Phyllosphere microflora of some Egyptian plants

The fungal and bacterial flora of the leaf surfaces of five plants growing in Egypt were studied. The fungal flora showed seasonal fluctuations with at least one peak. Twenty three genera with fifty three species were found,Aspergillus andPenicillium being most common. Other fungi showed variable percentages of the total count. Nitrogen-fixing bacteria were not isolated from the phyllosphere of the five plant species. Micrococci were most predominant among the epiphytic bacteria. Spore-forming bacteria and actinomycetes were less frequent on the leaf surfaces of the associated plants.     

The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves

Large populations of bacteria live on leaf surfaces and these phyllosphere bacteria can have important effects on plant health. However, we currently have a limited understanding of bacterial diversity on tree leaves and the inter‐ and intra‐specific variability in phyllosphere community structure. We used a barcoded pyrosequencing technique to characterize the bacterial communities from leaves of 56 tree species in Boulder, Colorado, USA, quantifying the intra‐ and inter‐individual variability in the bacterial communities from 10 of these species. We also examined the geographic variability in phyllosphere communities on Pinus ponderosa from several locations across the globe. Individual tree species harboured high levels of bacterial diversity and there was considerable variability in community composition between trees. The bacterial communities were organized in patterns predictable from the relatedness of the trees as there was significant correspondence between tree phylogeny and bacterial community phylogeny. Inter‐specific variability in bacterial community composition exceeded intra‐specific variability, a pattern that held even across continents where we observed minimal geographic differentiation in the bacterial communities on P. ponderosa needles.     

Leaf microbiota in an agroecosystem: spatiotemporal variation in bacterial community composition on field-grown lettuce

The presence, size and importance of bacterial communities on plant leaf surfaces are widely appreciated. However, information is scarce regarding their composition and how it changes along geographical and seasonal scales. We collected 106 samples of field-grown Romaine lettuce from commercial production regions in California and Arizona during the 2009–2010 crop cycle. Total bacterial populations averaged between 10⁵ and 10⁶ per gram of tissue, whereas counts of culturable bacteria were on average one (summer season) or two (winter season) orders of magnitude lower. Pyrosequencing of 16S rRNA gene amplicons from 88 samples revealed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were the most abundantly represented phyla. At the genus level, Pseudomonas, Bacillus, Massilia, Arthrobacter and Pantoea were the most consistently found across samples, suggesting that they form the bacterial ‘core'' phyllosphere microbiota on lettuce. The foliar presence of Xanthomonas campestris pv. vitians, which is the causal agent of bacterial leaf spot of lettuce, correlated positively with the relative representation of bacteria from the genus Alkanindiges, but negatively with Bacillus, Erwinia and Pantoea. Summer samples showed an overrepresentation of Enterobacteriaceae sequences and culturable coliforms compared with winter samples. The distance between fields or the timing of a dust storm, but not Romaine cultivar, explained differences in bacterial community composition between several of the fields sampled. As one of the largest surveys of leaf surface microbiology, this study offers new insights into the extent and underlying causes of variability in bacterial community composition on plant leaves as a function of time, space and environment.     

Effects of ecological environment and host genotype on the phyllosphere bacterial communities of cigar tobacco (Nicotiana tabacum L.)

Microorganisms of plant phyllosphere play an important role in plant health and productivity and are influenced by abiotic and biotic factors. In this study, we investigated the phyllosphere bacterial communities of three cigar tobacco varieties cultivated in Guangcun (GC) and Wuzhishan (WZS), Hainan, China. Metagenomic DNA was extracted from tobacco leaf samples and sequenced by 16S rDNA amplicon sequencing. Our results showed that bacterial communities of cigar tobacco phyllosphere in GC exhibited remarkably higher alpha diversity than that in WZS. There was slight effect of tobacco genotype variations on the alpha diversity in both cultivation sites, and beta diversity and structure of bacterial community were not influenced significantly by the cultivation sites and tobacco varieties. Statistical analyses of species diversity unraveled that the dominant species in bacterial communities of cigar tobacco phyllosphere among all these samples were phylogenetically affiliated to Proteobacteria and Cyanobacteria. At the genus level, the most abundant microorganism was Limnobacter, followed by Brevundimonas, unidentified_Cyanobacteria, and Pseudomonas. Additionally, environmental conditions except for humidity were negatively correlated with the relative abundance of bacterial genera. Further analyses revealed that influence of site‐specific factors on tobacco bacterial community was relatively higher than genotype‐specific factors. In short, this study may contribute to the knowledge base of practical applications of bacterial inoculants for tobacco leaf production.     

Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time

The phyllosphere is colonized by a wide variety of bacteria and fungi; it harbors epiphytes, as well as plant-pathogenic bacteria and even human pathogens. However, little is known about how the bacterial community composition on leafy greens develops over time. The bacterial community of the leafy-green phyllosphere obtained from two plantings of rocket salad (Diplotaxis tenuifolia) and three plantings of lettuce (Lactuca sativa) at two farms in Norway were profiled by an Illumina MiSeq-based approach. We found that the bacterial richness of the L. sativa samples was significantly greater shortly (3 weeks) after planting than at harvest (5 to 7 weeks after planting) for plantings 1 and 3 at both farms. For the second planting, the bacterial diversity remained consistent at the two sites. This suggests that the effect on bacterial colonization of leaves, at least in part must, be seasonally driven rather than driven solely by leaf maturity. The distribution of phyllosphere communities varied between D. tenuifolia and L. sativa at harvest. The variability between these species at the same location suggests that the leaf-dwelling bacteria are not only passive inhabitants but interact with the host, which shapes niches favoring the growth of particular taxa. This work contributes to our understanding of host plant-specific microbial community structures and shows how these communities change throughout plant development.     

Bacterial Succession on the Leaf Surface: A Novel System for Studying Successional Dynamics

Succession is a widely studied process in plant and animal systems, but succession in microbial communities has received relatively little attention despite the ubiquity of microorganisms in natural habitats. One important microbial habitat is the phyllosphere, or leaf surface, which harbors large, diverse populations of bacteria and offers unique opportunities for the study of succession and temporal community assembly patterns. To explore bacterial community successional patterns, we sampled phyllosphere communities on cottonwood (Populus deltoides) trees multiple times across the growing season, from leaf emergence to leaf fall. Bacterial community composition was highly variable throughout the growing season; leaves sampled as little as a week apart were found to harbor significantly different communities, and the temporal variability on a given tree exceeded the variability in community composition between individual trees sampled on a given day. The bacterial communities clearly clustered into early-, mid-, and late-season clusters, with early- and late-season communities being more similar to each other than to the mid-season communities, and these patterns appeared consistent from year to year. Although we observed clear and predictable changes in bacterial community composition during the course of the growing season, changes in phyllosphere bacterial diversity were less predictable. We examined the species–time relationship, a measure of species turnover rate, and found that the relationship was fundamentally similar to that observed in plant and invertebrate communities, just on a shorter time scale. The temporal dynamics we observed suggest that although phyllosphere bacterial communities have high levels of phylogenetic diversity and rapid turnover rates, these communities follow predictable successional patterns from season to season.     

Composition of the Phyllospheric Microbial Populations on Vegetable Plants with Different Glucosinolate and Carotenoid Compositions

The plant phyllosphere is intensely colonized by a complex and highly diverse microbial population and shows pronounced plant-species-specific differences. The mechanisms and influencing factors determining whether and in which density microorganisms colonize plant phyllosphere tissues are not yet fully understood. One of the key influencing factors is thought to be phytochemical concentration and composition. Therefore, correlations between various concentrations of individual glucosinolates and carotenoids in four different plant species-Brassica juncea, Brassica campestris, Cichorium endivia, and Spinacea oleracea-and the phyllospheric bacterial population size associated with the aerial parts of the same plants were analyzed. The concentration of various individual glucosinolates and carotenoids were measured using high-performance liquid chromatography. The phyllospheric bacterial population size including both nonculturable and culturable organisms was assessed using quantitative real-time polymerase chain reaction, and the physiological profile of the culturable microbial community was analyzed using the Biolog system. Results show significant differences between plant species in both concentration and composition of secondary metabolites, bacterial population size, and microbial community composition in three consecutively performed experiments. An interesting and underlying trend was that bacterial density was positively correlated to concentrations of beta-carotene in the plant phyllosphere of the four plant species examined. Likewise, the alkenyl glucosinolates, 2-propenyl, 3-butenyl, and 4-pentenyl, concentrations were positively correlated to the bacterial population density, whereas the aromatic glucosinolate 2-phenylethyl showed a negative correlation to the phyllospheric bacterial population size. Thus, we report for the first time the relationship between individual glucosinolate and carotenoid concentrations and the phyllospheric bacterial population size of nonculturable and culturable organisms and the phyllospheric microbial physiological profiles.     

Diversity and functional traits of culturable microbiome members,including cyanobacteria in the rice phyllosphere

The diversity and abundance of culturable microbiome members of the rice phyllosphere was investigated using cv. Pusa Punjab Basmati 1509. Both diversity and species richness of bacteria were significantly higher in plants in pots in a semi‐controlled environment than those in fields. Application of fertilisers reduced both diversity and species richness in field‐grown plants under a conventional flooded system of rice intensification (SRI) and in dry‐seeded rice (DSR) modes. Sequence analyses of 16S rDNA of culturable bacteria, those selected after amplified ribosomal DNA restriction analysis (ARDRA), showed the dominance of α‐proteobacteria (35%) and actinobacteria (38%); Pantoea, Exiguobacterium and Bacillus were common among the culturable phyllospheric bacteria. About 34% of 83 culturable bacterial isolates had higher potential (>2 μg·ml^?1) for indole acetic acid production in the absence of tryptophan. Interestingly, the phyllosphere bacterial isolates from the pot experiment had significantly higher potential for nitrogen fixation than isolates from the field experiment. Enrichment for cyanobacteria showed both unicellular forms and non‐heterocystous filaments under aerobic as well as anaerobic conditions. PCR‐DGGE analysis of these showed that aerobic and anaerobic conditions as well as the three modes of cultivation of rice in the field strongly influenced the number and abundance of phylotypes. The adaptability and functional traits of these culturable microbiome members suggest enormous diversity in the phyllosphere, including potential for plant growth promotion, which was also significantly influenced by the different methods of growing rice.     

Annual and Seasonal Variation in the Phyllosphere Bacterial Community Associated with Leaves of the Southern Magnolia (Magnolia grandiflora)

The phyllosphere contains a diverse bacterial community that can be intimately associated with the host plant; however, few studies have examined how the phyllosphere community changes over time. We sampled replicate leaves from a single magnolia (Magnolia grandiflora) tree in the winter of three consecutive years (2007?C2009) as well as during four seasons of 1?year (2008) and used molecular techniques to examine seasonal and year-to-year variation in bacterial community structure. Multivariate analysis of denaturing gradient gel electrophoresis profiles of 16S rRNA gene fragments revealed minimal leaf to leaf variation and much greater temporal changes, with the summer (August 2008) leaf community being most distinct from the other seasons. This was confirmed by sequencing and analysis of 16S rRNA gene clone libraries generated for each sample date. All phyllosphere communities were dominated by Alphaproteobacteria, with a reduction in the representation of certain Beijerinckiaceae during the summer and a concurrent increase in the Methylobacteriaceae being the most significant seasonal change. Other important components of the magnolia phyllosphere included members of the Bacteroidetes, Acidobacteria, and Actinobacteria, with the latter two lineages also showing differences in their representation in samples collected at different times. While the leaf-associated bacterial community sampled at the same time of year in three separate years showed some similarities, generally these communities were distinct, suggesting that while there are seasonal patterns, these may not be predictable from year to year. These results suggest that seasonal differences do occur in phyllosphere communities and that broad-leafed evergreen trees such as M. grandiflora may present interesting systems to study these changes in the context of changing environmental conditions.     

High Diversity Revealed in Leaf‐Associated Protists (Rhizaria: Cercozoa) of Brassicaceae

The largest biological surface on earth is formed by plant leaves. These leaf surfaces are colonized by a specialized suite of leaf‐inhabiting microorganisms, recently termed “phyllosphere microbiome”. Microbial prey, however, attract microbial predators. Protists in particular have been shown to structure bacterial communities on plant surfaces, but virtually nothing is known about the community composition of protists on leaves. Using newly designed specific primers targeting the 18S rDNA gene of Cercozoa, we investigated the species richness of this common protist group on leaves of four Brassicaceae species from two different locations in a cloning‐based approach. The generated sequences revealed a broad diversity of leaf‐associated Cercozoa, mostly bacterial feeders, but also including known plant pathogens and a taxon of potential endophytes that were recently described as algal predators in freshwater systems. This initial study shows that protists must be regarded as an integral part of the microbial diversity in the phyllosphere of plants.     

Changes amid constancy: Flower and leaf microbiomes along land use gradients and between bioregions

Microbial communities inhabiting above-ground parts of plants affect their host's development, fitness and function. Although studies on plant-associated microbes are of growing interest, environmental drivers of flower microbiomes in particular are poorly characterized. In this study, we investigated flower and leaf epiphytic bacterial microbiomes of Ranunculus acris and Trifolium pratense using metabarcoding of 16S ribosomal DNA in three German bioregions and along land-use intensity gradients. Our data suggests that the structures of bacterial communities clearly differed between plant species and tissue types. Also, floral bacterial communities of R. acris showed higher variability in comparison to T. pratense. Bacteria usually associated with pollinators were found solely in flower samples, while bacteria usually associated with the rhizosphere were only present in high abundances on leaves. We identified Pseudomonadaceae, Enterobacteriaceae and Sphingomonadaceae as the most abundant taxa on flowers, while Sphingomonadaceae, Methylobacteriaceae and Cytophagaceae dominated bacterial communities on leaves. We found that bacterial communities did not differ between long-distant regions. However, there was a turnover within each bioregion between short-distant locations. High land use intensity caused phylogenetically less diverse and more homogenous bacterial communities with an exception of T. pratense flowers. This was associated with a loss of rare bacterial families. Intensification of mowing affected the bacterial communities associated with leaves of T. pratense and fertilization led to more homogenous flower and leaf communities of R. acris, while grazing had no effects on the bacterial community composition. However, dominant taxa were not affected by land use intensification. Despite that, we identified indicator taxa for regularly disturbed environments in flower microbiomes. In conclusion, our study contributes to the knowledge about microbial community structures of the phyllosphere and extends the understanding of their community dynamics with respect to biogeographical separation and anthropogenic changes of the environment.     

Xanthomonas axonopodis pv. phaseoli var. fuscans Is Aggregated in Stable Biofilm Population Sizes in the Phyllosphere of Field-Grown Beans

The occurrence of “Xanthomonas axonopodis pv. phaseoli var. fuscans” (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10⁵ CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.     

不同生境中桂花和夹竹桃叶际细菌的群落结构

植物叶际微生物多样性是目前植物-微生物关系研究的热点之一,但影响叶际微生物群落结构的主要因素目前还存在很大争议.本研究以生长在3处生境的桂花和夹竹桃为对象,基于高通量测序技术,分析2种植物叶际细菌的群落结构,探讨影响植物叶际细菌群落结构的主要因素.结果表明:来自3处生境的2种植物叶际细菌多样性无显著差异,构成叶际细菌群落的优势门主要包括放线菌门、拟杆菌门、衣原体门、蓝细菌门、厚壁菌门和变形菌门,优势属主要包括甲基杆菌属、鞘氨醇单胞菌属、薄层杆属、Polaromonas和无毛螺旋体属.植物种类、生境及二者的交互作用均能显著影响叶际细菌群落结构,其中生境的影响最大.     

Both leaf properties and microbe-microbe interactions influence within-species variation in bacterial population diversity and structure in the lettuce (Lactuca Species) phyllosphere

Morphological and chemical differences between plant genera influence phyllosphere microbial populations, but the factors driving within-species variation in phyllosphere populations are poorly understood. Twenty-six lettuce accessions were used to investigate factors controlling within-species variation in phyllosphere bacterial populations. Morphological and physiochemical characteristics of the plants were compared, and bacterial community structure and diversity were investigated using terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA gene clone libraries. Plant morphology and levels of soluble carbohydrates, calcium, and phenolic compounds (which have long been associated with plant responses to biotic stress) were found to significantly influence bacterial community structure. Clone libraries from three representative accessions were found to be significantly different in terms of both sequence differences and the bacterial genera represented. All three libraries were dominated by Pseudomonas species and the Enterobacteriaceae family. Significant differences in the relative proportions of genera in the Enterobacteriaceae were detected between lettuce accessions. Two such genera (Erwinia and Enterobacter) showed significant variation between the accessions and revealed microbe-microbe interactions. We conclude that both leaf surface properties and microbial interactions are important in determining the structure and diversity of the phyllosphere bacterial community.     

Phyllosphere Bacterial Community of Floating Macrophytes in Paddy Soil Environments as Revealed by Illumina High-Throughput Sequencing

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments.     

Resilience of the Natural Phyllosphere Microbiota of the Grapevine to Chemical and Biological Pesticides

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.     

Diversity and Activity of PAH-Degrading Bacteria in the Phyllosphere of Ornamental Plants

Phyllosphere bacteria on ornamental plants were characterized based on their diversity and activity towards the removal of polycyclic aromatic hydrocarbons (PAHs), the major air pollutants in urban area. The amounts of PAH-degrading bacteria were about 1–10% of the total heterotrophic phyllosphere populations and consisted of diverse bacterial species such as Acinetobacter, Pseudomonas, Pseudoxanthomonas, Mycobacterium, and uncultured bacteria. Bacterial community structures analyzed by polymerase chain reaction–denaturing gradient gel electrophoresis from each plant species showed distinct band patterns. The uniqueness of these phyllosphere bacterial communities was partly due to the variation in leaf morphology and chemical properties of ornamental plants. The PAH degradation activity of these bacteria was monitored in gas-tight systems containing sterilized or unsterilized leaves. The results indicated that phyllosphere bacteria on unsterilized leaves were able to enhance the activity of leaves for phenanthrene removal. When compared between plant species, phenanthrene removal efficiency corresponded to the size of phenanthrene-degrading bacteria. In addition, phyllosphere bacteria on Wrightia religiosa were able to reduce other PAHs such as acenaphthylene, acenaphthene, and fluorine in 60-ml glass vials and in a 14-l glass chamber. Thus, phyllosphere bacteria on ornamental plants may play an important role in natural attenuation of airborne PAHs in urban areas.     

Distinct Phyllosphere Bacterial Communities on Arabidopsis Wax Mutant Leaves

The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed “core” community) while others were positively or negatively affected by the wax mutant phenotype (termed “plant line-specific“ community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria.     

Fitness of Salmonella enterica serovar Thompson in the Cilantro Phyllosphere

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22°C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30°C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.     

Geographical and land-use effects on seed-mass variation in common grassland plants

Seed mass is one of the most important plant traits. It is strongly related to plant fitness and life-history strategy, and is one of the key determinants of the ability of plants to spread and thus to respond to changing environments. While substantial empirical work has been devoted to understanding seed-mass variation across species, we know less about seed-mass variation within species, its geographical and ecological differentiation, and the degree to which it is influenced by environmental change. Here, we studied intraspecific variability in seed mass of six common grassland plants (Arrhenatherum elatius, Bromus hordeaceus, Cerastium holosteoides, Heracleum sphondylium, Trifolium repens and Veronica chamaedrys) across three regions in Germany and a broad range of land-use types and intensities. We found substantial seed-mass variation among regions, populations, and individuals within all of the studied species. In five species, seed mass had a strong and consistent geographic component, and in three species we found significant effects of land use – fertilisation, grazing intensity or mowing frequency – on seed mass. In several species, land use and geographic region not only affected mean seed mass, but also the variability of seed mass within populations. Our study demonstrates that seed mass is geographically and ecologically differentiated in common grassland species. It is likely that both phenotypic plasticity and genetic factors contribute to this differentiation. Our results also show that seed mass is a highly variable trait with typically around 10-fold variation within species.