Microscopic analysis of plant–bacterium interactions using auto fluorescent proteins

Plant growth promoting rhizobacteria (PGPR) include bacteria that fix nitrogen (e.g., Rhizobiaceae, Herbaspirillum, Azoarcus), produce phytohormones (e.g., Azospirillum) and provide protection against fungal and/or bacterial pathogens (e.g., Pseudomonas, Bacillus, Streptomyces). Interactions between PGPR and plants can be divided into different steps which include initial attraction, attachment, proliferation and colonization e.g., of roots, stem, leaves and flowers. At the genetic level the expression of many bacterial genes are altered during these processes. In addition to the interaction with the plant, PGPR interact and compete with the endogenous microflora, consisting of other bacteria, fungi and/or mycorrhizal fungi. In the case of biocontrol bacterial strains, a direct interaction with the pathogen is often required to suppress the disease. Microscopic analyses of plant growth promoting rhizobacteria (PGPR) in their natural environment and in specific during their interaction(s) with the host plant(s) and/or their target organism(s) is essential for the elucidation of their functioning and the successful application of commercial inoculants. With the discovery and development of auto fluorescent proteins (AFPs) as markers and the development of highly sophisticated fluorescence microscopes such as confocal laser scanning microscopes, a new dimension has been created for studying PGPR in their natural environment. This paper will give a short overview on available tools, the application of AFPs in PGPR research and some future perspectives. Several recent reviews will give the reader an option for further reading (Bloemberg and Lugtenberg 2004; Chalfie and Kain 2005; Larrainzar et al. 2005; Rediers et al. 2005; Bloemberg and Camacho 2006).     

Identification of isolate-specific and partial resistance to septoria tritici blotch in 238 European wheat cultivars and breeding lines

From a total of 238 European cultivars and breeding lines screened for isolate-specific resistance to septoria tritici blotch (STB) with eight Mycosphaerella graminicola isolates from five different countries, 142 lines were resistant to Ethiopian isolate IPO88004, and 43 lines were specifically resistant to IPO323, with little or no leaf area bearing pycnidia of M. graminicola . These lines probably all have the resistance gene Stb6 . Specific resistances to isolates CA30JI, IPO001, IPO89011, IPO92006 and ISR398 were less common. Seventy-three per cent of the lines were specifically resistant to at least one isolate and 36 lines were resistant to more than one isolate. The line with the greatest number of specific resistances was the spring cultivar Raffles, with five. The most resistant line in which no specific resistance was identified was the Italian landrace Rieti, an ancestor of many modern European wheat cultivars. There was also a wide range of partial resistance among the lines tested, expressed in detached seedling leaves. Information about the resistance of wheat lines to M. graminicola isolates will assist breeders to choose parents of crosses from which progeny with superior resistance to STB may be selected.     

Comparative epidemiology of Pyrenopeziza brassicae (light leaf spot) ascospores and conidia from Polish and UK populations

Despite differences in climate and in timing of light leaf spot epidemics between Poland and the UK, experiments provided no evidence that there are epidemiological differences between populations of Pyrenopeziza brassicae in the two countries. Ascospores of Polish or UK P. brassicae isolates germinated on water agar at temperatures from 8 to 24°C. After 12 h of incubation, percentages of ascospores that germinated were greatest at 16°C: 85% (Polish isolates) and 86% (UK isolates). The percentage germination reached 100% after 80 h of incubation at all temperatures tested. The rate of increase in germ tube length increased with increasing temperature from 8 to 20°C but decreased from 20 to 24°C, for both Polish and UK isolates. Percentage germination and germ tube lengths of UK P. brassicae ascospores were less affected by temperature than those of conidia. P. brassicae produced conidia on oilseed rape leaves inoculated with ascospores or conidia of Polish or UK isolates at 16°C with leaf wetness durations from 6 to 72 h, with most sporulation after 48 or 72 h wetness. Detection of both mating types of P. brassicae and production of mature apothecia on leaves inoculated with mixed Polish populations suggest that sexual reproduction does occur in Poland, as in the UK.     

The role of weeds in supporting biological diversity within crop fields*

Summary Weeds are major constraints on crop production, yet as part of the primary producers within farming systems, they may be important components of the agroecosystem. Using published literature, the role of weeds in arable systems for other above‐ground trophic levels are examined. In the UK, there is evidence that weed flora have changed over the past century, with some species declining in abundance, whereas others have increased. There is also some evidence for a decline in the size of arable weed seedbanks. Some of these changes reflect improved agricultural efficiency, changes to more winter‐sown crops in arable rotations and the use of more broad‐spectrum herbicide combinations. Interrogation of a database of records of phytophagous insects associated with plant species in the UK reveals that many arable weed species support a high diversity of insect species. Reductions in abundances of host plants may affect associated insects and other taxa. A number of insect groups and farmland birds have shown marked population declines over the past 30 years. Correlational studies indicate that many of these declines are associated with changes in agricultural practices. Certainly reductions in food availability in winter and for nestling birds in spring are implicated in the declines of several bird species, notably the grey partridge, Perdix perdix. Thus weeds have a role within agroecosystems in supporting biodiversity more generally. An understanding of weed competitivity and the importance of weeds for insects and birds may allow the identification of the most important weed species. This may form the first step in balancing the needs for weed control with the requirements for biodiversity and more sustainable production methods.     

Characterization of Venturia inaequalis pathogenicity on leaf discs of apple trees

Characterization of pathogenicity on whole plants is required to study host-pathogen interactions between Malus × domestica and Venturia inaequalis. We studied the reliability of an in vitro test of pathogenicity on leaf discs. Three strains of V. inaequalis (races 1, 6 and an English race) were inoculated in vitro onto a range of 16 Malus sp. clones including susceptible and resistant clones. The results were compared to those previously obtained in vivo. Resistant clones contained the main major known genes, i.e. Va, Vb, Vbj, Vf, Vg, Vm and Vr. Scab severity and the sporulation of the fungus were assessed 21 days after the inoculation date. The results indicated that it was possible to reproduce incompatible and compatible situations in vitro. A null severity corresponded to the avirulence of the strain for the clone considered. The resistance given by the Vb, Vbj, Vf, Vg, Vm and Vr genes were expressed in vitro. Only the clone carrying the Va gene and inoculated with the race 6 strain presented a compatible situation which was inconsistent with the observations on the whole plant. Improving this test will facilitate studies on the pathogenicity of V. inaequalis populations in relation to resistance genes of the host expressed in vitro as well as its genetic determinism.     

基于引力和场强模型的干旱内陆河流域城乡体系相互作用及空间表达

综合运用遥感数据和相关统计资料,在对石羊河流域城乡生态环境、人口分布、经济发展深入考察研究的基础上,结合环境生态学、物理学、数理统计学、城市规划学、区域分析及可持续发展理论,通过构建城乡规模评价指标反映城乡规模及辐射范围,结合流域内部相互辐射、引力及场强模型建立遥感与GIS模型。利用引力模型分析城乡间联系的疏密程度和城镇在流域体系中的集聚能力,利用场强模型分析城镇间的辐射作用强度。结果表明:对于干旱内陆河流域区域分析研究,能充分利用到遥感和GIS技术进行空间定位和空间表达,以此建立研究区城镇规模评价指标体系,对其相互作用及联系的紧密程度可以进行直观可视化表达,从而使评价指标体系更为完善。在上述研究成果的基础上深入探讨关于石羊河流域城乡体系协调发展的整合模式,从而为石羊河流域生态环境综合治理、城乡一体化发展及经济社会协调可持续发展提供参考依据。     

High montmorillonite content may affect soil microbial proteomic analysis

We studied the effects of high montmorillonite content in soil on the proteomic analysis of Cupriavidus metallidurans CH34 inoculated into model soils, containing a montmorilonite gradient. Bacterial proteomic analysis was conducted by two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry. The results showed that increasing the montmorillonite content in artificial soil the bacterial viability did not affect but the amount of extracted proteins and the number of protein spots in 2-DE decreased. Higher soil montmorillonite content also affected the protein identification, likely due to montomrillonite-induced conformational changes in proteins or degradation. Therefore the development of soil proteomics needs to increase the studies of interaction between protein and soil components as clays or humic substances. This experiment showed how the use of a model study can be an help to achieve more information about the complexity of soil and the fate of proteins in soil.     

Development of microbial community during primary succession in areas degraded by mining activities

Together with plants, soil microbial communities play an essential role in the development of stable ecosystems on degraded lands, such as postmining spoil heaps. Our study addressed concurrent development of the vegetation and soil fungal and bacterial communities in the course of primary succession in a brown coal mine spoil deposit area in the Czech Republic across a chronosequence spanning 54 years. During succession, the plant communities changed from sparse plants over grassland and shrubland into a forest, becoming substantially more diverse with time. Microbial biomass increased until the 21st year of ecosystem development and later decreased. Although there was a close association between fungi and vegetation, with fungi mirroring the differences in plant community assemblages, the development of the bacterial community was different. The early succession community in the barren nonvegetated soil largely differed from that in the older sites, especially in its high abundance of autotrophic and free‐living N₂‐fixing bacteria. Later in succession, bacterial community changes were minor and reflected the chemical parameters of the soil, including pH, which also showed a minor change with time. Our results show that complex forest ecosystems developed over 54 years on the originally barren soil of the temperate zone and indicate an important role of bacteria in the initial stage of soil development. Although the arrival of vegetation affects substantially fungal as well as bacterial communities, it is mainly fungi that respond to the ongoing development of vegetation.     

CO2 and inorganic N supply modify competition for N between co-occurring grass plants, tree seedlings and soil microorganisms

Plant-plant and plant-soil interactions play a key role in determining plant community structure and ecosystem function. However, the effects of global change on the interplay between co-occurring plants and soil microbes in successional communities are poorly understood. In this study, we investigated competition for nitrogen (N) between soil microorganisms, grass plants and establishing tree seedlings under factorial carbon dioxide (CO₂) and N treatments. Fraxinus excelsior seedlings were germinated in the presence or absence of grass competition (Dactylis glomerata) at low (380 μmol mol⁻¹) or high (645 μmol mol⁻¹) CO₂ and at two levels of N nutrition in a mesocosm experiment. Pulse ¹⁵N labelling was used to examine N partitioning among plant and soil compartments. Dactylis exerted a strong negative effect on Fraxinus biomass, N capture and ¹⁵N recovery irrespective of N and CO₂ treatment. In contrast, the presence of Dactylis had a positive effect on the microbial N pool. Plant and soil responses to N treatment were of a greater magnitude compared with responses to elevated CO₂, but the pattern of Fraxinus- and microbial-N pool response to N and CO₂ varied depending on grass competition treatment. Within the Dactylis competition treatment, decreases in Fraxinus biomass in response to N were not mirrored by decreases in tree seedling N content, suggesting a shift from below- to above-ground competition. In the Dactylis-sown pots, ¹⁵N recovery could be ranked Dactylis > microbial pool > Fraxinus in all N and CO₂ treatment combinations. Inequalities between Fraxinus and soil microorganisms in terms of ¹⁵N recovery were exacerbated by N addition. Contrary to expectations, elevated CO₂ did not increase plant-microbe competition. Nevertheless, microbial ¹⁵N recovery showed a small positive increase in the high CO₂ treatment. Overall, elevated CO₂ and N supply did not interact on plant/soil N partitioning. Our data suggest that the competitive balance between establishing tree seedlings and grass plants in an undisturbed sward is relatively insensitive to CO₂ or N-induced modifications in N competition between plant and soil compartments.     

Effects of community-level grassland management on the non-target rare annual Agalinis auriculata

The objective of grassland management is to maintain keystone species, as well as species diversity, to promote a particular community structure, or to preserve specific ecosystem processes. Studies of grassland management rarely ascertain the effects on rare plant species, although conservation plans for rare herbaceous plants often recommend habitat management as well as restoration activities. Indeed, conservation biology has evolved from a relatively species-specific discipline into one more focused on larger scale issues such as ecosystem function, community composition and habitat restoration. Few studies have tested whether species of concern are adequately managed with a community or ecosystem-level approach. In this study, we evaluate whether community-level management of white-tailed deer and woody brush improves the viability of the rare annual plant species, Agalinis auriculata (Earleaf false foxglove; Orobanchaceae). Reducing deer browse and removing brush each positively affect plant vital rates. Brush removal increases the proportion of plants that reach the largest size class. Reducing deer browse increases the fertility of plants, particularly those in the largest size class. We report on demographic matrix models created with data from five populations of A. auriculata studied across 4 years. We find that both management activities positively affect the non-target plant species and are, in fact, necessary for A. auriculata to persist. Conservation of A. auriculata at our study sites requires both reducing the density of white-tailed deer and brush removal. Our study demonstrates that management at the community and ecosystem levels can be compatible with conservation goals at the species and population levels.