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Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits.

The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of AGROSTIS: This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.

Journal Article↗

Root respiratory characteristics associated with plant adaptation to high soil temperature for geothermal and turf-type Agrostis species.

Respiration is a major avenue of carbohydrates loss. The objective of the present study was to examine root respiratory characteristics associated with root tolerance to high soil temperature for two Agrostis species: thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park, and two cultivars ('L-93' and 'Penncross') of a cool-season turfgrass species, A. stolonifera (creeping bentgrass), that differ in their heat sensitivity. Roots of thermal A. scabra and both creeping bentgrass cultivars were exposed to high (37 degrees C) or low soil temperature (20 degrees C). Total root respiration rate and specific respiratory costs for maintenance and ion uptake increased with increasing soil temperatures in both species. The increases in root respiratory rate and costs for maintenance and ion uptake were less pronounced for A. scabra than for both creeping bentgrass cultivars (e.g. respiration rate increased by 50% for A. scabra upon exposure to high temperature for 28 d, as compared with 99% and 107% in 'L-93' and 'Penncross', respectively). Roots of A. scabra exhibited higher tolerance to high soil temperature than creeping bentgrass, as manifested by smaller decreases in relative growth rate, cell membrane stability, maximum root length, and nitrate uptake under high soil temperature. The results suggest that acclimation of respiratory carbon metabolism plays an important role in root survival of Agrostis species under high soil temperatures, particularly for the thermal grass adaptation to chronically high soil temperatures. The ability of roots to tolerate high soil temperatures could be related to the capacity to control respiratory rates and increase respiratory efficiency by lowering maintenance and ion uptake costs.

Acclimatization↗

Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats.

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate-resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate-resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20,400 samples (0.04 +/- 0.01% SE). CP4 EPSPS-positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen-mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short-term production can result in establishment of transgenic plants at multi-kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.

Agriculture↗

Assimilation and allocation of carbon and nitrogen of thermal and nonthermal Agrostis species in response to high soil temperature.

We studied whether changes in the assimilation and allocation of carbon and nitrogen are associated with plant tolerance to high soil temperatures. Two Agrostis species, thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park (USA), and two cultivars of a cool-season species, Agrostis stolonifera, L-93 and Penncross, were exposed to soil temperatures of 37 or 20 degrees C, while shoots were exposed to 20 degrees C. Net photosynthesis rate, photochemical efficiency, NO(3) (-)-assimilation rate and root viability decreased with increasing soil temperatures in both species. However, the decreases were less pronounced for A. scabra than for both A. stolonifera cultivars. Carbon investment in growth of plants exposed to 37 degrees C decreased more dramatically in both A. stolonifera cultivars than in A. scabra. Nitrogen allocation to shoots was greater in A. scabra than in both creeping bentgrass cultivars at 37 degrees C soil temperature. Our results demonstrate that plant tolerance to high soil temperature is related to efficient expenditure and adjustment of C- and N-allocation patterns between growth and respiration.

Agrostis↗

Comparison of early development of three grasses: Lolium perenne, Agrostis stolonifera and Poa pratensis.

BACKGROUND AND AIMS: To improve the management of grass communities, early plant development was compared in three species with contrasting growth forms, a caespitose (Lolium perenne), a rhizomatous (Poa pratensis) and a caespitose-stoloniferous species (Agrostis stolonifera). METHODS: Isolated seedlings were grown in a glasshouse without trophic constraints for 37 d (761 degrees Cd). The appearance of leaves and their location on tillers were recorded. Leaf appearance rate (LAR) on the tillers and site-filling were calculated. Tillering was modelled based on the assumption that tiller number increases with the number of leaves produced on the seedling main stem. Above- and below-ground parts were harvested to compare biomass. KEY RESULTS: Lolium perenne and A. stolonifera expressed similar bunch-type developments. However, root biomass was approx. 30 % lower in A. stolonifera than in L. perenne. Poa pratensis was rhizomatous. Nevertheless, the ratio of above-ground : below-ground biomass of P. pratensis was similar to that of L. perenne. LAR was approximately equal to 0.30 leaf d(-1) in L. perenne, and on the main stem and first primary tillers of A. stolonifera. LAR on the other tillers of A. stolonifera was 30 % higher than on L. perenne. For P. pratensis, LAR was 30 % lower than on L. perenne, but the interval between the appearance of two successive shoots from rhizomes was 30 % higher than the interval between two successive leaf stages on the main stem. Above-ground parts of P. pratensis first grew slower than in the other species to the benefit of the rhizomes, whose development enhanced tiller production. CONCLUSIONS: Lolium perenne had the fastest tiller production at the earliest stages of seedling development. Agrostis stolonifera and P. pratensis compensated almost completely for the delay due to higher LAR on tillers or ramets compared with L. perenne. This study provides a basis for modelling plant development.

Agrostis↗

Identification of a gene in the process of being lost from the genus Agrostis.

Lineage-specific gene loss is considered one of the processes contributing to speciation and genome diversity. Such gene loss has been inferred from interspecies comparisons of orthologous DNA segments. Examples of intraspecific gene loss are rare. Here we report identification of a gene, designated Crs-1 (creeping specific-1), that appears to be in the process of being lost from heterozygous populations of the species creeping bentgrass (Agrostis stolonifera). The Crs-1 gene encodes a protein with an N-terminal dirigent protein domain and a C-terminal lectin domain and is similar to the maize (Zea mays) beta-glucosidase aggregating factor. Most individual creeping bentgrass plants examined are lacking Crs-1. Some individuals are hemizygous for the Crs-1 locus, indicating major haplotype noncolinearity at that locus. Crs-1 was not detected in several other Agrostis species, indicating it is being lost from the genus. The Crs-1 locus in creeping bentgrass provides a rare example of the evolutionary process of gene loss occurring within a plant species.

Agrostis↗

Genetic diversity in colonial bentgrass (Agrostis capillaris L.) revealed by EcoRI-MseI and PstI-MseI AFLP markers.

Colonial bentgrass (Agrostis capillaris L.) is a potential source for genetic improvement of resistance to environmental stress and disease for other bentgrass species (Agrostis spp.). To conserve and study the existing genetic resources of colonial bentgrass for use in breeding, genetic diversity was investigated using amplified fragment length polymorphism (AFLP) markers. Included in this study were 22 accessions from US Department of Agriculture germplasm collected from 11 countries, in conjunction with 14 accessions from northern Spain and 3 commercial cultivars. Ten EcoRI-MseI and 6 PstI-MseI AFLP primer combinations produced 181 and 128 informative polymorphic bands, respectively. Cluster analysis of genetic similarity estimates revealed a high level of diversity in colonial bentgrass species with averages of 0.51 (EcoRI-MseI) and 0.63 (PstI-MseI). Greater genetic diversity was detected by the EcoRI-MseI AFLP primer combinations. A low but significant positive correlation (r = 0.44, p = 0.0099) between the 2 Jaccard similarity matrices was obtained by the Mantel test. Commercial cultivars of bentgrass showed a narrow genetic background. The assessment of genetic diversity among colonial bentgrass accessions suggested the potential value of the colonial bentgrass germplasm in turfgrass cultivar improvement.

Agrostis↗

Polarography as a tool in peptide and protein analysis: studies on metal-chelating substances induced by cadmium in the algae Pheodactylum tricornutum and the graminae Agrostis capillaris.

Methods to determine characteristics and quantities of metal-binding substances are needed in order to identify phytochemical systems for use in agriculture and waste treatment. A metal-binding polypeptide (phytochelatin) was prepared by liquid chromatography from the algae Pheodactylum tricornutum and roots of the graminae Agrostis capillaris after exposure to cadmium. Polarographic (voltammetric) analysis indicated binding of cadmium by the polypeptide at different stabilities. No catalytic hydrogen peak at -1.7 V appeared in the polarogram of plant and algae metal-binding substance, but did appear in the polarogram of the metal-chelating protein metallothionein. In all polarograms, two significant peaks were detected. One at approximately -520 mV probably represents the reduction of single cadmium-sulfur bonds, and one at -930 mV the reduction of cadmium-thiolate complexes. A dose-response relationship between cadmium concentration and peak current at -930 mV was observed after exposure of roots of Agrostis at 5 and 10 microM.

Cadmium↗

Responses of the plant parasitic nematodes Rotylenchulus reniformis, Anguina agrostis and Meloidogyne javanica to chemical attractants.

Rotylenchulus reniformis, Anguina agrostis and Meloidogyne javanica respond differently to gradients of chemical attractants. In chemotaxis assays performed on agarose plates, R. reniformis L2 larvae oriented their movement to several common inorganic salts, cyclic AMP and AMP, as well as to germinated host plant seeds. M. javanica L2 larvae were attracted to germinated seeds, but not to the salts tested, and A. agrostis dauer larvae were not strongly attracted to any of 12 different tested agents, including host root or shoot tips. Attraction of R. reniformis to salts was measured by comparing different pairwise combinations of ions at equivalent concentrations. The indicated order of attractiveness was: Cl- greater than Na+ greater than C2H3O2- greater than Mg2+, NH4+, SO4(2-). The least attractive salts, (NH4)2 SO4 and MgSO4, were weakly attractive at an orientation threshold of 1 mM, whereas the most attractive salt, MgCl2, was strongly attractive at a threshold of 0.2 mM Cl-. 3',5' cyclic AMP was strongly attractive at a threshold of 0.05 mM, whereas 5'-AMP was a weak attractant. Some of these responses may affect the distribution of R. reniformis in its natural environment.

Adenosine Monophosphate↗

Linkage map construction in allotetraploid creeping bentgrass (Agrostis stolonifera L.).

Creeping bentgrass (Agrostis stolonifera L.) is one of the most adapted bentgrass species for use on golf course fairways and putting greens because of its high tolerance to low mowing height. It is a highly outcrossing allotetraploid species (2n=4x=28, A(2) and A(3) subgenomes). The first linkage map in this species is reported herein, and it was constructed based on a population derived from a cross between two heterozygous clones using 169 RAPD, 180 AFLP, and 39 heterologous cereal and 36 homologous bentgrass cDNA RFLP markers. The linkage map consists of 424 mapped loci covering 1,110 cM in 14 linkage groups, of which seven pairs of homoeologous chromosomes were identified based on duplicated loci. The numbering of all seven linkage groups in the bentgrass map was assigned according to common markers mapped on syntenous chromosomes of ryegrass and wheat. The number of markers linked in coupling and repulsion phase was in a 1:1 ratio, indicating disomic inheritance. This supports a strict allotetraploid inheritance in creeping bentgrass, as suggested by previous work based on chromosomal pairing and isozymes. This linkage map will assist in the tagging and eventually in marker-assisted breeding of economically important quantitative traits like disease resistance to dollar spot (Sclerotinia homoeocarpa F.T. Bennett) and brown patch (Rhizoctonia solani Kuhn).

Agrostis↗

Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment.

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.

Agrostis↗

The influence of season and leaf age on concentrations of radiocaesium (137Cs), stable caesium (133Cs) and potassium in Agrostis capillaris.

The transfer of radioactive caesium from soils to plants has been well researched. In contrast there is limited knowledge on natural stable 133Cs and its potential role as a predictor for radiocaesium behaviour. In a pot experiment with Agrostis capillaris close correlations were found between plant 137Cs and plant 133Cs concentrations (R2 90-96%). Season and leaf age had significant effects with concentrations increasing 10-30-fold between June and December. Simultaneously the plant concentrations of K, the nutrient analogue of Cs, decreased to around one third. In the soil the exchangeable fractions of K and 137Cs declined. No clear relationships were found between 137+133Cs in the plant and exchangeable K in the soil. However, at the end of the experiment the K content of the above-ground biomass was higher than the exchangeable pool in the soil, suggesting that depletion of soil K could be a key factor in the observed increase of plant 137+133Cs over time.

Agrostis↗

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris).

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.

Agrostis↗

Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene.

This study investigated whether pre-treating plants with specific putative signaling components and heat acclimation would induce tolerance of a cool-season grass, creeping bentgrass (Agrostis stolonifera var. palustris), to subsequent heat stress and whether thermotolerance induction of those pretreatments was associated with the regulation of antioxidant regenerating enzymes. The treatments included foliar application of salicylic acid (SA), abscisic acid (ABA), calcium chloride (CaCl2), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene prior to the exposure of plants to heat stress (35 degrees C) in a growth chamber. Physiological measurements including turf quality, leaf photosynthetic rate, and levels of oxidative damage demonstrated that all treatments increased heat tolerance. The better heat tolerance for pre-treated plants as compared to controls was related to the protection of oxidative damage under heat stress. APX activity increased over the first 2 days and 5 days of heating for ACC and CaCl2 respectively, but for only 12 h for H2O2. SA and ABA pre-treatments had no effects on APX activity earlier, but maintained APX activity at a significantly higher level than in controls after 24 h of heating. SA and ABA pre-treatments had no effects on POX activity. ACC treatment significantly increased POX activity. Pre-treatment with CaCl2, H2O2, and HA reduced POX activity, particularly during the later phase of heating. Plants treated with SA, CaCl2, H2O2 and HA had lower CAT activity than their control plants prior to heating and within 48 h of heat stress. ABA and ACC pre-treatments maintained higher CAT activity than the controls after 48 h of heating. ACC, CaCl2, or HA pre-treatments increased SOD activity only before 5 days of heat stress. SA and ABA pre-treatments had less effect on APX activity earlier under heat stress. These results suggest that specific groups of potential signaling molecules may induce tolerance of creeping bentgrass to heat stress by reducing oxidative damage.

Abscisic Acid↗

Population structure and mating-type genes of Colletotrichum graminicola from Agrostis palustris.

Eighty-seven isolates of Colletotrichum graminicola, mostly from Agrostis palustris, were collected in grass fields, most of which were in Ontario, Canada. Specific primers were designed to amplify the mating-type (MAT) genes and, among 35 isolates tested, all yielded a band of the expected size for MAT2. For six isolates, the MAT2 PCR products were sequenced and found to be similar to that reported for MAT2 of C. graminicola from maize. Based on 119 polymorphic bands from 10 random amplified polymorphic DNA primers, analyses of genetic distances were found to generally cluster isolates by host and geographic origin. Among 42 isolates from a grass field in Ontario, significant spatial autocorrelation was found to occur within a 20-m distance, implying that this is the effective propagule dispersal distance. Although clonal propagation was observed in the 87 isolates with 67 unique genotypes, the extent of genetic variation in local populations implies some occurrence of sexual or asexual recombination.

Agrostis↗

Studies on the surface coat (glycocalyx) of the dauer larva of Anguina agrostis.

Imprints of the surface coat (glycocalyx) from the cuticles of living second stage dauer larvae (DL2) of Anguina agrostis (syn. A. funesta) have been examined using incident light fluorescence microscopy and scanning electron microscopy. These surface coats contain residues of N-acetyl-D-glucosamine which were detected by treatment with wheat germ agglutinin labelled with either fluorescein or rhodamine. They also contain protein which was demonstrated by treatment with either pepsin or trypsin. These enzymes inhibited the attachment of the coryneform bacterium Clavibacter sp. to the surface coat, indicating that proteins play a crucial role in the adhesion of these bacteria to the nematode. This inhibition of attachment was reversed within 18 h after removal of the DL2 from the enzymes, indicating that the nematode was capable of renewing its surface proteins.

Actinomycetales↗

Heat-Shock Response in Heat-Tolerant and Nontolerant Variants of Agrostis palustris Huds.

The heat-shock response in heat-tolerant variants (SB) and non-tolerant variants (NSB) of creeping bentgrass (Agrostis palustris Huds.) was investigated. Both variants were derived from callus initiated from a single seed of the cultivar Penncross. SB and NSB synthesized heat-shock proteins (HSPs) of 97, 83, 70, 40, 25, and 18 kD. There were no major differences between SB and NSB in the time or temperature required to induce the heat-shock response. When the HSPs synthesized by SB and NSB were analyzed by two-dimensional gel electrophoresis, it was apparent that SB synthesized two to three additional members of the HSP27 family, which were smaller (25 kD) and more basic than those synthesized by NSB. Analysis of F1 progeny of NSB x SB indicated that 7 of the 20 progeny did not synthesize the additional HSP25 polypeptides. These progeny were significantly less heat tolerant than progeny that did synthesize the additional HSP25 polypeptides. The X2 test of independence (X2 = 22.45, P < 0.001) indicated that heat tolerance and the presence of the additional HSP25 polypeptides are linked traits.

Journal Article↗

Isolation and Partial Purification of Cadmium-Binding Protein from Roots of the Grass Agrostis gigantea.

A cadmium-binding protein was isolated from roots of the grass Agrostis gigantea Roth. Heat-stable proteins were chromatographed on the anion exchanger QAE-Sephadex A-25. The major cadmium fraction was purified further by gel filtration on Sephadex G-75 in 1 molar KCl buffer. The resulting protein preparation was light brown, had an apparent molecular weight of 3700, contained 29% cysteine and close to 4 gram atoms cadmium/mole. The cadmium:cysteine ratio was 1:2.7. Spectroscopic measurements indicated cadmium-thiolate coordination. The roots produced the metallothionein-like protein when they were exposed to cadmium for 7 days.

Journal Article↗