Effects of fungal endophytes on the seed and seedling biology of Lolium perenne and Festuca arundinacea

Summary Many grasses are infected by endophytic fungi that grow intercellularly in leaves, stems, and flowers and are transmitted maternally by hyphal growth into ovules and seeds. The seed biology and seedling growth of endophyte-infected and uninfected perennial ryegrass (Lolium perenne) and tall fescue (Festuca arundinacea) were investigated under controlled environmental conditions. The percentage of filled seeds produced by infected tall fescue was over twice of uninfected tall fescue; infected and uninfected perennial reegrass had similar percentages. Weights of seeds from infected and uninfected plants were similar in both species. Seeds from infected plants of both species exhibited a higher rate of germination than seeds from uninfected plants. Shoot growth in the greenhouse was compared by making three sequential harvests of above-ground plant parts from infected and uninfected plants of both species. Infected perennial ryegrass plants produced significantly more biomass and tillers than uninfected plants after 6 and 10 weeks of growth and significantly more biomass after 14 weeks of growth. Infected tall fescue plants produced significantly more biomass and tillers than uninfected plants after 10 and 14 weeks of growth. The physiological mechanism of enhancement of growth is not known. The results of this study suggest that infected plants may have a selective advantage in populations with uninfected members.     

Phylogeny of tall fescue and related species using RFLPs

The wild species of tall fescue (Festuca arundinacea var.genuina Schreb.) represent a wide range of genetic variation and constitute potential germplasm for tall fescue improvement. Our objective was to evaluate genome specificity of the previously-identified DNA probes and to examine the phylogenetic relationship of tall fescue with six related species by using RFLP data. A total of 29 DNA probes from aPstI-genomic library of tall fescue were hybridized toEcoRI-orHindIII-digested DNA of 32 plants from sixFestuca species and fromLolium perenne L. Fifteen probes hybridized to all seven species. The remaining 14 probes showed differential hybridization patterns (i.e., ±), especially at the diploid and tetraploid levels. This hybridization pattern reflected genome divergence in these species. The DNA probes will be useful markers in breeding programs involving interspecific and intergeneric hybridization. Cluster analyses were performed using the average genetic distances calculated with the RFLP data from 53 probe-enzyme combinations. Generally, genotypes from the same species were grouped in the same cluster. These data indicated that tall fescue has a close relationship withF. pratensis Huds. (diploid),F. arundinacea var.glaucescens Boiss. (tetraploid), andL. perenne L. (diploid) and thatFestuca pratensis andL. perenne had the closest degree of relationship.This paper is a contribution of the Missouri Agricultural Experimental Station, Journal Series no. 11,798     

SSRs transferability and genetic diversity of Tunisian Festuca arundinacea and Lolium perenne

SSR primers specific to Lolium perenne generated a total of 96 alleles and 124 genotypes within Festuca arundinacea and Lolium perenne accessions. Their highly transferability (100 %) across genera was evidenced. Six alleles specific to loci H01F02, H02C11 and K01A03 and only 5/96 common alleles between both species (60, 140, 144, 190 and 192) expressed the differentiation between species. Besides, based on the Wrights fixation indices, the genetic variation within each species was attributable to differences within populations with a significant deficiency of heterozygous. The unweighted pair group method with arithmetic averaging dendrogram based on the Nei’s distances and the principal coordinate analysis based on Jaccard coefficient similarity distinguished each genus independently of the geographical origin. However, typically continuous genetic diversity and a low level of gene flow (N_m: 0.29–2.47) expressed the relatively closely relationships of both genera and suggest a possible hybridization in nature.     

Preferential uptake of soil nitrogen forms by grassland plant species

In this study, we assessed whether a range of temperate grassland species showed preferential uptake for different chemical forms of N, including inorganic N and a range of amino acids that commonly occur in temperate grassland soil. Preferential uptake of dual-labelled (¹³C and ¹⁵N) glycine, serine, arginine and phenylalanine, as compared to inorganic N, was tested using plants growing in pots with natural field soil. We selected five grass species representing a gradient from fertilised, productive pastures to extensive, low productivity pastures (Lolium perenne, Holcus lanatus, Anthoxanthum odoratum, Deschampsia flexuosa, and Nardus stricta). Our data show that all grass species were able to take up directly a diversity of soil amino acids of varying complexity. Moreover, we present evidence of marked inter-species differences in preferential use of chemical forms of N of varying complexity. L. perenne was relatively more effective at using inorganic N and glycine compared to the most complex amino acid phenylalanine, whereas N. stricta showed a significant preference for serine over inorganic N. Total plant N acquisition, measured as root and shoot concentration of labelled compounds, also revealed pronounced inter-species differences which were related to plant growth rate: plants with higher biomass production were found to take up more inorganic N. Our findings indicate that species-specific differences in direct uptake of different N forms combined with total N acquisition could explain changes in competitive dominance of grass species in grasslands of differing fertility.     

A novel method of quantifying root exudation in the presence of soil microflora

A microcosm is described in which root exudation may be estimated in the presence of microorganisms. Ryegrass seedlings are grown in microcosms in which roots were spatially separated from a microbial inoculant by a Millipore membrane. Seedlings grown in the microcosms were labelled with [¹⁴C]-CO₂, and the fate of the label within the plant and rhizosphere was determined. Inoculation of the microcosms with Cladosporium resinae increased net fixation of the [¹⁴C] label compared to plants grown under sterile conditions. Inoculation also increased root exudation. The use of the microcosm was illustrated and its applications discussed.     

A morphological change in the fungal symbiont Neotyphodium lolii induces dwarfing in its host plant Lolium perenne

The endophytic fungus Neotyphodium lolii forms symbiotic associations with perennial ryegrass (Lolium perenne) and infection is typically described as asymptomatic. Here we describe a naturally occurring New Zealand N. lolii isolate that can induce dwarfing of L. perenne and suppress floral meristem development in the dwarfed plants. Further to this we demonstrate that the observed host dwarfing correlates with a reversible morphological change in the endophyte that appears associated with colony age. Mycelium isolated from normally growing plants had a typical cottony appearance in culture whereas mycelium from dwarfed plants appeared mucoid. Cottony colonies could be induced to turn mucoid after prolonged incubation and seedlings inoculated with this mucoid mycelium formed dwarfed plants. Mucoid colonies on the other hand could be induced to form cottony colonies through additional further incubation and these did not induce dwarfing. The reversibility of colony morphology indicates that the mucoid dwarfing phenotype is not the result of mutation. Ten isolates from other locations in New Zealand could also undergo the reversible morphological changes in culture, induce dwarfing and had the same microsatellite genotype as the original isolate, indicating that a N. lolii genotype with the ability to dwarf host plants is common in New Zealand.     

The influence of some soil and plant factors on the concentration of copper in perennial ryegrass

Summary The absorption and transport of Cu were studied in perennial ryegrass grwon on 21 soils under controlled environment conditions. Neither the concentration, nor the total amount, of Cu in the shoots was related to available Cu in the soils as assessed by extraction with 0.05M EDTA, 0.005M DTPA, or 1.95 per cent HNO₃. The concentration in the roots and, more especially, absorption per unit weight of root (i.e. μg Cu g dry wt⁻¹) were, however, highly correlated with available soil Cu. This suggests that, unless the extent of exploitation of the soil by roots is taken into account, measurements of available Cu will not be effective in predicting uptake by plants. On average, 63 per cent of the Cu absorbed by the roots was retained in the roots, and variation in the proportion retained was related to the transport of nitrogen from roots to shoots. On some soils the concentrations of N and Cu in the shoots were significantly correlated, and variation in N concentration accounted for a considerable proportion of the variance in the Cu concentration at later harvests. The relative importance of the measured soil (pH, organic matter) and plant (dry weight, N content) factors changed markedly over 6 successive harvests.     

Influence of dissimilarities in temporal and spatial N-uptake patterns on15N-based estimates of fixation and transfer of N in ryegrass-clover mixtures

The temporal N-uptake patterns of white clover (Trifolium repens L.) mixed with perennial ryegrass (Lolium perenne L.) and of red clover (Trifolium pratense L.) mixed with Italian ryegrass (Lolium multiflorum Lam.) were determined in successive harvests of herbage within the growth cycles of a ley established near Zürich (Switzerland). Rooting patterns were examined by injecting¹⁵N-fertilizer at soil depths ranging from 10 to 40 cm. The results were analyzed to determine the effect of variations in time and depth of N-uptake on the¹⁵N-based measurement of N from symbiosis (N_sym) and N from transfer (N_trans).Grasses in mixture appeared to have deeper rooting systems than grass monocultures, which led to an overestimation of N transfer from white clover to perennial ryegrass if¹⁵N was spread on the soil surface.White clover generally lagged behind grass in soil N- uptake. Soil N-uptake of red clover slowed down before that of the grass because % N_sym almost reached 100% during the second half of each growth cycle. However, the effect of these dissimilarities on the seasonal average of %N_sym did not exceed 2%.It is concluded that at the observed high levels of N₂ fixation, failure to account for the N-uptake patterns of the test and reference crops only slightly affected the estimates of % N_sym and % N_trans, and did not invalidate the observed differences between species.     

Temporal and spatial patterns in the recruitment of Gibbula umbilicalis

Size/frequency samples have been taken from populations of the trochid gastropod Gibbula umbilicalis at a number of sites around the British Isles each year since 1978. In the North of Scotland, close to the limit of the species distribution, recruitment was generally poor and populations were sparse and dominated by large old individuals. While such poor recruitment may reflect the low density of adults and the shortage of nursery areas, a distinct temporal pattern of recruitment was also evident. At the start of our study, the majority of sites had population structures biased towards young animals implying that conditions for the settlement of larvae or their subsequent survival had been favourable over a wide area. In the years that followed only the enclosed Loch Eriboll regularly received substantial recruitment. Further to the south, in Wales and S.W. England, recruitment was usually more regular, populations were more dense and individuals smaller.     

Availability of cadmium and zinc accumulated in the leaves of Thlaspi caerulescens incorporated into soil

When grown on contaminated soil, hyperaccumulator plants contain high concentrations of metals which may return to the soil after senescence. This work was undertaken to assess the availability of Cd and Zn associated to the leaves of the hyperaccumulator Thlaspi caerulescens after incorporation into an uncontaminated soil. A Zn- and Cd- accumulator population of T. caerulescens was grown on a Cd- and Zn- contaminated soil previously labelled with ¹⁰⁹Cd. Leaves (TCL) were harvested, dried, ground and incorporated into the soil at a rate of 2.07 mg Cd kg⁻¹ and 51.9 mg Zn kg⁻¹. Then a pot experiment was conducted for 3 months with rye grass (Lolium perenne) and T. caerulescens. Rye grass was harvested monthly and T. caerulescens at the end of the experiment. Plant biomass was measured, along with the concentration of Cd, Zn and ¹⁰⁹Cd. Results showed that water-extractable metals in TCL were 69% for Zn and 33% for Cd. Addition of TCL to soil, depleted growth of rye grass, and improved that of T. caerulescens. At harvest, concentrations of both metals were increased in plants by TCL. Concentrations of Cd in rye grass increased with the cut number, while that of Zn decreased slightly. Rye grass extracted 1.6% of the total Cd and 0.9% of the total Zn, and T. caerulescens extracted up to 22.4% of the Cd and 7% of the Zn. About 94% of the Cd in rye grass and 86% in T. caerulescens was derived from TCL. In conclusion, metals associated with leaves of the hyperaccumulator T. caerulescens were very mobile after incorporation into the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Net carbon exchange and evapotranspiration in postfire and intact sagebrush communities in the Great Basin

Invasion of non-native annuals across the Intermountain West is causing a widespread transition from perennial sagebrush communities to fire-prone annual herbaceous communities and grasslands. To determine how this invasion affects ecosystem function, carbon and water fluxes were quantified in three, paired sagebrush and adjacent postfire communities in the northern Great Basin using a 1-m³ gas exchange chamber. Most of the plant cover in the postfire communities was invasive species including Bromus tectorum L., Agropyron cristatum (L.) Gaertn and Sisymbrium altissimum L. Instantaneous morning net carbon exchange (NCE) and evapotranspiration (ET) in native shrub plots were greater than either intershrub or postfire plots. Native sagebrush communities were net carbon sinks (mean NCE 0.2–4.3 μmol m⁻² s⁻¹) throughout the growing season. The magnitude and seasonal variation of NCE in the postfire communities were controlled by the dominant species and availability of soil moisture. Net C exchange in postfire communities dominated by perennial bunchgrasses was similar to sagebrush. However, communities dominated by annuals (cheatgrass and mustard) had significantly lower NCE than sagebrush and became net sources of carbon to the atmosphere (NCE declined to −0.5 μmol m⁻² s⁻¹) with increased severity of the summer drought. Differences in the patterns of ET led to lower surface soil moisture content and increased soil temperatures during summer in the cheatgrass-dominated community compared to the adjacent sagebrush community. Intensive measurements at one site revealed that temporal and spatial patterns of NCE and ET were correlated most closely with changes in leaf area in each community. By altering the patterns of carbon and water exchange, conversion of native sagebrush to postfire invasive communities may disrupt surface-atmosphere exchange and degrade the carbon storage capacity of these systems.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.     

Spatial patterns and species performances in experimental plant communities

The roles of co-occurring herbivores that modify habitat structure and ecosystem processes have seldom been examined in manipulative experiments or explored in early successional communities. In a created marsh in southern California (USA), we tested the individual and combined effects of two epibenthic invertebrates on nutrient and biomass pools, community structure, and physical habitat features. We manipulated snail (Cerithidea californica) and crab (Pachygrapsus crassipes) presence in field enclosures planted with pickleweed (Salicornia virginica) at elevations matching the plant’s lower extent in an adjacent natural marsh. In the 4-month experiment, C. californica altered habitat structure by reducing sediment surface heterogeneity and shear strength (a measure of sediment stability) markedly throughout the enclosures. Both invertebrates had strong negative effects on a group of correlated sediment physicochemical characteristics, including nitrogen and organic matter concentrations and soil moisture. In addition, both invertebrates greatly reduced benthic chlorophyll a, a proxy for biomass of microphytobenthos. Compared to controls, macroalgal cover was up to sixfold lower with crabs present, while snails increased cover at low elevations of enclosures. Unexpectedly, macroalgal cover was eliminated with both species present, perhaps through P. crassipes consumption of larger thalli and C. californica reduction in cover of recruits. Neither species influenced the S. virginica canopy (quantified with an index of branch length and number); however, at the lower elevation of enclosures, the two species together negatively impacted the plant canopy. The two invertebrates’ modifications to our experimental marshes led to distinct suites of biotic and physicochemical features depending on their presence or co-occurrence, with the latter producing several unexpected results. We propose that the roles and interactions of habitat-modifying fauna deserve further attention, particularly in the context of efforts to conserve and restore the processes found in natural systems.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.     

Seasonal changes in respiration of halophytes in salt playas in the Great Basin,U.S.A.

Many desert playas are covered with water in the early spring. As theweather becomes warmer and drier, water evaporates, increasing saltcontent of the soil from 7,000 to almost 16,000 mmol NaCl Kg^-1. Changes in respiratory metabolism during the growing season of fourhalophytes characteristic of cold desert playas was followed usingcalorimetry. In order of decreasing salt tolerance, the species examinedwere: the forbs Salicornia rubra, S. utahensis; the grass Distichlisspicata; and the shrub Allenrolfea occidentalis. Tissue collected in thefield from sites of low and high salinity in a single playa during May, June,and August of 1997 was put in isothermal calorimeters and the metabolicheat rate (q) and respiration rate (R_CO2) measured. Efficiency ofsubstrate carbon conversion (q/R_CO2) and predicted specific growthrate (H_BR_SG) were calculated. These species are allwell-adapted to the environment in which they are found. Highestmetabolism, respiration, efficiency and growth are found during May andJune and are lowest during the hot, dry month of August. Differencesbetween the species are also noted.     

Association of a fungal endophyte in perennial ryegrass with antibiosis to larvae of the southern armyworm, Spodoptera eridania

Larvae of the Southern armyworm Spodoptera eridania Cramer (Lepidoptera:Noctuidae), feeding on perennial ryegrasses (Lolium perenne L.) infected with an endophytic fungus (Acremonium loliae Latch, Christensen and Samuels), had a much lower survival rate (7–13%) than larvae feeding on endophyte-free ryegrasses (82–90%). Death of the larvae on endophyte-infected entries occurred rapidly between 144 h and 168 h of feeding. This corresponded with armyworms feeding on the base of the plant, where endophyte concentration is highest. Twenty-four hours after the start of the bioassay the larval mass and rate of larval development were significantly higher on endophytic entries. From 48–144 h no differences were seen, but after 144 h the mass of larvae on endophyte-infected grasses sharply declined. Observations from this bioassay further substantiate the association between A. loliae-infected ryegrass and antibiosis to several lepidopterous and coleopterous insect pests.

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Résumé Le ray-grass vivace (Lolium perenne), contaminé par le champignon endophyte Acremonium loliae Latch, Christensen & Samuels, a présenté une augmentation de la resistance à de nombreux coléoptères et lépidoptères nuisibles. Cette note examine les réactions de Spodoptera eridania Cramer (Lépido., Noctuidae) alimenté sur trois lignées de ray-grass contaminées par le champignon et trois lignées saines. Après 168 h d'alimentation sur ray-grass contaminé, les chenilles presentent une très forte mortalité; la survie n'est que de 7 à 13% contre 82 à 90% pour le ray-grass sain. Le décès brutal des chenilles correspond à leur alimentation sur la base de la plante ou la concentration du champignon est la plus forte. Les chenilles consomment constamment, broutant plus des 2/3 du feuillage du ray-grass; les broutements des six séries ne différaient pas significativement.Au bout de 24 h, la nombre de chenilles passées du 3ème au 4ème stade, et l'augmentation de poids sont plus élevés pour les séries sur plante contaminée, ce qui suggère un avantage initial pour les chenilles en présence de champignon endophyte, l'analyse en poids sec a montré que l'augmentation de poids initial est réel. Entre 48 et 144 h, cependant, le nombre de 4ème stade et le poids des chenilles sont les mêmes dans les deux séries. Après 144 h, le poids des chenilles sur ray-grass contaminé diminue significativement; aucune n'était parvenue au 5ème stade, contre 11% sur ray-grass sain. Nous n'avons pas observé de signes apparents de neurotoxicité. Au lieu de cela, il ya a eu interaction avec un processus physiologique fondamental, ce qui a provoqué une forte perte de poids larvaire et la mort, indiquant l'intervention d'inhibiteurs métaboliques.

     

Transformation of recalcitrant turfgrass cultivars through improvement of tissue culture and selection regime

Tissue culture techniques, medium composition, pH value and targeted tissues, agroinfection and co-culture conditions, selection process were optimized for efficient turfgrass transformation. A highly regenerable callus lines were produced in callus induction medium modified from N6 basal medium. Six-week-old calluses were cultured on Pre-regeneration medium I for 4 days and then subjected to Agrobacterium tumefaciens. After co-cultivation at 20±1 °C in a 16 h light/8 h darkness for 3 days, the calluses were cultured on non-selective Pre-regeneration medium II supplemented with 400 mg l⁻¹ l-cysteine for 7 days. Plantlets were regenerated on the Regeneration medium without selection pressure. A selection pressure was given to the regenerated plantlets when they were rooted on the Plantlet rooting medium. Roots appeared within 8–12 days in putative transformed plantlets. Resistant plants obtained were phenotypically normal and fully fertile. Chemical and molecular analyses confirmed that foreign genes were successfully introduced into the genome of perennial ryegrass or tall fescue. The transformation efficiency can attain 23.3% in perennial ryegrass.