Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides

Current knowledge of resistance (R) genes and their use for genetic improvement in buffalograss (Buchloe dactyloides [Nutt.] Engelm.) lag behind most crop plants. This study was conducted to clone and characterize cDNA encoding R gene-like (RGL) sequences in buffalograss. This report is the first to clone and-characterize of buffalograss RGLs. Degenerate primers designed from the conserved motifs of known R genes were used to amplify RGLs and fragments of expected size were isolated and cloned. Sequence analysis of cDNA clones and analysis of putative translation products revealed that most encoded amino acid sequences shared the similar conserved motifs found in the cloned plant disease resistance genes RPS2, MLA6, L6, RPM1, and Xa1. These results indicated diversity of the R gene candidate sequences in buffalograss. Analysis of 5' rapid amplification of cDNA ends (RACE), applied to investigate upstream of RGLs, indicated that regulatory sequences such as TATA box were conserved among the RGLs identified. The cloned RGL in this study will further enhance our knowledge on organization, function, and evolution of R gene family in buffalograss. With the sequences of the primers and sizes of the markers provided, these RGL markers are readily available for use in a genomics-assisted selection in buffalograss

Development and Characterization of Texas Bluegrass x Kentucky Bluegrass Interspecific Hybrids for the Southern United States

Kentucky bluegrass (Poa pratensis L.) and tall fescue (Festuca arundinacea Schreb.) are commonly grown cool-season grasses in the United States for home lawns and various sports turf applications. However, environmental conditions such as prolonged periods of drought and heat as well as heavy shade often result in a loss of visual appeal, early dormancy, or death of the turf stand. Hybrid bluegrass (Poa spp.) cultivars derived from Texas bluegrass (P. arachnifera Torr.) x Kentucky bluegrass interspecific crosses may serve as viable alternatives for perennial turfgrass in the southern United States. Methods to aid in the breeding and selection of Texas x Kentucky bluegrasses for these environmental conditions would be useful. The goals of this research were to 1) develop new interspecific hybrid bluegrasses and optimize a seed germination technique, 2) use flow cytometry and molecular markers for the characterization of hybrid progeny, and 3) evaluate the growth response and performance of hybrid bluegrasses maintained in shade. In 2012 and 2013, controlled pollinations were made between different Poa species. Seed from these crosses were germinated through two different techniques that resulted in 61 new interspecific hybrids. A higher percentage of individuals (89%) were recovered through germination on nutrient agar medium than soil. In addition to these hybrids, 52 Texas x Kentucky hybrids created in 2001 were tested for three years in multiple locations across the southern states and the transition zone. Experimental hybrid TAES 5653, registered as DALBG 1201, was the superior genotype. Flow cytometry was used to estimate the DNA content of 19 Texas bluegrass genotypes, and four Texas x Kentucky bluegrass hybrids from two different pedigrees. Variability in DNA content suggested variability in the ploidy levels of Texas bluegrass. Texas x Kentucky bluegrass interspecific hybrids had an intermediate DNA content demonstrating flow cytometry can identify true hybrids between parents with vastly different DNA content. A thioredoxin-like (trx) nuclear gene was also useful in identifying true Texas x Kentucky bluegrass hybrids. A distinctive 851 bp trx allele with a 163 bp insertion site was also identified. The insertion is evidently a transposable element that may help to elucidate ancestors of Texas bluegrass. Another study was conducted to develop a method of comparing Texas x Kentucky bluegrass hybrids to commercial cultivars for shade tolerance. It was determined that evaluations should be conducted in the spring to early summer months using moderate shade (50%) levels, and high turfgrass quality and slower leaf elongation rates as selective traits. Trinexapac-ethyl was not necessary to reduce the leaf elongation rate or increase quality of dwarf hybrids under shade. In summary, several methods have been tested to generate and evaluate new Texas x Kentucky bluegrass hybrids. Seed germination conducted under controlled conditions using agar medium, and flow cytometry and the trx nuclear gene can be employed in bluegrass breeding programs to identify true interspecific hybrids. Finally, improved dwarf-type hybrid bluegrasses, such as DALBG 1201 and TAES 5654, seem to have enhanced shade tolerance and potential for use as perennial turfgrass alternatives in the southern and transition zone regions of the United States

Transcriptome analysis of two buffalograss cultivars

Background: Buffalograss [Buchloë dactyloides (Nutt.) Engel. syn. Bouteloua dactyloides (Nutt.) Columbus] is a United States native turfgrass species that requires less irrigation, fungicides and pesticides compared to more commonly used turfgrass species. In areas where water is limited, interest in this grass species for lawns is increasing. While several buffalograss cultivars have been developed through buffalograss breeding, the timeframe for new cultivar development is long and is limited by a lack of useful genetic resources. Two high throughput next-generation sequencing techniques were used to increase the genomic resources available for buffalograss. Results: Total RNA was extracted and purified from leaf samples of two buffalograss cultivars. ‘378’ and ‘Prestige’ cDNA libraries were subjected to high throughput sequencing on the Illumina GA and Roche 454 Titanium FLX sequencing platforms. The 454 platform (3 samples) produced 1,300,885 reads and the Illumina platform (12 samples) generated approximately 332 million reads. The multiple k-mer technique for de novo assembly using Velvet and Oases was applied. A total of 121,288 contigs were assembled that were similar to previously reported Ensembl commelinid sequences. Original Illumina reads were also mapped to the high quality assembly to estimate expression levels of buffalograss transcripts. There were a total of 325 differentially expressed genes between the two buffalograss cultivars. A glycosyl transferase, serine threonine kinase, and nb-arc domain containing transcripts were among those differentially expressed between the two cultivars. These genes have been previously implicated in defense response pathways and may in part explain some of the performance differences between ‘Prestige’ and ‘378’. Conclusions: To date, this is the first high throughput sequencing experiment conducted on buffalograss. In total, 121,288 high quality transcripts were assembled, significantly expanding the limited genetic resources available for buffalograss genetic studies. Additionally, 325 differentially expressed sequences were identified which may contribute to performance or morphological differences between ‘Prestige’ and ‘378’ buffalograss cultivars

Buffalograss Diversity Assessment

Buffalograss [Buchloë dactylodes (Nutt.) Engelm. syn. Bouteloua dactyloides (Nutt.) Columbus] is a low input alternative turfgrass for the Great Plains region. Buffalograss is a dioecious stoloniferous warm season grass with wide variation of valuable traits and is well adapted for use in minimal maintenance landscapes. The obligate outcrossing nature of the species and wide variation of potentially valuable traits make buffalograss a prime candidate for cultivar improvement and genetic study. Limited information is available regarding buffalograss genetics. In effort to expand genetic resources around buffalograss, we developed buffalograss derived simple sequence repeat markers from previously available transcriptomes of buffalograss cultivars ‘378’ and ‘Prestige’. Simple sequence repeat markers were developed using the microsatellite identification tool (MiSa). We developed 139 simple sequence repeat markers. A panel of 24 of the markers were confirmed for amplification and validated on a set of seven buffalograss cultivars. A collection of 96 new buffalograss genotypes were collected from throughout the Great Plains region and genetically characterized using the panel of SSR markers. The collection was clustered by similarity using the unweighted pair-group arithmetic means (UGMA) produced by the sequential, agglomerative, hierarchical, and nested clustering methods (SAHN) program in the Numerical Taxonomy (NTSYS) system of software, genetic similarity coefficients ranged from 0.46 to 1.00. The collection was also observed for visual quality, establishment, stolon count, stolon width, genetic color, fall color, and spring green up. Phenotypic differences were observed between genotypes and by geographic location that the genotype was collected. Advisor: Keenan Amundse

Characterization and Control of Ophiosphaerella spp. Causing Spring Dead Spot of Bermudagrass in South Carolina, USA and Buenos Aires, Argentina

Spring dead spot (SDS) disease is an important root disease of bermudagrass (Cynodon spp.) in transition zone habitats around the world. Causal agents have been identified in the U.S. as Ophiosphaerella korrae (OK), O. herpotricha (OH), and O. narmari, but they have not been identified in Argentina (ARG)

Ameliorating Drought-Induced Stress in Turfgrass through Genetic Manipulation

To delineate the major processes associated with short water scarcity in four tall fescue species, we examined their enzymatic and nonenzymatic antioxidant activity and FaSGR expression levels. Moreover, we examined the possibility of Agrobacterium-mediated transformation of Arabidopsis P5CS1 gene in tall fescue. According to the results, proline has been introduced as an important compatible osmolyte, so as to protect enzymes and cellular structures under water scarcity. In addition to that, superoxide dismutase (SOD) along with proline can be used as a core physiological indicator for the assessment of adaptability to environmental conditions. Results indicated that most of the superoxide that was produced as a result of drought stress was converted to H2O2 by SOD and subsequently detoxified by ascorbate peroxidase (APX) into H2O. Notably, the FaSGR transcript increased drastically over the course of the drought stress in Pixie and Mini-mustang, in contrast to jaguar and h–d, supporting the notion of Stay GReen (SGR)-mediated chlorophyll degradation in the less drought-tolerant cultivars. Different modulations of ROSs quenching system in tall fescue genotypes suggest that even one stress signal causes different signaling responses in different cultivars. The heterologous transformation of P5CS1 in Festuca arundinacea background, confirmed by PCR and transient GUS assay, most probably can improve tall fescue tolerance to drought stress

Physiological and genetic control of water stress tolerance in zoysiagrass

Significant cultivar difference in many water stress responses of zoysiagrass (Zoysia japonica (Steud.) and Zoysia matrella (L.) Merr.) are shown in this study. Of the four cultivars, Palisades was the most water stress tolerant, had the most negative turgor loss point, and leaf rolled after loss of full turgor pressure. On the other end of the spectrum, Diamond was the least water stress tolerant, had the lowest full turgor pressure, the least negative turgor loss point, and leaf rolled at full turgor. Differences between Diamond, Cavalier, Palisades, and DALZ 8504 in leaf rolling, loss of full turgor, water release curve parameters, root characteristics and gene expression make zoysiagrass a prime candidate for further investigation into the mechanisms of water stress avoidance/tolerance. Enhanced antioxidant activity and stomatal control, along with root characteristics, most likely explain the cultivar difference in water stress tolerance of zoysiagrass. Palisades and DALZ 8504 maintained full turgor for significantly longer than Diamond and Cavalier, which may be associated with root characteristics and/or enhanced stomatal control as only those two cultivars showed enhanced expression of a stomatal control gene (phospholipase D). The apparent response (most apparent in turgid weight/dry weight ratios (TWDW)) of well watered plants to water stressed neighbor plants will likely be the most novel finding of this study. Well watered zoysiagrass and Kentucky bluegrass responded to water stressed neighbors by reducing TWDW. Significant increases in gene expression of a systemin degrading enzyme and of an integral membrane protein (signal receptor) were also observed in well watered plants. Results from this study indicate that this phenomenon is occurring and expose a dearth in scientific understanding that must be filled. Improving water stress tolerance through breeding for parameters like those discussed in this paper (delayed leaf rolling or loss of full turgor, enhanced stomatal control, enhanced antioxidant activity, deep rooting characteristics, etc.) may very likely produce turfgrasses that can survive and maintain desired aesthetic qualities using significantly less water

Etiology and Epidemiology of Mini-ring in Ultradwarf Bermudagrass Putting Greens

Mini-ring is a disease in ultradwarf bermudagrass (UDBG) [Cynodon dactylon (L.) Pers. × C. transvaalensis (Burtt-Davy)] putting greens caused by Waitea zeae (Voorhees) J.A. Crouch & Cubeta, (formerly Rhizoctonia zeae). Symptoms typically resemble frog-eye patches that are 10 to 40 cm in diameter with a bronze to orange outer ring and green center. In the southeastern United States, mini-ring symptoms appear in late-summer and generally persist until UDBG dormancy in late-fall. Mini-ring is often problematic in UDBG when nitrogen (N) fertility is reduced to manage organic matter production and improve putting green performance and perceived green speed. While W. zeae is most frequently reported as the causal agent, other species of Waitea have been isolated from UDBG exhibiting mini-ring symptoms. Waitea zeae causes visible leaf lesions and basal sheath rot in other turfgrasses; however, in UDBG, dieback of leaf tissue occurs in the absence of leaf lesions and sheath rot. Although W. zeae has been isolated from UDBG leaf tissue throughout the growing season, it is unclear if other plant tissues—e.g., root, rhizomes, and stolons—may be possible infection courts and when W. zeae infection most likely occurs. The objectives of these studies were to: I) investigate the impact of N source and N rate on mini-ring disease development and severity in UDBG; II) determine what plant tissues W. zeae can infect and when infection is most likely to occur; and III) collect and characterize isolates of Waitea spp. recovered from symptomatic UDBG putting greens. To study the impact of N on mini-ring disease severity, ammonium sulfate (AMS) [(NH4)2SO4] and urea (CH4N2O) were applied weekly to ‘P18’ (MiniVerde) and ‘TifEagle’ UDBG at rates of 4.9, 9.8, and 14.7 kg N ha-1. Mini-ring severity increased with increasing rates of AMS whereas disease symptoms in plots treated with urea remained relatively low. Cores from a UDBG putting green located in Florence, SC were collected monthly from June to October in 2016 and 2017. Isolation of W. zeae occurred in all months, isolation frequencies were greatest in August and September. In a growth chamber bioassay, UDBG roots, stolons, and leaves were infected by W. oryzae or W. zeae when inoculum when growing medium was infested with representative isolates. Nineteen Waitea species isolates were recovered from UDBG putting greens expressing mini-ring symptoms in North Carolina and South Carolina. Isolates of W. prodiga, W. oryzae, and W. zeae represented 5, 16, 79% of isolates collected. Isolates were characterized by sequencing the rDNA-internal transcribed spacer region, and these sequences clustered with Waitea species isolate sequences deposited in GenBank and previously described. These studies demonstrate the primary causal agent of mini-ring in UDBG is W. zeae, while other species of Waitea are likely involved to a lesser extent. An effective mini-ring management strategy should include regular N applications using N sources other than AMS, such as urea, to promote UDBG growth and recuperative potential and applications of fungicides mid-summer before symptom development. Fungicides should be applied in a manner that encourages movement of active ingredients into the putting green rootzone to reduce W. zeae infection of UDBG roots and stolons

Agricultural Research Division 108th Annual Report 1994

It is a pleasure to provide the 108th Annual Report of the UNL Agricultural Research Division (ARD). This report contains lists of current faculty, visiting scientists and research associates; active research projects; refereed journal articles, books, and book chapters published; theses and dissertations written; germplasm/cultivars released; and awards received by faculty. Also included are brief descriptions of accomplishments from selected research projects and the financial report for the period July 1, 1993, to June 30, 1994. This report was compiled in compliance with the intent of the law of the State of Nebraska that established the Nebraska Agricultural Experiment Station on March 31, 1887. Research Impacts ... 4 Foreword ... 5 Research Highlights ... 6 Faculty Awards and Recognition ... 14 Variety and Germplasm Releases ... 16 Patents ... 18 Administration ... 19 … Administrative Personnel ... 19 … Organizational Chart ... 20 … Administrative Units ... 21 … IANR Research Facilities ... 22 Faculty ... 23 … Agricultural/Natural Resources Departments ... 23 … College of Human Resources and Family Sciences ... 29 … Off-Campus Research Centers ... 30 Visiting Scientists/Research Associates ... 32 Research Projects ... 34 Publications ... 43 Research Expenditures ... 6

Population Diversity and Fungicide Sensitivity of Gaeumannomyces graminis var. graminis from St. Augustinegrass in Texas

Gaeumannomyces graminis var. graminis (Ggg) is an ectotrophic root infecting fungus that causes root-decline of warm-season turfgrasses and black sheath rot of rice (Oryza sativa). A total of 76 Ggg isolates were collected from St. Augustinegrass (Stenotaphrum secundatum) throughout Texas. Variations of colony morphology, hyphopodia production, teleomorphic and anamorphic structures, and growth rate of isolates were examined on potato dextrose agar (PDA), and rice seedling pathogenicity assays were performed. A multilocus phylogeny reconstruction was developed to support phenotypic data and to resolve the phylogeny of Ggg. Fungicide sensitivities were also evaluated. Azoxystrobin 50% effective concentration (EC50) values of nine isolates were determined in the presence and absence of salicylhydroxamic acid (SHAM) for determination of alternative respiration. Tebuconazole and azoxystrobin EC50 values from 15 and 20 isolates, respectively, were used to establish single discriminatory concentrations for resistance screening and EC50 determination of remaining isolates. Isolates were also screened for thiophanate-methyl resistance. Three phenotypic groups were found on PDA: (i) highly melanized with round colony formation (termed M group), (ii) non to slightly melanized with round colony formation (termed L group), and (iii) highly melanized with irregular colony formation (termed H group). All isolates produced lobed hyphopodia and a Phialophora-like anamorph, where phialospore lengths were significantly longer among M and H groups compared to the L group. M group isolates were adapted to warmer temperatures compared to L and H group isolates, and groups were differentiated based on growth rates at 35°C. The M group was most aggressive. A multilocus phylogeny reconstruction supported the differentiation between phenotypic groups and distinguished Ggg and G. graminis varieties avenae and tritici. EC50 values from azoxystrobin-amended PDA were significantly lower in the presence of SHAM, indicating utilization of alternative respiration. Azoxystrobin and tebuconazole discriminatory concentrations were 0.1 μg a.i. ml-1 and were effective for rapid EC50 determination. Azoxystrobin EC50 values for 75 isolates ranged from <0.014 to 0.399. Tebuconazole EC50 values for 76 isolates ranged from 0.021 to 0.296 with a mean of 0.049. All isolates were completely inhibited by thiophanate-methyl at 500 μg a.i. ml^-1