Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.     

Fine-scale spatial genetic structure and gene dispersal in Silene latifolia

Plants are sessile organisms, often characterized by limited dispersal. Seeds and pollen are the critical stages for gene flow. Here we investigate spatial genetic structure, gene dispersal and the relative contribution of pollen vs seed in the movement of genes in a stable metapopulation of the white campion Silene latifolia within its native range. This short-lived perennial plant is dioecious, has gravity-dispersed seeds and moth-mediated pollination. Direct measures of pollen dispersal suggested that large populations receive more pollen than small isolated populations and that most gene flow occurs within tens of meters. However, these studies were performed in the newly colonized range (North America) where the specialist pollinator is absent. In the native range (Europe), gene dispersal could fall on a different spatial scale. We genotyped 258 individuals from large and small (15) subpopulations along a 60 km, elongated metapopulation in Europe using six highly variable microsatellite markers, two X-linked and four autosomal. We found substantial genetic differentiation among subpopulations (global F_ST=0.11) and a general pattern of isolation by distance over the whole sampled area. Spatial autocorrelation revealed high relatedness among neighboring individuals over hundreds of meters. Estimates of gene dispersal revealed gene flow at the scale of tens of meters (5–30 m), similar to the newly colonized range. Contrary to expectations, estimates of dispersal based on X and autosomal markers showed very similar ranges, suggesting similar levels of pollen and seed dispersal. This may be explained by stochastic events of extensive seed dispersal in this area and limited pollen dispersal.     

The occurrence ofFusarium andAlternaria on faba bean seed

The studies on the occurrence ofFusarium andAlternaria in faba bean seed have been conducted for three years. The fungal flora in seed lots from healthy plant and from plant inoculated withAscochyta fabae were determined. In addition the occurrence ofFusarium andAlternaria in three parts of seed: testa, cotyledones, and embryo axis were investigated. It was found thatFusarium andAlternaria were present in faba bean seed, but the frequency of occurrence varied depending upon cultivar and experimental year.     

Insect pest damage increases faba bean (Vicia faba) yield components but only in the absence of insect pollination

Identifying and quantifying crop stressors interactions in agroecosystems is necessary to guide sustainable crop management strategies. Over the last 50 years, faba bean cropping area has been declining, partly due to yield instabilities associated with uneven insect pollination and herbivory. Yet, the effect of interactions between pollinators and a key pest, the broad bean beetle Bruchus rufimanus (florivorous and seed predating herbivore) on faba bean yield has not been investigated. Using a factorial cage experiment in the field, we investigated how interactions between two hypothesized stressors, lack of insect pollination by bumblebees and herbivory by the broad bean beetle, affect faba bean yield. Lack of bumblebee pollination reduced bean weight per plant by 15%. Effects of the broad bean beetle differed between the individual plant and the plant‐stand level (i.e., when averaging individual plant level responses at the cage level), likely due to high variation in the level of herbivory among individual plants. At the individual plant level, herbivory increased several yield components but only in the absence of pollinators, possibly due to plant overcompensation and/or pollination by the broad bean beetle. At the plant‐stand level, we found no effect of the broad bean beetle on yield. However, there was a tendency for heavier individual bean weight with bumblebee pollination, but only in the absence of broad bean beetle herbivory, possibly due to a negative effect of the broad bean beetle on the proportion of legitimate flower visits by bumblebees. This is the first experimental evidence of interactive effects between bumblebees and the broad bean beetle on faba bean yield. Our preliminary findings of negative and indirect associations between the broad bean beetle and individual bean weight call for a better acknowledgment of these interactions in the field in order to understand drivers of crop yield variability in faba bean.     

Cleistogamy as a bet‐hedging strategy in Oxalis acetosella, a perennial herb

1 Phenology and reproduction were studied in three populations of the cleistogamous herb Oxalis acetosella during three growing seasons, in order to see how the balance between chasmogamous (CH) and cleistogamous (CL) reproduction varies with temporal and spatial environmental variation and with plant size. The numbers of CH and CL flower buds, flowers, immature capsules and mature capsules were counted per ramet, ramet sizes were estimated, and mature capsules were collected for seed counts.
2 Both CH and CL flower numbers were positively correlated with ramet size expressed as leaf number, but the correlation was much stronger in the CL phase. CL flower production also varied significantly between years and sites. Thus, CL production in O. acetosella was not independent of resources and climatic variation.
3 When the effects of year and site were taken into account, the probability of an individual flower developing into a mature fruit was not higher in the CL phase than in the CH phase.
4 CL production was affected by fertilization success in the CH phase. Ramets with one or more CH flowers left unfertilized generally produced more CL buds than ramets with all their CH flowers fertilized. The former group also tended to have more seeds per CL capsule.
5 Since reproductive success in the two phases varies in different temporal and spatial patterns, cleistogamy in O. acetosella is considered to be a bet‐hedging strategy optimizing reproductive output in fluctuating environments.
6 The findings of this study are in conflict with the general view on cleistogamy as a fail‐safe mechanism for back‐up seed production, unaffected by variations in resource supply and environmental conditions.     

Biocontrol Agent Talaromyces flavus Stimulates the Growth of Cotton and Potato

Beneficial plant-microbe interactions in the rhizosphere are primary determinants of plant health and soil fertility. Some antagonistic fungi have shown great effects toward the growth of plant crops. In this study, two major crops, cotton and potato, were selected to evaluate their growth promotion by the antagonistic fungus Talaromyces flavus. For each plant, five T. flavus isolates were selected from our fungal collection which had shown the highest antagonistic activities against the causal agent of wilt diseases on these plants. In the next step, for every crop, five isolates were used under greenhouse conditions. For evaluation of the plant growth promotion ability of T. flavus isolates, a split-plot trial was arranged in a randomized complete block design with four replications. The main factor was the method of application of T. flavus as a soil treatment, a seed treatment, and a combination of both methods. The subfactor was the use of different fungal isolates. Measured parameters were root length, crown length, plant height, plant fresh weight, and plant dry weight. Results showed that the maximum increase in the above parameters was mediated by the seed treatment method. The most effective isolate for cotton plants was TF-Co-M-23, which increased root length, plant height, plant fresh weight, and plant dry weight by 1.80-, 2.26-, 1.23-, and 1.19-fold, respectively. There were no significant differences among the various treatments affected by T. flavus in terms of crown length. The most effective isolate for potato plants was TF-Po-V-50, which increased root length, crown length, plant height, and plant dry weight by 1.71-, 1.09-, 1.45-, and 3.75-fold, respectively. The overall results of this study suggest that it may be possible to promote cotton and potato growth characteristics by using the antagonistic fungus T. flavus.     

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.     

Size,structure and nitrogen content of seeds of Fabaceae in relation to nodulation

Looking for clues to explain the low rate and unpredictability of the pollination of allogamous, epiphytic orchids in the context of the success of the orchid family in general, we compared the pollination of two miniature twig epiphytes in Mexico: Notylia barkeri, a ‘weedy’, rewarding species, and Erycina crista‐galli, a rare, deceptive species. We measured the effects of the spatial organization of the flowers and various parameters of visibility and height above the ground in relation to the spatial organization of the seed capsules (which retrospectively measures the activity of the pollinators). The spatial presentation of the flowers of E. crista‐galli explained only 5% of the distribution of the seed capsules. For N. barkeri, with compound flowers, the spatial presentation, considering individual flowers or inflorescences, explained 12% and 45% of the distribution of the seed capsules, respectively, and all other interactions between the variables were insignificant. Both the deceptive and reward strategies resulted in the production of large numbers of seeds despite a low pollination percentage (1–5%). Notylia barkeri produced 11.8 (2005–6) and 53.7 (2007–8) times more seeds in total than E. crista‐galli. Furthermore, unlike E. crista‐galli, N. barkeri responded to loss of individuals after high winds with an increased production of flowers per plant. We suggest that orchids have evolved to specialize in chance, and instead of maximizing pollinator attraction, they maximize the seed production resulting from every casual encounter. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 251–266.     

Factors affecting germinable seed yield in Cypripedium calceolus var. pubescens (Willd.) Correll and Epipactis helleborine (L.) Crantz (Orchidaceae)

One of the challenges facing orchid conservationists is to get a better understanding of seed parent fecundity. This can be especially laborious for orchids whose seed is difficult to germinate. Mature seeds ofCypripedium calceolusvar.pubescens(Willd.) Correll (Orchidaceae) have been reported to require 8 weeks pre-chilling at 4°C and a further month at 20°C to induce germination, but seeds prematurely harvested at 7–8 weeks post-pollination germinate asymbiotically without chilling in less than 1 month. Mature seeds ofEpipactis helleborine(L.) Crantz (Orchidaceae) have also presented a challenge but the identification of plants whose seeds are quick and easy to germinate has provided us with a tool to study patterns of production of this seed class. Preliminary field experiments suggest that timing of pollination in these two taxa is critical for the development of seed. When seeds from capsules ofC. calceolusvar.pubescensharvested 7 weeks after pollination were examined, the proportion of fully mature embryos increased with increasing anthesis-pollination interval over the first 10 days. Paired flowers from five genets xenogamously hand-pollinated at 2-day intervals from the second day after anthesis until the flowers wilted were significantly less likely to produce fruits as the flowers aged but the flower age at which this occurred varied between genets. The germinability of seeds from different pollen and seed parent combinations was tested on eight different media formulations and over a range of post-anthesis and post-pollination intervals. Different aged seeds germinated variably, the post-anthesis interval being the most reliable predictor of germination outcomes. Intercrossing of plants ofC. calceolusvar.pubescenssuggests that seed production and germinability are maternally mediated traits. WithE. helleborine, germinable seed can be produced both by self- and by cross-pollination of selected plants but we have observed an influence of flower position in the inflorescence on self-compatibility and on the production of the easy-to-germinate seed class. The timing of pollination in these two unrelated orchids has a considerable influence upon seed production and seed quality in plants of established fecundity. These findings suggest that flower age at pollination must be considered when interpreting similar studies in the Orchidaceae.     

No Effect of Host Species on Phenoloxidase Activity in a Mycophagous Beetle

Ecological immunology is an interdisciplinary field that helps elucidate interactions between the environment and immune response. The host species individuals experience have profound effects on immune response in many species of insects. However, this conclusion comes from studies of herbivorous insects even though species of mycophagous insects also inhabit many different host species. The goal of this study was to determine if fungal host species as well as individual, sex, body size, and host patch predict one aspect of immune function, phenoloxidase activity (PO). We sampled a metapopulation of Bolitotherus cornutus, a mycophagous beetle in southwestern Virginia. B. cornutus live on three species of fungus that differ in nutritional quality, social environment, and density. A filter paper phenoloxidase assay was used to quantify phenoloxidase activity. Overall, PO activity was significantly repeatable among individuals (0.57) in adult B. cornutus. While there was significant variance among individuals in PO activity, there were surprisingly no significant differences in PO activity among subpopulations, beetles living on different host species, or between the sexes; there was also no effect of body size. Our results suggest that other factors such as age, genotype, disease prevalence, or natal environment may be generating variance among individuals in PO activity.     

The Influence of Ustilago maydis Upon Free Radicals Concentration in the Reproduction Organs of Maize

The reproduction organs of maize are those most often attacked by Ustilago maydis. In the male organs it produces a decline in free radicals concentration and, conversely, a rise of concentration in the female organs (in ears). The smut proper, grown on the flower, exhibits an essentially higher free radicals concentration than the smut grown out on the ear of that plant. A smut mixture from flower and ear has only a slightly higher free radicals concentration than the separate ear smut, but an essentially lower one than the smut of the flower.     

Competition and facilitation among plants for pollination: can pollinator abundance shift the plant–plant interactions?

We hypothesize interactions among plants for pollination may depend on pollinator abundance, which always varies among years and habitats and has different effects on plant reproductive success. Honeybee-pollinated plants, Lotus corniculatus, and its commonly coflowering neighbor, Potentilla reptans var. sericophylla, were used in a two-year project. We designed six types of plant combinations with different conspecific and interspecific flower densities in 2011 and repeated this in the same site in 2012. Meanwhile, we artificially increased pollinator abundance by hiring beehives only in 2011. Pollinator abundance as well as flower density significantly affected pollination of L. corniculatus plants from both the conspecific and interspecific plots. Total number of bees visiting a plot was enhanced by an increase in both the conspecific and interspecific flower densities regardless of high or low pollinator abundance. However, changes in visitation rates and fruit sets in the focal plants when flower densities were increased depended on pollinator abundance. Under high pollinator abundance, an increase in both the conspecific and interspecific flower densities significantly enhanced pollinator visits to L. corniculatus. However, under low pollinator abundance, the pollinator visitation rate remained unchanged as conspecific flower density increased, but decreased when there was an increase in interspecific flower density. Coflowering plants enhanced fruit sets of L. corniculatus only when the pollinator abundance was high. The findings suggest that the interactions among plants for pollination are influenced not only by a plant density threshold, but also by a pollinator abundance threshold.     

Increasing frequency of low summer precipitation synchronizes dynamics and compromises metapopulation stability in the Glanville fritillary butterfly

Climate change is known to shift species'' geographical ranges, phenologies and abundances, but less is known about other population dynamic consequences. Here, we analyse spatio-temporal dynamics of the Glanville fritillary butterfly (Melitaea cinxia) in a network of 4000 dry meadows during 21 years. The results demonstrate two strong, related patterns: the amplitude of year-to-year fluctuations in the size of the metapopulation as a whole has increased, though there is no long-term trend in average abundance; and there is a highly significant increase in the level of spatial synchrony in population dynamics. The increased synchrony cannot be explained by increasing within-year spatial correlation in precipitation, the key environmental driver of population change, or in per capita growth rate. On the other hand, the frequency of drought during a critical life-history stage (early larval instars) has increased over the years, which is sufficient to explain the increasing amplitude and the expanding spatial synchrony in metapopulation dynamics. Increased spatial synchrony has the general effect of reducing long-term metapopulation viability even if there is no change in average metapopulation size. This study demonstrates how temporal changes in weather conditions can lead to striking changes in spatio-temporal population dynamics.     

Is the Pathogenic Ergot Fungus a Conditional Defensive Mutualist for Its Host Grass?

It is well recognized, that outcomes of mutualistic plant-microorganism interactions are often context dependent and can range from mutualistic to antagonistic depending on conditions. Instead, seemingly pathogenic associations are generally considered only harmful to plants. The ergot fungus (Claviceps purpurea) is a common seed pathogen of grasses and cereals. Ergot sclerotia contain alkaloids which can cause severe toxicity in mammals when ingested, and thus the fungal infection might provide protection for the host plant against mammalian herbivores. Theoretically, the net effect of ergot infection would positively affect host seed set if the cost is not too high and the defensive effect is strong enough. According to our empirical data, this situation is plausible. First, we found no statistically significant seed loss in wild red fescue (Festuca rubra) inflorescences due to ergot infection, but the seed succession decreased along increasing number of sclerotia. Second, in a food choice experiment, sheep showed avoidance against forage containing ergot. Third, the frequency of ergot-infected inflorescences was higher in sheep pastures than surrounding ungrazed areas, indicating a protective effect against mammalian grazing. We conclude that, although ergot can primarily be categorized as a plant pathogen, ergot infection may sometimes represent indirect beneficial effects for the host plant. Ergot may thus serve as a conditional defensive mutualist for its host grass, and the pathogenic interaction may range from antagonistic to mutualistic depending on the situation.     

Isolation and Characterization of Antagonistic Bacillus Strains Capable to Degrade Ethylenethiourea

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.     

Patchy population structure in a short-distance migrant: evidence from genetic and demographic data

Species often occur in subdivided populations as a consequence of spatial heterogeneity of the habitat. To describe the spatial organization of subpopulations, existing theory proposes three main population models: patchy population, metapopulation and isolated populations. These models differ in their predicted levels of connectivity among subpopulations, and in the risk that a subpopulation will go extinct. However, spatially discrete subpopulations are commonly considered to be organized as metapopulations, even though explicit tests of metapopulation assumptions are rare. Here, we test predictions of the three models on the basis of demographic and genetic data, a combined approach so far surprisingly little used in mobile organisms. From 2002 to 2005, we studied nine subpopulations of the wetland-restricted reed bunting ( Emberiza schoeniclus ) in the southeastern part of the Canton Zurich (Switzerland), from which local declines of this species have been reported. Here, wetlands are as small as 2.7 ha and separated through intensively used agricultural landscapes. Demographic data consisted of dispersal of colour-banded individuals among subpopulations, immigration rates and extinction-/recolonization dynamics. Genetic data were based on the distribution of genetic variability and gene flow among subpopulations derived from the analysis of nine microsatellite loci. Both demographic and genetic data revealed that the patchy population model best described the spatial organization of reed bunting subpopulations. High levels of dispersal among subpopulations, high immigration into the patchy population, and genetic admixture suggested little risk of extinction of both subpopulations and the entire patchy population. This study exemplifies the idea that spatially discrete subpopulations may be organized in ways other than a metapopulation, and hence has implications for the conservation of subpopulations and species.     

Spatial variation in breeding phenology at small spatial scales: A stochastic effect of population size

Spatial variation in phenology can occur at small spatial scales over which individuals can disperse or forage within one generation. Previous studies have assumed that variations in phenological peaks are caused by differences in abiotic environmental characteristics. However, environments should generally be similar among local habitats over small spatial scales. When the local population size is small, the phenological peak of the local population should be strongly affected by the variation in timing expressed by individuals. If a regional population consists of small local subpopulations (e.g., a metapopulation), the stochastic processes regulated by population sizes may explain the spatial variation in phenology. In this study, we quantitatively evaluated the extent of the spatial and annual variations in the breeding phenology of the forest green tree frog, Rhacophorus arboreus habiting a small area (<10 km²). The spatial variation in phenological peaks among 25 breeding sites was large over 6 years. This spatial variation was not explained by differences in air temperature or water depth. Randomization tests revealed that a large portion of the spatial variation could be explained by differences in population size, without considering site-specific factors. Annual variations in phenological peaks tended to be greater for smaller populations. These results imply that the stochastic process might have caused the spatial and annual variations in the phenological peaks of R. arboreus observed in the study region. Understanding spatiotemporal variation in phenology determined by stochastic process would be important to better predict interspecific interactions and (meta)population dynamics at small spatial scales.     

Culturable Endophytes Associated with Soybean Seeds and Their Potential for Suppressing Seed-Borne Pathogens

Seed-borne pathogens in crops reduce the seed germination rate and hamper seedling growth, leading to significant yield loss. Due to the growing concerns about environmental damage and the development of resistance to agrochemicals among pathogen populations, there is a strong demand for eco-friendly alternatives to synthetic chemicals in agriculture. It has been well established during the last few decades that plant seeds harbor diverse microbes, some of which are vertically transmitted and important for plant health and productivity. In this study, we isolated culturable endophytic bacteria and fungi from soybean seeds and evaluated their antagonistic activities against common bacterial and fungal seed-borne pathogens of soybean. A total of 87 bacterial isolates and 66 fungal isolates were obtained. Sequencing of 16S rDNA and internal transcribed spacer amplicon showed that these isolates correspond to 30 and 15 different species of bacteria and fungi, respectively. Our antibacterial and antifungal activity assay showed that four fungal species and nine bacterial species have the potential to suppress the growth of at least one seed-borne pathogen tested in the study. Among them, Pseudomonas koreensis appears to have strong antagonistic activities across all the pathogens. Our collection of soybean seed endophytes would be a valuable resource not only for studying biology and ecology of seed endophytes but also for practical deployment of seed endophytes toward crop protection.     

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship

Main conclusion

Orchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.