Occurrence and Genetic Structure of the Systemic Grass Endophyte <Emphasis Type="Italic">Epichloë festucae</Emphasis> in Fine Fescue Populations

Epichloë species are systemic fungal endophytes that usually specialize in a certain group of related grass species. We examined the infection frequency of Epichloë festucae in populations of two fine fescue species (Festuca rubra and F. ovina) in natural and seminatural habitats at 86 study sites (total = 2514 plants) across Finland and northern Norway. Infection incidence varied significantly among grass species and populations. A substantial number of the F. rubra and F. ovina populations (53 out of 77 and 25 out of 30, respectively) were either endophyte-free or had very low (<20%) infection frequencies. The highest infection frequencies were found in subarctic areas. Moreover, infection incidence differed between habitats. In the area with the highest infection frequencies, we used microsatellite markers to study genetic diversity and the rates of gene flow of E. festucae among 12 F. rubra populations. Twenty out of the 25 fungal genotypes detected with four microsatellite markers were carrying multiple alleles in at least one locus, indicating multiple infections or vegetative hybridization of the fungus. One dominant genotype occurred in all 12 populations, representing 63.5% of all isolates. We found a moderate level of average genotypic variation and a low level of genetic differentiation (F _st = 0.0814). There was no correlation between infection frequency and genotypic diversity. Although the existence of a dominant genotype and the detected linkage disequilibrium suggest that the fungus is mainly asexual and vertically transmitted, the multiallelic loci and variation of genetic diversity among populations indicate occasional contagious spread and sexual or parasexual recombination of the fungus in some populations. Furthermore, the genotypes carrying multiallelic loci suggest the possibility of multiple infections or hybridization of the endophyte.     

<Emphasis Type="Italic">Epichloë occultans</Emphasis> enhances micropropagation efficiency in <Emphasis Type="Italic">Lolium multiflorum</Emphasis>

Italian ryegrass (Lolium multiflorum) is an annual grass considered as one of the most important temperate forage grasses in the world. However, it is recalcitrant to plant tissue culture techniques hindering its genetic manipulation. Epichloë occultans is an endophytic fungus associated with L. multiflorum. This symbiosis causes improvements in physiological and ecological traits of the host plants. The objective of this work was to study the effect of E. occultans on L. multiflorum micropropagation. We compared the response of endophyte-infected (E+) and endophyte-free (E?) seeds in different micropropagation stages. The E+ seeds were more successful than E? seeds in in vitro germination (83?±?5 vs. 63?±?6%), callus induction (78?±?5 vs. 57?±?6%), callus proliferation (average diameter of 21.5?±?1.3 mm in two subcultures vs 17.3?±?0.8 mm in three subcultures) and plant regeneration from callus (83?±?7 vs. 30?±?8%). These results indicate that E. occultans enhances significantly L. multiflorum micropropagation. The use of endophyte-infected (E+) seeds can be a solution to make this grass more amenable to different biotechnological tools, such as the genetic transformation.     

Variation in mycorrhizal performance in the epiphytic orchid <Emphasis Type="Italic">Tolumnia variegata in vitro</Emphasis>: the potential for natural selection

Symbiotic seed germination is a critical stage in orchid life histories. Natural selection may act to favor plants that efficiently use mycorrhizal fungi. However, the necessary conditions for natural selection – variation, heritability, and differences in fitness – have not been demonstrated for either orchid or fungus. With the epiphytic orchid Tolumnia variegata as a model system, we ask the following questions: (1) Do seeds from different individuals in a population differ in germination and seedling development in the presence of the same fungi? (2) Do different mycorrhizal fungi (Ceratobasidium spp.) differ in ability to stimulate seed germination and growth in T. variegata? And (3) are the Ceratobasidium isolates that best induce seed germination and seedling development more closely related to each other than to isolates that are less effective? We performed symbiotic seed germination experiments in vitro. The experiments were done using mycorrhizal fungi isolated from T. variegata; relationships among the fungi were inferred from nuclear ribosomal ITS sequences. We found significant variation for both symbiotic germination and seedling growth among biparental seed crops obtained from a population of T. variegata plants. Differences among Ceratobasidium fungi in seed germination were significant. The fungi that induced highest seed germination and seedling development belonged to two of four clades of Ceratobasidium. The two experiments show that there is potential for natural selection to act on orchid–fungus relationships. Given that orchids vary in performance, and that mycorrhizal fungi are not geographically distributed homogeneously, mycorrhizae may affect population size, distribution and evolution of orchids.     

Correction to: Neighbourhood stories: role of neighbour identity,spatial location and order of arrival in legume and non-legume initial interactions

Background and aims

We characterized fungal endophytes of seeds of invasive, non-native Phragmites from three sites in the Great Lakes region to determine if fungal symbiosis could contribute to invasiveness through their effects on seed germination and seedling growth.

Methods

Field-collected seeds were surface sterilized and plated on agar to culture endophytes for ITS sequencing. Prevalence of specific endophytes from germinated and non-germinated seeds, and from seedlings, was compared.

Results

One-third of 740 seeds yielded endophyte isolates. Fifteen taxa were identified with Alternaria sp. representing 54% of all isolates followed by Phoma sp. (21%) and Penicillium corylophilum (12%). Overall germination of seeds producing an isolate (36%) was significantly higher than seeds not producing an isolate (20%). Penicillium in particular was strongly associated with increased germination of seeds from one site. Sixty-three isolates and 11 taxa were also obtained from 30 seedlings where Phoma, Penicillium and Alternaria respectively were most prevalent. There was a significant effect of isolating an endophyte from the seed on seedling growth.

Conclusions

These results suggest that many endophyte taxa are transmitted in seeds and can increase seed germination and seedling growth of invasive Phragmites. The role of fungal endophytes in host establishment, growth and invasiveness in nature requires further research.

     

Mechanisms of influence of invasive grass litter on germination and growth of coexisting species in California

In grasslands, litter has been recognized as an important factor promoting grass persistence and the suppression of forbs. The invasive European annual grass Bromus diandrus (ripgut brome) is widespread throughout California, where it produces a persistent and thick litter layer. The native grass, Stipa pulchra, is also common in some grassland settings and can also produce persistent litter, yet it is typically associated with more forbs. Very little is known about the mechanisms through which these two common grass species influence seedling establishment of both exotic invasive and native herbs. Here, we evaluated the effect of B. diandrus and S. pulchra litter on seedling establishment of two invasive (the grass B. diandrus and the forb Centaurea melitensis) and two native (the grass S. pulchra, and the forb Clarkia purpurea) herbaceous plants in a greenhouse setting. Our results showed that B. diandrus litter cover hindered seedling establishment of the four species tested, but that the degree and mechanism of inhibition was dependent on which species was tested, life form (e.g. monocot/dicot) and seed size. Seedling emergence of the two forb species was more vulnerable to litter cover than either grass species and both forbs had smaller seed size. After germination, only seedling biomass of B. diandrus itself was reduced by litter (both B. diandrus and S. pulchra). We found no significant effects of leachate of either grass species on seedling emergence of any species, while a high concentration of B. diandrus leachates inhibited root growth of all species including B. diandrus seedlings. Stipa pulchra litter leachates did not affect S. pulchra or C. melitensis seedlings although it did suppress B. diandrus and C. purpurea seedling growth. Our findings provide direct experimental evidence for the mechanism of effect of litter on these coexisting invasive and native species. Such evidence helps advance our understanding of role of B. diandrus and S. pulchra litter in California grassland.     

Moisture conditions and the presence of bryophytes determine fescue species abundance in a dry calcareous grassland

Festuca ovina is the abundant matrix-forming species and F. rubra a subordinate species in shallow-soil calcareous grasslands. F. pratensis is a transient species, occurring sparsely in this community. We hypothesised that the different abundances of these three species are primarily due to the differential effect of moisture conditions on their germination and early establishment, and that the effect of the pattern of rainfall intensity depends on the presence or absence of a bryophyte layer. We studied the dependence of the germination and establishment of the three fescue species on the moisture conditions both in the laboratory and in the patches of intact grassland community (microcosms). In a laboratory germination experiment, F. pratensis showed the highest, F. rubra , the intermediate and F. ovina, the lowest drought tolerance. In microcosms, the establishment of F. ovina was the highest. At the same time, the annual mortality of seedlings of F. ovina was the lowest. All three species responded positively to an increasing irrigation level. Differently from F. ovina, F. rubra showed a positive response only in plots from which the bryophyte layer had been removed, while F. pratensis responded positively to both irrigation and bryophyte removal. We conclude that moisture conditions have a differential effect on the three fescue species mainly in the seedling establishment, not in the germination phase. For the successful establishment of F. rubra and F. pratensis, the coincidence of high rainfall and local disturbance, removing bryophytes, is required. The presence or absence of bryophytes had no effect on establishment in dry years, while in rainy years the removal of bryophytes has a clear positive effect.     

Using Illumina-Based Sequence Analysis to Guide Probiotic Candidate Selection and Isolation

Selection for probiotic candidates by in vivo experimental trials is time and labor consuming; more informed strategy is needed to select successful probiotic candidates. The aim of the study was to elucidate the microbial taxa transmitted from maize seeds to seedlings during the germination process of maize and their probiotic effects. The bacterial and fungal taxa in kernel germs and sprouts were analyzed by Illumina-based sequencing. The sprouts contained more diverse fungi than those in germs. The bacterial species (OTUs) declined with the germination from germs to the sprouts. However, the endophytic fungal diversity increased during the germination process. Seed-borne dominant bacterial genera Bacillus, Halomonas, and Shewanella and dominant fungal genera Aspergillus were also detected in sprouts. The spore-forming bacteria BS3 isolated directly from sprouts could promote growth of maize seedling and resistance to F. verticillioides under F. verticillioides-infested soils. The results suggested that maize contained core bacterial and fungal taxa during the development from seeds to sprouts, and the core endophytes showed more intimate correlation with host plants than did other microbial taxa. Illumina-based sequence analysis is feasible to guide probiotic candidate selection and isolation.     

Efficacy of bacterial seed treatments for the control of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> in maize

Maize colonization by the phytopathogenic fungi Fusarium verticillioides leads to economical and food quality losses and also implicates potential health risks. In order to control this fungal species different strategies are being considered. In the present work we investigated the in situ effects of the use of two in vitro proven bacterial biological control agents against Fusarium verticillioides, using maize seedlings grown in greenhouse conditions. The treatment of maize seeds with Fusarium verticillioides 10⁷ spores ml^?1 negatively affected the length of the stem and the weight of the root systems of resultant seedlings, and also reduced the numbers of non-rhizospheric organisms with ammonification and nitrification activities. The addition of Bacillus amyloliquefaciens or Microbacterium oleovorans, at a concentration of 10⁹ colony forming units ml^?1, to the seeds significantly reduced Fusarium verticillioides count at the root inner tissues of resultant seedlings. When testing the impact of bacterial treatments on soil populations, no alterations with respect to control numbers of organisms with nitrification, ammonification or cellulolytic potentials were observed. Culturable bacterial richness and diversity calculated at the rhizoplane and root inner tissues of maize seedlings neither changed in bacterized treatments when compared to control treatment. Our study showed that the Fusarium verticillioides in vitro proven antagonists, Bacillus amyloliquefaciens and Microbacterium oleovorans (at 10⁹ colony forming units ml^?1), were also effective at greenhouse conditions without causing major changes in culturable rhizospheric and endophytic microbial richness and diversity.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

Does dormancy protect seeds against attack by the pathogenic fungus <Emphasis Type="Italic">Fusarium tricinctum</Emphasis> in a semiarid grassland of Northwest China?

Aims

Soil fungal pathogens can result in the failure of seedling establishment, but the effects of fungicide applications on seed/seedling survival have differed among studies. We assumed that the variation may relate to seed dormancy/germination characteristics and hypothesized that nondormant germinating seeds are more likely to be killed by fungal pathogens than dormant seeds.

Methods

Dormant and nondormant seeds of Stipa bungeana and Lespedeza davurica were inoculated with a pathogenic fungus Fusarium tricinctum under laboratory and field conditions. The outcomes of seed/seedling fate and other parameters were evaluated.

Results

In the laboratory, nondormant seeds inoculated with F. tricinctum developed white tufts of mycelium on the radicles of germinating seeds causing them to quickly die, but dormant seeds remained intact. In contrast, in the field inoculation with F. tricinctum did not cause higher mortality of nondormant than dormant seeds but resulted in higher percentages of seedling death before they emerged from soil than the controls.

Conclusions

Our results suggest that dormancy protects seeds from being attacked by some pathogens by preventing germination, but the protection is lost once germination has commenced. Further study involving various plant species with more seeds is needed to assess the generality of this pathogen-seed interaction hypothesis.

     

Disease protection and allelopathic interactions of seed-transmitted endophytic pseudomonads of invasive reed grass (<Emphasis Type="Italic">Phragmites australis</Emphasis>)

Background and aims

Non-native Phragmites australis (haplotype M) is an invasive grass that decreases biodiversity and produces dense stands. We hypothesized that seeds of Phragmites carry microbes that improve seedling growth, defend against pathogens and maximize capacity of seedlings to compete with other plants.

Methods

We isolated bacteria from seeds of Phragmites, then evaluated representatives for their capacities to become intracellular in root cells, and their effects on: 1.) germination rates and seedling growth, 2.) susceptibility to damping-off disease, and 3.) mortality and growth of competitor plant seedlings (dandelion (Taraxacum officionale F. H. Wigg) and curly dock (Rumex crispus L.)).

Results

Ten strains (of 23 total) were identified and characterized; seven were identified as Pseudomonas spp. Strains Sandy LB4 (Pseudomonas fluorescens) and West 9 (Pseudomonas sp.) entered root meristems and became intracellular. These bacteria improved seed germination in Phragmites and increased seedling root branching in Poa annua. They increased plant growth and protected plants from damping off disease. Sandy LB4 increased mortality and reduced growth rates in seedlings of dandelion and curly dock.

Conclusions

Phragmites plants associate with endophytes to increase growth and disease resistance, and release bacteria into the soil to create an environment that is favorable to their seedlings and less favorable to competitor plants.

     

Sexual reproduction of clonal dwarf shrubs in a forest–tundra ecotone

Successful sexual reproduction may be more important for regeneration of clonal species in high-latitude and -altitude areas than has been previously suggested. We investigated the potential of Vaccinium myrtillus, V. vitis-idaea and Empetrum nigrum ssp. hermaphroditum (E. hermaphroditum) for sexual reproduction at three sites in the forest–tundra ecotone in Finnish Lapland. We studied whether the potential differs between plant communities, whether disturbance enhances germination, and whether seedling emergence is limited by seed availability. We established a field experiment with disturbance and sowing treatments, and monitored seed and seedling numbers and survival rates for two years. The number of mature seeds of V. myrtillus was higher in plants from the tundra heath than in those from the coniferous and mountain birch forests. The number of mature seeds and seedlings emerging from the seed bank of E. hermaphroditum tended also to be higher in the tundra heath. Disturbance marginally increased the seedling emergence of V. myrtillus and E. hermaphroditum, whereas sowing generally increased the seedling numbers. The seedling number of V. myrtillus was lower in the tundra heath and that of E. hermaphroditum was lower in the coniferous forest than at the other sites. Seedling survival was equal for all plant species at all sites. We conclude that the capacity for sexual reproduction varies among plant communities, and seed availability is a stronger constraint than microsite availability for the studied species. Once the crucial early phase of seedling establishment is overcome, seedling survival enables successful recruitment of V. myrtillus, V. vitis-idaea and E. hermaphroditum in the subarctic area.     

Colonization by nitrogen-fixing <Emphasis Type="Italic">Frankia</Emphasis> bacteria causes short-term increases in herbivore susceptibility in red alder (<Emphasis Type="Italic">Alnus rubra</Emphasis>) seedlings

Carbon allocation demands from root-nodulating nitrogen-fixing bacteria (NFB) can modulate the host plant’s chemical phenotype, with strong bottom–up effects on herbivores. In contrast to well-studied rhizobia, the effects of other important NFB on plant chemistry and herbivory are much less understood. Here, combining field surveys in the Oregon Coast Range, USA with laboratory experiments, we analyzed how N₂-fixing Frankia bacteria influenced plant growth, chemistry, and herbivory on Alnus rubra (red alder) seedlings. In the field, we quantified Frankia nodulation, herbivore damage, and plant size. In the laboratory, we grew seedlings with Frankia (F+), Frankia-free but nitrogen-fertilized (N+), or both uncolonized and unfertilized (F?N?) and assessed growth and leaf chemistry. We further conducted choice trials with black slugs, Arion rufus, a natural red alder herbivore. In the field, Frankia nodulation was significantly positively correlated with herbivory and negatively with seedling height. In contrast, in the lab, F+ as well as N+ seedlings were significantly taller than the F?N? controls. Seedlings from both treatments also had significantly increased leaf protein concentration compared to controls, whereas carbon-based nutritive compounds (carbohydrates) as well as leaf palatability-decreasing condensed tannins, lignin, and fiber were decreased in F+ but not in N+ treatments. In the choice assays, slugs preferred leaf material from F+ seedlings, but the effects were only significant in young leaves. Our study indicates that colonization by Frankia causes short-term ecological costs in terms of susceptibility to herbivory. However, the ubiquity of this symbiosis in natural settings suggests that these costs are outweighed by benefits beyond the seedling stage.     

Mutualistic fungus promotes plant invasion into diverse communities

Reducing the biological diversity of a community may decrease its resistance to invasion by exotic species. Manipulative experiments typically support this hypothesis but have focused mainly on one trophic level (i.e., primary producers). To date, we know little about how positive interactions among species may influence the relationship between diversity and invasibility, which suggests a need for research that addresses the question: under what conditions does diversity affect resistance to invasion? We used experimental manipulations of both plant diversity and the presence of an endophytic fungus to test whether a fungal mutualist of an invasive grass species (Lolium arundinaceum) switches the relationship between plant community diversity and resistance to invasion. Association with the fungal endophyte (Neotyphodium coenophialum) increased the ability of L. arundinaceum to invade communities with greater species diversity. In the absence of the endophyte, the initial diversity of the community significantly reduced the establishment of L. arundinaceum. However, establishment was independent of initial diversity in the presence of the endophyte. Fungal symbionts, like other key species, are often overlooked in studies of plant diversity, yet their presence may explain variation among studies in the effect of diversity on resistance to invasion.     

Differences in reward removal efficiency and its consequences for seed germination in the ant-dispersed sedge <Emphasis Type="Italic">Carex tristachya</Emphasis> (Cyperaceae)

Reward removal is an essential step for seed dispersal mutualism because residual rewards inhibit germination. Nevertheless, variation in the reward removal efficiency (RRE) among dispersers and its consequences for germination have rarely been reported. In this study, we compared the RREs of two sympatric seed-dispersing ants, Formica japonica and Pheidole noda, using seeds of the ant-dispersed sedge Carex tristachya. Then, we conducted seed sowing experiments in a non-heated glasshouse to evaluate the effect of RRE on the percentage and speed of germination. The majority (85%) of seeds handled by F. japonica had residual elaiosomes, while elaiosomes were completely removed from all seeds handled by P. noda, demonstrating that P. noda has much higher RRE than F. japonica. The seed sowing experiments revealed that RRE, defined by the presence or absence of residual elaiosomes, was not associated with the percentage germination within a year. However, high RRE seeds with no residual elaiosomes germinated significantly faster than low RRE seeds with residual elaiosomes. Similarly, artificial removal of elaiosomes from C. tristachya seeds accelerated germination speed without affecting germination percentage. These results suggest that RRE is one of the most important parameters determining the effectiveness of a seed dispersal agent.     

Biocontrol of a root rot of kale by <Emphasis Type="Italic">Muscodor albus</Emphasis> strain MFC2

Pythium ultimum is an oomycetous root rot pathogen that causes significant crop production losses on many crops including kale (Brassica oleracea), an economically important vegetable in Thailand. An endophytic fungus from Thailand designated Muscodor albus MFC2 controlled P. ultimum both in vitro and on kale seedlings grown under outdoor conditions via the production of volatile antibiotics. Ten-day old M. albus MFC2 PDA cultures killed P. ultimum in vitro. Thoroughly mixing three PDA plates of 10-day old M. albus MFC2 into a 500 g mixture of commercial soil and field soil did not adversely affect kale seed germination. The same amount of M. albus MFC2 could restore seedling emergence in P. ultimum inoculated soil to a level close to that of a non-infested control. In addition, M. albus MFC2 did not cause any disease symptoms, but rather seemed to promote the growth of kale in the presence or absence of P. ultimum for up to eight weeks after planting.