Location: Floral and Nursery Plants Research2020 Annual Report
1a. Objectives (from AD-416):
1. Enhance marker resources and diversity assessment in the genus Danthonia. (NP215 2D) Sub-objective 1.A: Utilize induced mutations to study D.spicata reproductive biology. Sub-objective 1.B: Evaluate native Danthonia species genetic diversity and ploidy. 2. Characterize the phytobiome of native and newly established Danthonia populations and correlate them with biotic and abiotic stress resistance. (NP215 2D) Sub-objective 2.A: Evaluate the genetic diversity of Atkinsonella hypoxylon associated with D. spicata. Sub-objective 2.B: Utilize culture-dependent fungal isolation to identify the endophytic fungal populations of D. spicata from diverse locations. Subobjective 2.C: Determine the contribution of the Danthonia phytobiome on ecosystem services in the DC metropolitan urban environment. 3. Determine the impacts of stress (heat and drought) on Danthonia gene expression and reproductive biology. (NP215 2D) 4. Characterize the phytobiome of turf species and determine their role in disease resistance in the warm humid climate of the mid-Atlantic U.S. The research will investigate the contribution of the turf phytobiome to ecosystem services and disease resistance.
1b. Approach (from AD-416):
The native grass Danthonia spicata has a number of characteristics that make it well suited for use as a low input turf in the U.S. mid-Atlantic region. The primary drawback for utilization is poor seed production potential and very limited genetic diversity. For example, D. spicata seed production suffers from seed shattering, a common characteristic of unimproved grasses, but its lack of genetic diversity combined with its unusual reproductive characteristics do not provide options for improving seed production through conventional breeding approaches. The unusual reproductive characteristics of D. spicata include anther developmental arrest at a very early stage and precocious seed production without any evidence of fertilization. Progeny plants exhibit extreme uniformity, and SSR markers scored on progeny populations are monomorphic. Although apomixis would be a possible reproductive strategy, apomicts typically exhibit high levels of fixed heterozygosity due to premeiotic embryo formation. Post-meiotic automixis, either through the formation of a restitution nucleus or an endomitotic event, are additional possible mechanisms. To test the various reproductive strategies genetically and to generate variation for plant improvement requires the creation of polymorphisms that can be followed through meiosis; this will be done through mutation breeding. The genus Danthonia includes a number of species that have been recognized as native grasses of the U.S. The existence of an unusual reproductive biology in the genus and the fact that a number of the proposed species grow in close proximity suggests that some of the species may simply be variants of a single species. One mechanism that would support the current species designations is polyploidy. Currently no data is available on the ploidy levels of the native Danthonia species. A second factor that would support the species designations is large amounts of genetic variation between the described species; this will be tested using Simple Sequence Repeat (SSR) markers. Atkinsonella hypoxylon (Clavicipitaceae, tribe Balansieae) is an Ascomycete that has been reported to grow epiphytically and endophytically on grasses in the genus Danthonia. Genetic diversity assessments of A. hypoxylon have been completed; however, they were conducted using isozyme technology and no Internal Transcribed Spacer (ITS) data was obtained to confirm isolate taxonomy. The genome sequence of A. hypoxylon has been determined, making Simple Sequence Repeat (SSR) marker development possible; currently our lab has approximately 20 functional SSR primer pairs that have been tested on isolates from 4 locations. Additionally, all isolates we have examined exhibit multiple loci with 2 alleles. This multiallelic state in what is expected to be haploid hyphae has also been reported in Epichloe festucae and could be due to multistrain infections of the plants. ITS cloning will be utilized to determine if Danthonia plants harbor multiple symbiotic fungi.
3. Progress Report:
Sequencing of the Atkinsonella hypoxylon internal transcribed spacer 1 and 2 regions of the 5.8S rRNA gene and analysis of ten SSR loci from 27 A. hypoxylon isolates collected at five different locations has been completed. The results of this study indicated that the diversity of A. hypoxylon can be evaluated by simply scoring SNPs in the ITS-1 spacer region and that two distinct groups of isolates exist within natural populations. The ITS-1 sequence was utilized to design A. hypoxylon-specific PCR primers that allow the fungus to be identified and grouped using D. spicata plant tissue or collected A. hypoxylon choke fruiting bodies. The primers were used to screen 20 D. spicata seed heads from plants that have never exhibited choke, and all samples tested positive for A. hypoxylon. These results indicate that the fungus may be associated with all D. spicata plants not just those exhibiting choke, providing further evidence that A. hypoxylon may influence the reproductive biology of D. spicata. Creeping bentgrass, Agrostis stolonifera, and colonial bentgrass, A. capillaris, are cool-season turfgrass species well adapted for utilization on golf courses. Natural stands of creeping bentgrass are often found in wetland areas and exhibit very poor tolerance to dry soils. Colonial bentgrass is frequently found in dryer habitats and has the ability to go dormant and recover quickly under water deficit stress. Hybridization between creeping and colonial bentgrass is possible, and a better understanding of gene regulation under water deficit conditions could allow bentgrasses with improved water deficit stress tolerance to be developed. A greenhouse dry-down study was conducted using two creeping bentgrass clones with differing water use profiles and one colonial bentgrass clone. Three replicates of each genotype were treated, and well-watered control clones included for a total of 18 experimental units. Gravimetric Evapotranspiration (ET) was determined daily during the dry-down with control plants watered at 80% ET and deficit irrigation plants watered at 50% ET. Electrolyte leakage was monitored at the beginning and end of the dry-down with no differences detected. ET rates were very similar among all plants, with the creeping genotypes exhibiting stress earlier and recovering more slowly than the colonial genotype. At the conclusion of the experiment all above ground plant material was harvested and RNA-seq analysis was conducted; approximately 10,000 differentially expressed contigs were identified. A high number of the differentially regulated water deficit genes are present in the colonial bentgrass entry.