Effects of shade stress on turfgrasses morphophysiology and rhizosphere soil bacterial communities.

BACKGROUND: The shade represents one of the major environmental limitations for turfgrass growth. Shade influences plant growth and alters plant metabolism, yet little is known about how shade affects the structure of rhizosphere soil microbial communities and the role of soil microorganisms in plant shade responses. In this study, a glasshouse experiment was conducted to examine the impact of shade on the growth and photosynthetic capacity of two contrasting shade-tolerant turfgrasses, shade-tolerant dwarf lilyturf (Ophiopogon japonicus, OJ) and shade-intolerant perennial turf-type ryegrass (Lolium perenne, LP). We also examined soil-plant feedback effects on shade tolerance in the two turfgrass genotypes. The composition of the soil bacterial community was assayed using high-throughput sequencing. RESULTS: OJ maintained higher photosynthetic capacity and root growth than LP under shade stress, thus OJ was found to be more shade-tolerant than LP. Shade-intolerant LP responded better to both shade and soil microbes than shade-tolerant OJ. The shade and live soil decreased LP growth, but increased biomass allocation to shoots in the live soil. The plant shade response index of LP is higher in live soil than sterile soil, driven by weakened soil-plant feedback under shade stress. In contrast, there was no difference in these values for OJ under similar shade and soil treatments. Shade stress had little impact on the diversity of the OJ and the LP bacterial communities, but instead impacted their composition. The OJ soil bacterial communities were mostly composed of Proteobacteria and Acidobacteria. Further pairwise fitting analysis showed that a positive correlation of shade-tolerance in two turfgrasses and their bacterial community compositions. Several soil properties (NO3--N, NH4+-N, AK) showed a tight coupling with several major bacterial communities under shade stress. Moreover, OJ shared core bacterial taxa known to promote plant growth and confer tolerance to shade stress, which suggests common principles underpinning OJ-microbe interactions. CONCLUSION: Soil microorganisms mediate plant responses to shade stress via plant-soil feedback and shade-induced change in the rhizosphere soil bacterial community structure for OJ and LP plants. These findings emphasize the importance of understanding plant-soil interactions and their role in the mechanisms underlying shade tolerance in shade-tolerant turfgrasses.

Structural and metabolic alterations in root systems under limited water conditions in forage grasses of Lolium-Festuca complex.

Drought resistance is a crucial attribute of plants and to properly decipher its mechanisms, a valuable plant model is required. Lolium multiflorum is a forage grass characterized by a low level of abiotic stress resistance, whereas Festuca arundinacea is recognized as a species with drought resistance, including both stress avoidance and tolerance strategies. These two species can be crossed with each other. Two closely related L. multiflorum/F. arundinacea introgression forms with distinct levels of field drought resistance were involved, thus enabling the dissection of this complex trait into its crucial components. The processes occurring in roots were shown to be the most significant for the expression of drought resistance. Thus, the analysis was focused on the root architecture and the accumulation of selected hormones, primary metabolites and glycerolipids in roots. The introgression form, with a higher resistance to field water deficit was characterized by a deeper soil penetration by its roots, and it had a higher accumulation level of primary metabolites, including well recognized osmoprotectants, such as proline, sucrose or maltose, and an increase in phosphatidylcholine to phosphatidylethanolamine ratio compared to the low resistant form. A comprehensive model of root performance under water deficit conditions is presented here for the first time for the grass species of the Lolium-Festuca complex.

Leaf Length Tracker: a novel approach to analyse leaf elongation close to the thermal limit of growth in the field.

Leaf growth in monocot crops such as wheat and barley largely follows the daily temperature course, particularly under cold but humid springtime field conditions. Knowledge of the temperature response of leaf extension, particularly variations close to the thermal limit of growth, helps define physiological growth constraints and breeding-related genotypic differences among cultivars. Here, we present a novel method, called 'Leaf Length Tracker' (LLT), suitable for measuring leaf elongation rates (LERs) of cereals and other grasses with high precision and high temporal resolution under field conditions. The method is based on image sequence analysis, using a marker tracking approach to calculate LERs. We applied the LLT to several varieties of winter wheat (Triticum aestivum), summer barley (Hordeum vulgare), and ryegrass (Lolium perenne), grown in the field and in growth cabinets under controlled conditions. LLT is easy to use and we demonstrate its reliability and precision under changing weather conditions that include temperature, wind, and rain. We found that leaf growth stopped at a base temperature of 0°C for all studied species and we detected significant genotype-specific differences in LER with rising temperature. The data obtained were statistically robust and were reproducible in the tested environments. Using LLT, we were able to detect subtle differences (sub-millimeter) in leaf growth patterns. This method will allow the collection of leaf growth data in a wide range of future field experiments on different graminoid species or varieties under varying environmental or treatment conditions.

Natural variation of salinity response, population structure and candidate genes associated with salinity tolerance in perennial ryegrass accessions.

Natural variation in salinity response, effects of population structure on growth and physiological traits and gene-trait association were examined in 56 global collections of diverse perennial ryegrass (Lolium perenne L.) accessions. Three population structure groups were identified with 66 simple sequence repeat markers, which on average accounted for 9 and 11% of phenotypic variation for the control and salinity treatment at 300 mm NaCl. Group 1 (10 accessions) had greater plant height, leaf dry weight and water content, chlorophyll index, K(+) concentration and K(+) /Na(+) than group 2 (39 accessions) and group 3 (7 accessions) under salinity stress, while group 3 had higher Na(+) than groups 1 and 2. Eighty-seven single nucleotide polymorphisms were detected from four partial candidate genes encoding aquaporin and Na(+) /H(+) antiporter in both plasma and tonoplast membranes. Overall, rapid decay of linkage disequilibrium was observed within 500 bp. Significant associations were found between the putative LpTIP1 and Na(+) for the control and between the putative LpNHX1 and K(+) /Na(+) under the control and salinity treatments after controlling population structure. These results indicate that population structure influenced phenotypic traits, and allelic variation in LpNHX1 may affect salinity tolerance of perennial ryegrass.

Leaf Rubisco turnover in a perennial ryegrass (Lolium perenne L.) mapping population: genetic variation, identification of associated QTL, and correlation with plant morphology and yield.

This study tested the hypotheses that: (i) genetic variation in Rubisco turnover may exist in perennial ryegrass (Lolium perenne L.); (ii) such variation might affect nitrogen use efficiency and plant yield; and (iii) genetic control of Rubisco turnover might be amenable to identification by quantitative trait loci (QTL) mapping. A set of 135 full-sib F1 perennial ryegrass plants derived from a pair cross between genotypes from the cultivars 'Grasslands Impact' and 'Grasslands Samson' was studied to test these hypotheses. Leaf Rubisco concentration at different leaf ages was measured and modelled as a log-normal curve described by three mathematical parameters: D (peak Rubisco concentration), G (time of D), and F (curve standard deviation). Herbage dry matter (DM) yield and morphological traits (tiller weight (TW), tiller number (TN), leaf lamina length (LL), and an index of competitive ability (PI)) were also measured. The progeny exhibited continuous variation for all traits. Simple correlation and principal component analyses indicated that plant productivity was associated with peak Rubisco concentration and not Rubisco turnover. Lower DM was associated with higher leaf Rubisco concentration indicating that Rubisco turnover effects on plant productivity may relate to energy cost of Rubisco synthesis rather than photosynthetic capacity. QTL detection by a multiple QTL model identified seven significant QTL for Rubisco turnover and nine QTL for DM and morphological traits. An indication of the genetic interdependence of DM and the measures of Rubisco turnover was the support interval overlap involving QTL for D and QTL for TN on linkage group 5 in a cluster involving QTL for DM and PI. In this region, alleles associated with increased TN, DM, and PI were associated with decreased D, indicating that this region may regulate Rubisco concentration and plant productivity via increased tillering. A second cluster involving QTL for LL, TN, PI and DM was found on linkage group 2. The two clusters represent marker-trait associations that might be useful for marker-assisted plant breeding applications. In silico comparative analysis indicated conservation of the genetic loci controlling Rubisco concentration in perennial ryegrass and rice.

Effect of perennial ryegrass (Lolium perenne L.) cultivars on the milk yield of grazing dairy cows.

The objective of this experiment was to investigate the effect of four perennial ryegrass cultivars: Bealey, Astonenergy, Spelga and AberMagic on the milk yield and milk composition of grazing dairy cows. Two 4 × 4 latin square experiments were completed, one during the reproductive and the other during the vegetative growth phase of the cultivars. Thirty-two Holstein-Friesian dairy cows were divided into four groups, with each group assigned 17 days on each cultivar during both experiments. Within each observation period, milk yield and milk composition, sward morphology and pasture chemical composition were measured. During the reproductive growth phase, organic matter digestibility (OMD) was greater for Bealey and Astonenergy (P < 0.001; +1.6%). AberMagic contained a higher stem proportion (P < 0.01; +0.06) and a longer sheath height (P < 0.001; +1.9 cm). Consequently, cows grazing AberMagic recorded a lower milk yield (P < 0.001; -1.5 kg/day) and a lower milk solids yield (P < 0.001; -0.13 kg/day). During the vegetative growth phase, OMD was greater (P < 0.001; +1.1%) for Bealey, whereas the differences between the cultivars in terms of sward structure were smaller and did not appear to influence animal performance. As a result, cows grazing Bealey recorded a higher milk yield (P < 0.001; +0.9 kg/day) and a higher milk solids yield (P < 0.01; +0.08 kg/day). It was concluded that grass cultivar did influence milk yield due to variations in sward structure and chemical composition.

PAHs pass through the cell wall and partition into organelles of arbuscular mycorrhizal roots of ryegrass.

The subcellular process and distribution of polycyclic aromatic hydrocarbons (PAHs) in arbuscular mycorrhizal plants remains to be elucidated. This work used a greenhouse experiment to show that, accompanied by the apoplastic and symplastic water movement through the root, acenaphthene (ACE) as a representative PAH passed through the cell-wall boundary, dissolved in the cell solution, and partition organelles in arbuscular mycorrhizal roots of ryegrass (Lolium multiflorum Lam.). The observed concentrations of ACE in organelles were 0.6 to 4.4 times higher than in the cell walls. The cell wall and organelles were the dominant storage domains for ACE in the root, and the distribution of ACE in cells of mycorrhizal ryegrass roots was, in descending order, cell organelles (40.8-70.8%) > cell wall (19.7-3.8%) cell solution (9.6-20.5%).

Identification of QTLs for morphological traits influencing waterlogging tolerance in perennial ryegrass (Lolium perenne L.).

Perennial ryegrass is a globally cultivated obligate outbreeding diploid species (2n = 2x = 14) which is subjected to periods of waterlogging stress due to flood irrigation during winter and the lead-up to summer. Reduction of oxygen supply to root systems due to waterlogging produces consequent deleterious effects on plant performance. Framework genetic maps for a large-scale genetic mapping family [F1(NA(x) × AU6)] were constructed containing 91 simple sequence repeat and 24 single nucleotide polymorphism genetic markers. Genetic trait dissection using both control and waterlogging treatments was performed in the glasshouse, a total of 143 maximally recombinant genotypes being selected from the overall sib-ship and replicated threefold in the trial. Analysis was performed for nine quantitative morphological traits measured 8 weeks after stress treatments were applied. A total of 37 quantitative trait loci (QTLs) were identified; 19 on the NA(x) parental genetic map, and 18 on the AU6 parental genetic map. Regions of particular interest were identified on linkage groups (LGs) 4 and 3 of the respective maps, which have been targeted for further analysis by selection of critical recombinants. This first study of genetic control of waterlogging tolerance in ryegrasses has important implications for breeding improvement of abiotic stress adaptation.

A comparative analysis of the temperature response of leaf elongation in Bromus stamineus and Lolium perenne plants in the field: intrinsic and size-mediated effects.

BACKGROUND AND AIMS: Growth of grass species in temperate-humid regions is restricted by low temperatures. This study analyses the origin (intrinsic or size-mediated) and mechanisms (activity of individual meristems vs. number of active meristems) of differences between Bromus stamineus and Lolium perenne in the response of leaf elongation to moderately low temperatures. METHODS: Field experiments were conducted at Balcarce, Argentina over 2 years (2003 and 2004) using four cultivars, two of B. stamineus and two of L. perenne. Leaf elongation rate (LER) per tiller and of each growing leaf, number of growing leaves and total leaf length per tiller were measured on 15-20 tillers per cultivar, for 12 (2003) or 10 weeks (2004) during autumn and winter. KEY RESULTS: LER was faster in B. stamineus than in L. perenne. In part, this was related to size-mediated effects, as total leaf length per tiller correlated with LER and B. stamineus tillers were 71% larger than L. perenne tillers. However, accounting for size effects revealed intrinsic differences between species in their temperature response. These were based on the number of leaf meristems simultaneously active and not on the (maximum) rate at which individual leaves elongated. Species differences were greater at higher temperatures, being barely notable below 5 degrees C (air temperature). CONCLUSIONS: Bromus stamineus can sustain a higher LER per tiller than L. perenne at air temperatures > 6 degrees C. In the field, this effect would be compounded with time as higher elongation rates lead to greater tiller sizes.

Linkage disequilibrium in synthetic varieties of perennial ryegrass.

Synthetic varieties obtained after three to four panmictic generations are variable, not structured and so can be used for association studies. The pattern of linkage disequilibrium (LD) decay determines whether a genome scan or a candidate gene approach can be used for an association study between genotype and phenotype. Our goal was to evaluate the effect of the number of parents used to build the synthetic varieties on the pattern of LD decay. LD was investigated in the gibberelic acid insensitive gene (GAI) region in three synthetic varieties of perennial ryegrass (Lolium perenne L.) chosen for their contrasted number of parents in the initial polycrosses. Results were compared with those obtained from a core collection. STS and SSR markers were used to evaluate variation, structuration and LD in each variety. As expected, the varieties variability increased with the number of parents almost up to the core collection variability. No structuration was observed in the varieties. Significant LDs were observed up to 1.6 Mb in a variety originated from six related parents and not above 174 kb in a variety originated from 336 parents. These results suggest that a candidate gene approach can be used when varieties have a large number of parents and a genome scan approach can be envisaged in specific regions when varieties have a low number of parents. Nevertheless, we strongly recommend to estimate the pattern of LD decay in the population and in the genomic region studied before performing an association study.

Effect of spring grazing date and stocking rate on sward characteristics and dairy cow production during midlactation.

The objective of this study was to investigate the effect of initial spring grazing date and subsequent stocking rate on sward characteristics, grazing behavior, milk yield, and dry matter intake of spring-calving dairy cows during the main grazing season. Sixty-four spring-calving Holstein-Friesian dairy cows (58 +/- 9 d in milk) were balanced and randomly assigned to 1 of 4 grazing treatments (n = 16) between April 12, and July 3, 2004. Two swards, an early-grazed (E) sward and a late-grazed (L) sward had 2 stocking rates, high and medium, imposed across them. Cows grazing the E swards were stocked at 4.5 cows/ha (E4.5) and 5.5 cows/ha (E5.5), whereas cows grazing the L sward were stocked at 5.5 cows/ha (L5.5) and 6.4 cows/ha (L6.4). Sward characteristics, grazing behavior, and grass dry matter intake (GDMI) were investigated during the second (R2) and fourth grazing rotations (R4). Total dry matter yield was greater on L swards in R2. In R2, the E swards had a greater proportion of leaf as well as a lesser stem and dead dry matter yield. During R2, organic matter digestibility and crude protein content were greater on the E sward than the L sward. Pre-and postgrazing heights were greater for the L swards in R2 and R4. In R4, there was a larger leaf allowance on the E swards. Grazing time was greater and ruminating time lesser for animals grazing the E sward in R2. During R4, intake per bite was greater for the E5.5 and E4.5 treatments. Milk and solids-corrected milk yields as well as GDMI were greater for animals grazing the E sward in both R2 and R4. The results of the present study suggest that early grazing initially had a positive effect on sward quality and structure, which resulted in improved grazing behavior characteristics, increased GDMI, and increased milk production. During R4, sward quality and structure were similar between swards; thus, differences in grazing behavior were due to divergent daily herbage allowances. These results suggest that sward structure and quality as well as daily herbage allowance are important factors that influence animal performance and grazing behavior.

Phosphorus nutrition and mycorrhiza effects on grass leaf growth. P status- and size-mediated effects on growth zone kinematics.

This study tested whether leaf elongation rate (LER, mm h(-1)) and its components--average relative elemental growth rate (REGRavg, mm mm(-1) h(-1)) and leaf growth zone length (L(LGZ), mm)--are related to phosphorus (P) concentration in the growth zone (P(LGZ) mg P g(-1) tissue water) of Lolium perenne L. cv. Condesa and whether such relationships are modified by the arbuscular mycorrhizal fungus (AMF) Glomus hoi. Mycorrhizal and non-mycorrhizal plants were grown at a range of P supply rates and analysed at either the same plant age or the same tiller size (defined by the length of the sheath of the youngest fully expanded leaf). Both improved P supply (up to 95%) and AMF (up to 21%) strongly increased LER. In tillers of even-aged plants, this was due to increased REGRavg and L(LGZ). In even-sized tillers, it was exclusively due to increased REGRavg. REGRavg was strictly related to P(LGZ) (r2 = 0.95) and independent of tiller size. Conversely, L(LGZ) strictly depended on tiller size (r2 = 0.88) and not on P(LGZ). Hence, P status affected leaf growth directly only through effects on relative tissue expansion rates. Symbiosis with AMF did not modify these relationships. Thus, no evidence for P status-independent effects of AMF on LER was found.

Nutrient availability and atmospheric CO2 partial pressure modulate the effects of nutrient heterogeneity on the size structure of populations in grassland species.

BACKGROUND AND AIMS: Size-asymmetric competition occurs when larger plants have a disproportionate advantage in competition with smaller plants. It has been hypothesized that nutrient heterogeneity may promote it. Experiments testing this hypothesis are inconclusive, and in most cases have evaluated the effects of nutrient heterogeneity separately from other environmental factors. The aim of this study was to test, using populations of Lolium perenne, Plantago lanceolata and Holcus lanatus, two hypotheses: (a) nutrient heterogeneity promotes size-asymmetric competition; and (b) nutrient heterogeneity interacts with both atmospheric CO2 partial pressure (P(CO2)) and nutrient availability to determine the magnitude of this response. METHODS: Microcosms consisting of monocultures of the three species were grown for 90 d in a factorial experiment with the following treatments: P(CO2) (37.5 and 70 Pa) and nutrient availability (NA; 40 and 120 mg of N added as organic material) combined with different spatial distribution of the organic material (NH; homogeneous and heterogeneous). Differences in the size of individual plants within populations (size inequality) were quantified using the coefficient of variation of individual above-ground biomass and the combined biomass of the two largest individuals in each microcosm. Increases in size inequality were associated with size-asymmetric competition. KEY RESULTS: Size inequality increased when the nutrients were heterogeneously supplied in the three species. The effects of NH on this response were more pronounced under high nutrient supply in both Plantago and Holcus (significant NA x NH interactions) and under elevated P(CO2) in Plantago (significant P(CO2) x NA x NH interaction). No significant two- and three-way interactions were found for Lolium. CONCLUSIONS: Our first hypothesis was supported by our results, as nutrient heterogeneity promoted size-asymmetric competition in the three species evaluated. Nutrient supply and P(CO2) modified the magnitude of this effect in Plantago and Holcus, but not in Lolium. Thus, our second hypothesis was partially supported.

Three-dimensional microorganization of the soil-root-microbe system.

Soils contain the greatest reservoir of biodiversity on Earth, and the functionality of the soil ecosystem sustains the rest of the terrestrial biosphere. This functionality results from complex interactions between biological and physical processes that are strongly modulated by the soil physical structure. Using a novel combination of biochemical and biophysical indicators and synchrotron microtomography, we have discovered that soil microbes and plant roots microengineer their habitats by changing the porosity and clustering properties (i.e., spatial correlation) of the soil pores. Our results indicate that biota act to significantly alter their habitat toward a more porous, ordered, and aggregated structure that has important consequences for functional properties, including transport processes. These observations support the hypothesis that the soil-plant-microbe complex is self-organized.

Impact of defoliation intensity and frequency on N uptake and mobilization in Lolium perenne.

The aim of the study was to evaluate the impact of defoliation intensity, defoliation frequency, and interactions with N supply on N uptake, N mobilization from and N allocation to roots, adult leaves, and growing leaves. Plants of Lolium perenne were grown under two contrasted N regimes. Defoliation intensity treatments consisted of a range of percentage leaf area removal (0, 25, 50, 75, or 100%). These treatments were applied in parallel to a set of plants previously undefoliated, and to a second set of plants which had been defoliated several times at a constant height. A (15)N tracer technique was used to quantify N uptake, mobilization, and allocation over a 7 d period. A significant reduction in plant N uptake was observed with the removal of more than 75% of lamina area, but only with high N supply. As defoliation intensity increased, the amount of N taken up and subsequently allocated to growing leaves during the labelling period was maintained at the expense of N allocation to roots and adult leaves. Increasing defoliation intensity increased the relative contribution of roots supplying mobilized N to growing leaves and decreased the relative contribution of adult leaves. Defoliation frequency did not substantially alter N uptake, mobilization, and allocation between roots, adult and growing leaves on a plant basis. However, tiller number per plant was largely increased under repeated defoliation, hence indicating that allocation and mobilization of N to growing leaves, on the basis of individual tillers, was decreased by defoliation frequency.

Tracing field hybridization in Ryegrass species using microsatellite and morphological markers.

We have assessed the utility of morphological and microsatellite markers for tracing field hybridization between Lolium multiflorum and Lolium perenne in cereal-enclosed gene flow plots. The presence of awns on the inflorescence of F(1) hybrids was found to be a reliable, but underscoring, indicator of L. multiflorum paternity in L. perenne derived seed as determined by inheritance of species-specific alleles at the microsatellite locus 'H01 H06' in these progeny. A positive correlation was evident in the experimental treatment between the number of pollen donor plants in a given plot and the frequency of hybrid F(1) seed harvested from pollen receptor plants in that plot. These experiments have established the utility of naturally occurring heritable markers for the measurement of gene flow rates in field Ryegrass populations, with particular significance for risk assessment modeling of potential gene flow from transgenic grass cultivars.

Phosphorus nutrition-mediated effects of arbuscular mycorrhiza on leaf morphology and carbon allocation in perennial ryegrass.

The aim of this work was to disentangle phosphorus status-dependent and -independent effects of arbuscular mycorrhizal fungus (AMF) on leaf morphology and carbon allocation in perennial ryegrass (Lolium perenne). To this end, we assessed the P-response function of morphological components in mycorrhizal and nonmycorrhizal plants of similar size. AMF (Glomus hoi) stimulated relative P-uptake rate, decreased leaf mass per area (LMA), and increased shoot mass ratio at low P supply. Lower LMA was caused by both decreased tissue density and thickness. Variation in tissue density was almost entirely caused by variations in soluble C, while that in thickness involved structural changes. All effects of AMF were indistinguishable from those mediated by increases in relative P-uptake rate through higher P-supply rates. Thus the relationships between relative P-uptake rate, leaf morphology and C allocation were identical in mycorrhizal and nonmycorrhizal plants. No evidence was found for AMF effects not mediated by changes in plant P status.

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium.

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.

The effect of ozone on pollen development in Lolium perenne L.

Perennial ryegrass plants (Lolium perenne L.) were exposed in "Closed-Top Chambers" to different ozone concentrations and to charcoal filtered ambient air to study the effect of ozone on the development of pollen. Ozone at ambient (65 nl l(-1), 8h) and elevated (110 nl l(-1), 4h) concentrations affected the maturing of pollen by inhibiting starch accumulation in pollen throughout the anther. Affected pollen persisted in the vacuolated state while normal pollen in the same anther were filled with amyloplasts. The percentage of underdeveloped pollen-determined in transversal sections-was significantly higher in exposed plants than in plants grown in filtered air. Results indicate that ozone stress was responsible for the disrupted development of pollen in L. perenne.

Morphological compatibility of white clover and perennial ryegrass cultivars grown under two nitrate levels in flowing solution culture.

The effects of nitrate (NO3-) supply on shoot morphology, vertical distribution of shoot and root biomass and total nitrogen (N) acquisition by two perennial ryegrass (Lolium perenne L.) cultivars (AberElan and Preference) and two white clover (Trifolium repens L.) cultivars (Grasslands Huia and AberHerald) were studied in flowing nutrient culture. Cultivars were grown from seed as monocultures and the clovers inoculated with Rhizobium. The 6-week measurement period began on day 34 (grasses) and day 56 (clovers) when the NO3- supply was adjusted to either 2 mmol m-3 (low nitrogen, LN) or 50 mmol m-3 (high nitrogen, HN). These treatments were subsequently maintained automatically. Plants were harvested at intervals to measure their morphology and N content. Cultivars of both species differed significantly in several aspects of their response to NO3- supply. In the grasses, the LN treatment increased the root : shoot ratio of AberElan but did not affect the distribution of root length in the root profile. In contrast, this treatment changed the root distribution of Preference compared with HN, resulting in a larger proportion of root length being distributed further down the root profile. The morphology of white clover Grasslands Huia was for the most part unaffected by the level of NO3- supply. In contrast, AberHerald exhibited different growth strategies, with LN plants increasing their stolon weight per unit length at the expense of leaf production, leaf area and stolon length, whereas HN plants showed reduced stolon thickness, greater leaf area production and stolon length per plant. Cultivars with different morphological/physiological strategies in response to NO3- supply may be of value in the construction of 'compatible mixtures' aimed at reducing oscillations in sward clover content by extending the range of conditions that allow balanced coexistence of species to occur.