Angela Sessitsch

Microbial population structures in soil particle size fractions of a long-term fertilizer field experiment.

ABSTRACT Soil structure depends on the association between mineral soil particles (sand, silt, and clay) and organic matter, in which aggregates of different size and stability are formed. Although the chemistry of organic materials, total microbial biomass, and different enzyme activities in different soil particle size fractions have been well studied, little information is available on the structure of microbial populations in microhabitats. In this study, topsoil samples of different fertilizer treatments of a long-term field experiment were analyzed. Size fractions of 200 to 63 μm (fine sand fraction), 63 to 2 μm (silt fraction), and 2 to 0.1 μm (clay fraction) were obtained by a combination of low-energy sonication, wet sieving, and repeated centrifugation. Terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes were used to compare bacterial community structures in different particle size fractions. The microbial community structure was significantly affected by particle size, yielding higher diversity of microbes in small size fractions than in coarse size fractions. The higher biomass previously found in silt and clay fractions could be attributed to higher diversity rather than to better colonization of particular species. Low nutrient availability, protozoan grazing, and competition with fungal organisms may have been responsible for reduced diversities in larger size fractions. Furthermore, larger particle sizes were dominated by α-Proteobacteria, whereas high abundance and diversity of bacteria belonging to the Holophaga/Acidobacteriumdivision were found in smaller size fractions. Although very contrasting organic amendments (green manure, animal manure, sewage sludge, and peat) were examined, our results demonstrated that the bacterial community structure was affected to a greater extent by the particle size fraction than by the kind of fertilizer applied. Therefore, our results demonstrate specific microbe-particle associations that are affected to only a small extent by external factors.

Endophytic colonization of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN.

ABSTRACT Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.

Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense

ABSTRACT Thlaspi goesingense is able to hyperaccumulate extremely high concentrations of Ni when grown in ultramafic soils. Recently it has been shown that rhizosphere bacteria may increase the heavy metal concentrations in hyperaccumulator plants significantly, whereas the role of endophytes has not been investigated yet. In this study the rhizosphere and shoot-associated (endophytic) bacteria colonizing T. goesingense were characterized in detail by using both cultivation and cultivation-independent techniques. Bacteria were identified by 16S rRNA sequence analysis, and isolates were further characterized regarding characteristics that may be relevant for a beneficial plant-microbe interaction—Ni tolerance, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and siderophore production. In the rhizosphere a high percentage of bacteria belonging to the Holophaga/Acidobacterium division and α-Proteobacteria were found. In addition, high-G+C gram-positive bacteria, Verrucomicrobia, and microbes of the Cytophaga/Flexibacter/Bacteroides division colonized the rhizosphere. The community structure of shoot-associated bacteria was highly different. The majority of clones affiliated with the Proteobacteria, but also bacteria belonging to the Cytophaga/Flexibacter/Bacteroides division, the Holophaga/Acidobacterium division, and the low-G+C gram-positive bacteria, were frequently found. A high number of highly related Sphingomonas 16S rRNA gene sequences were detected, which were also obtained by the cultivation of endophytes. Rhizosphere isolates belonged mainly to the genera Methylobacterium, Rhodococcus, and Okibacterium, whereas the majority of endophytes showed high levels of similarity to Methylobacterium mesophilicum. Additionally, Sphingomonas spp. were abundant. Isolates were resistant to Ni concentrations between 5 and 12 mM; however, endophytes generally tolerated higher Ni levels than rhizosphere bacteria. Almost all bacteria were able to produce siderophores. Various strains, particularly endophytes, were able to grow on ACC as the sole nitrogen source.

The Hidden World within Plants: Ecological and Evolutionary Considerations for Defining Functioning of Microbial Endophytes

SUMMARY All plants are inhabited internally by diverse microbial communities comprising bacterial, archaeal, fungal, and protistic taxa. These microorganisms showing endophytic lifestyles play crucial roles in plant development, growth, fitness, and diversification. The increasing awareness of and information on endophytes provide insight into the complexity of the plant microbiome. The nature of plant-endophyte interactions ranges from mutualism to pathogenicity. This depends on a set of abiotic and biotic factors, including the genotypes of plants and microbes, environmental conditions, and the dynamic network of interactions within the plant biome. In this review, we address the concept of endophytism, considering the latest insights into evolution, plant ecosystem functioning, and multipartite interactions.

Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

ß-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria.

Summary: A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes

Abstract Endophytic bacteria are ubiquitous in most plants and colonise plants without exhibiting pathogenicity. Studies on the diversity of bacterial endophytes have been mainly approached by characterisation of isolates obtained from internal tissues. Despite the broad application of culture-independent techniques for the analysis of microbial communities in a wide range of natural habitats, little information is available on the species diversity of endophytes. In this study, microbial communities inhabiting stems, roots and tubers of three potato varieties were analysed by 16S rRNA-based techniques such as terminal restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis as well as 16S rDNA cloning and sequencing. Two individual plant experiments were conducted. In the first experiment plants suffered from light deficiency, whereas healthy and robust plants were obtained in the second experiment. Plants obtained from both experiments showed comparable endophytic populations, but healthy potato plants possessed a significantly higher diversity of endophytes than stressed plants. In addition, plant tissue and variety specific endophytes were detected. Sequence analysis of 16S rRNA genes indicated that a broad phylogenetic spectrum of bacteria is able to colonise plants internally including alpha-, beta-, and gamma-Proteobacteria, high-GC Gram-positives, microbes belonging to the Flexibacter/Cytophaga/Bacteroides group and Planctomycetales. Group-specific analysis of Actinomycetes indicated a higher abundance and diversity of Streptomyces scabiei-related species in the variety Mehlige Mühlviertler, which is known for its resistance against potato common scab caused by S. scabiei.

Climate change effects on beneficial plant–microorganism interactions

It is well known that beneficial plant-associated microorganisms may stimulate plant growth and enhance resistance to disease and abiotic stresses. The effects of climate change factors such as elevated CO(2), drought and warming on beneficial plant-microorganism interactions are increasingly being explored. This now makes it possible to test whether some general patterns occur and whether different groups of plant-associated microorganisms respond differently or in the same way to climate change. Here, we review the results of 135 studies investigating the effects of climate change factors on beneficial microorganisms and their interaction with host plants. The majority of studies showed that elevated CO(2) had a positive influence on the abundance of arbuscular and ectomycorrhizal fungi, whereas the effects on plant growth-promoting bacteria and endophytic fungi were more variable. In most cases, plant-associated microorganisms had a beneficial effect on plants under elevated CO(2). The effects of increased temperature on beneficial plant-associated microorganisms were more variable, positive and neutral, and negative effects were equally common and varied considerably with the study system and the temperature range investigated. Moreover, numerous studies indicated that plant growth-promoting microorganisms (both bacteria and fungi) positively affected plants subjected to drought stress. Overall, this review shows that plant-associated microorganisms are an important factor influencing the response of plants to climate change.

Advances in Rhizobium Research

Referee: Prof. Dr. Dietrich Werner, FG Zellbiologie und Angewandte Botanik, Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-35032 Marburg, Germany Rhizobia are well known for their capacity to establish a symbiosis with legumes. They inhabit root nodules, where they reduce atmospheric nitrogen and make it available to the plant. Biological nitrogen fixation is an important component of sustainable agriculture, and rhizobial inoculants have been applied frequently as biofertilizers. In this review we present recently developed technologies and strategies for selecting quality inoculant strains by taking into consideration the complex interaction between the edaphic environment with the genotypes of both the legume and its microsymbiont. Enhanced competitive ability in an inoculant strain is a key requirement for successful colonization of plant roots, nodule formation, and subsequent N2-fixation. We discuss several avenues for the management and manipulation of rhizobial competition as well as genes that influence competition in the rhizosphere. The use of molecular techniques has greatly contributed to our knowledge of nodule-bacterial diversity and phylogeny. Approaches to the study of rhizobial diversity as well as mechanisms for the evolutionary diversification of nodulating bacteria are presented. Rhizobium genomes ranging from 5.5 to 9 Mb have been sequenced recently and deposited in public databases. A comparison of sequence data has led to a better understanding of genes involved in the symbiotic process as well as possible mechanisms responsible for horizontal transfer of genetic elements and symbiosis genes among rhizobia. Furthermore, rhizobia are frequent rhizosphere colonizers of a wide range of plants and may also inhabit nonleguminous plants endophytically. In these rhizospheric and endophytic habitats they may exhibit several plant growth-promoting effects, such as hormone production, phosphate solubilization, and the suppression of pathogens.

Comparison of Diversities and Compositions of Bacterial Populations Inhabiting Natural Forest Soils

ABSTRACT The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the α-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the α-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.