus controls whether sorghum stalks are dry or juicy, which can improve post-harvest drying of forage. The responses of plants with either stalk type are being assessed for responses to stalk pathogens along with sugar concentration and composition. This research will determine whether stalk type affects the susceptibility to stalk pathogens, which is critical information for developing forage sorghum lines. Beneficial microbes associated with sorghum are potentially effective ways to control stalk pathogens without using fungicides. To characterize the microbial genes associated with sorghum colonization, fungal inhibition and growth promotion, the genomes of beneficial bacteria were sequenced with next-generation technology through a USDA Alternatives to Antibiotics Program funded grant. The gene identification will increase our understanding of how beneficial microbes prevent plant disease and promote plant growth. Together these projects will determine how common stalk traits affect susceptibility to stalk rots and which microbe stains may limit these diseases.
Fungal pathogens also infect sorghum grain, which may render it unusable as food or feed due to the presence of fungal toxins. The grain pigments of sorghum may protect against grain mold pathogens. Objective 4: we grew sorghum with red, yellow or unpigmented grain in both Texas (high disease pressure) and Nebraska (moderate disease pressure) to assess the role of grain pigments in grain mold resistance. We are currently determining the amount of pathogens in these samples. Sorghum lines with and without tannins, a lignin like compound that may be present in the outer layer of the grain, were grown in Nebraska. This outer layer was removed through decortication in collaboration with ARS scientists in Manhattan, Kansas, and we will be able to determine how far different pathogens can grow into the grain if the tannins are present or absent. Understanding whether grain pigments or tannins affect grain molds is critical information for the development of food-grade and specialty sorghums where pigments may be undesirable.
Accomplishments
1. Demonstrated a lignin-related enzyme changes lignin composition of sorghum biomass. Sorghum biomass serves as an important forage crop for livestock, and it is being developed as a bioenergy crop. The ferulate 5-hydroxylase (F5H) gene encodes an enzyme involved in the synthesis of the biomass component lignin. To understand the role of this enzyme in lignin synthesis and its effect on cell wall composition, researchers in Lincoln, Nebraska, and their collaborators used biotechnology to greatly elevate expression of the F5H gene in sorghum plants. F5H alone and in combination with brown midrib 12 (bmr12) changed the lignin composition within cell walls, which was observable through the microscope. This work was published in the journal Plant Molecular Biology, which featured it on the cover of the June issue. This research demonstrated new ways to change the lignin composition of sorghum biomass, which may lead to the production of renewable chemicals that require specific lignin composition.
2. Characterization of the changes responsible for increased stalk pathogen- and drought-resistant in brown midrib 12 (bmr12) sorghum. Researchers in Lincoln, Nebraska, examined the responses of two brown midrib lines (bmr6 and bmr12) and the corresponding normal line to two stalk diseases (Fusarium stalk rot and charcoal rot) under drought stress and adequate water conditions. Although bmr12 plants are impaired in lignin synthesis, which is a cell wall component that is thought to play a role in plant defenses against both drought and pathogen attack, surprisingly bmr12 plants had less disease symptoms under drought conditions compared to normal plants or even bmr12 plants under adequate water conditions. Further analyses show that bmr12 had increased defense signals under drought conditions, which suggested these plants were already prepared for a pathogen attack. This research showed that bmr12 can effectively reduce lignin to improve forage and bioenergy sorghum, and it may even increase disease and drought resistance. The identification of genes and pathways affected in bmr12 plants may lead to the development of more climate and disease resilient sorghum hybrids.
Review Publications
Funnell-Harris, D.L., Graybosch, R.A., O'Neill, P.M., Duray, Z.T., Wegulo, S.N. 2019. Amylose-free (“waxy”) wheat colonization by fusarium spp. and response to fusarium head blight. Plant Disease. 103(5):972-983. https://doi.org/10.1094/PDIS-05-18-0726-RE.
Funnell-Harris, D.L., Sattler, S.E., O'Neill, P.M., Gries, T.L., Tetreault, H.M., Clemente, T.E. 2019. Response of sorghum enhanced in monolignol biosynthesis to stalk pathogens. Plant Disease. 103(9):2277-2287. https://doi.org/10.1094/PDIS-09-18-1622-RE.
Tetreault, H.M., Gries, T.L., Palmer, N.A., Funnell-Harris, D.L., Sarath, G., Sattler, S.E., Sato, S., Ge, Z. 2020. Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor. Plant Molecular Biology. 103(3):269-285. https://doi.org/10.1007/s11103-020-00991-3.
Bolanos-Carriel, C., Wegulo, S.N., Hallen-Adams, H., Baenziger, P.S., Eskridge, K.M., Funnell-Harris, D.L. 2020. Effects of field-applied fungicides, grain moisture, and time on deoxynivalenol during postharvest storage of winter wheat grain. Canadian Journal of Plant Science. 100(3):304-313. https://doi.org/10.1139/cjps-2019-0075.
