Jan Dirk van Elsas

Diversity of Endophytic Bacterial Populations and Their Interaction with Xylella fastidiosa in Citrus Plants

ABSTRACT Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.

Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis.

Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then amplified by PCR with bacterial primers, resulting in a mixture of amplicons separable via denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of FSL soil were indicative of dominant soil bacterial types, as evidenced by assessing the amplification of Enterobacter cloacae and Arthrobacter sp. targets in a soil DNA background. These targets produced barely detectable bands when present in soil DNA at roughly 5 x 10(6) genome equivalents per g dry soil, and strong bands at 27-fold higher levels. The PCR-DGGE analysis of the FSL soil was highly reproducible. Furthermore, different single versus composite topsoil samples yielded similar DGGE profiles with respect to major bands. In addition, samples taken along vertical soil cores (0-45 cm depth) revealed relative stability of the DGGE profiles. The profiles produced with DNA obtained from different aggregate size fractions of this soil were also similar with respect to the main bands. Moreover, FSL topsoil samples taken over a 1-year period (fallow soil) yielded stable profiles. These data suggested that the soil bacterial communities thus determined were dominated by a limited number of stable and ubiquitous types. The 16 soils, representing varying types and geographical locations, were assessed for differences in their bacterial DGGE profiles. There were striking differences between the profiles obtained for these soils. Evidence was found for the hypothesis that similar soil types tend to contain similar structures of the dominating bacterial types as revealed by the DGGE profiles.

Analysis of Bacterial Communities in the Rhizosphere of Chrysanthemum via Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA as Well as DNA Fragments Coding for 16S rRNA

ABSTRACT The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such asPseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root.

Microbial diversity determines the invasion of soil by a bacterial pathogen

Natural ecosystems show variable resistance to invasion by alien species, and this resistance can relate to the species diversity in the system. In soil, microorganisms are key components that determine life support functions, but the functional redundancy in the microbiota of most soils has long been thought to overwhelm microbial diversity–function relationships. We here show an inverse relationship between soil microbial diversity and survival of the invading species Escherichia coli O157:H7, assessed by using the marked derivative strain T. The invaders fate in soil was determined in the presence of (i) differentially constructed culturable bacterial communities, and (ii) microbial communities established using a dilution-to-extinction approach. Both approaches revealed a negative correlation between the diversity of the soil microbiota and survival of the invader. The relationship could be explained by a decrease in the competitive ability of the invader in species-rich vs. species-poor bacterial communities, reflected in the amount of resources used and the rate of their consumption. Soil microbial diversity is a key factor that controls the extent to which bacterial invaders can establish.

Survival of Escherichia coli in the environment: fundamental and public health aspects.

In this review, our current understanding of the species Escherichia coli and its persistence in the open environment is examined. E. coli consists of six different subgroups, which are separable by genomic analyses. Strains within each subgroup occupy various ecological niches, and can be broadly characterized by either commensalistic or different pathogenic behaviour. In relevant cases, genomic islands can be pinpointed that underpin the behaviour. Thus, genomic islands of, on the one hand, broad environmental significance, and, on the other hand, virulence, are highlighted in the context of E. coli survival in its niches. A focus is further placed on experimental studies on the survival of the different types of E. coli in soil, manure and water. Overall, the data suggest that E. coli can persist, for varying periods of time, in such terrestrial and aquatic habitats. In particular, the considerable persistence of the pathogenic E. coli O157:H7 is of importance, as its acid tolerance may be expected to confer a fitness asset in the more acidic environments. In this context, the extent to which E. coli interacts with its human/animal host and the organisms survivability in natural environments are compared. In addition, the effect of the diversity and community structure of the indigenous microbiota on the fate of invading E. coli populations in the open environment is discussed. Such a relationship is of importance to our knowledge of both public and environmental health.

Effects of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L)

The effects of genotype, plant growth and experimental factors (soil and year) on potato-associated bacterial communities were studied. Cultivars Achirana Inta, Désirée, Merkur and transgenic Désirée line DL12 (containing T4 lysozyme gene) were assessed in two field experiments. Cross-comparisons between both experiments were made using Désirée plants. Culture-dependent and -independent approaches were used to demonstrate effects on total bacterial, actinobacterial and Pseudomonas communities in bulk and rhizosphere soils and endospheres. PCR-denaturing gradient gel electrophoresis fingerprints prepared with group-specific primers were analyzed using multivariate analyses and revealed that bacterial communities in Achirana Inta plants differed most from those of Désirée and Merkur. No significant effects were found between Désirée and DL12 lines. Plant growth stage strongly affected different plant-associated communities in both experiments. To investigate the effect of plant-associated communities on plant health, 800 isolates from rhizospheres and endospheres at the flowering stage were tested for suppression of Ralstonia solanacearum biovar 2 and/or Rhizoctonia solani AG3. A group of isolates closely resembling Lysobacter sp. dominated in young plants. Its prevalence was affected by plant growth stage and experiment rather than by plant genotype. It was concluded that plant growth stage overwhelmed any effect of plant genotype on the bacterial communities associated with potato.

Structural diversity of microorganisms in chemically perturbed soil assessed by molecular and cytochemical approaches.

Until recently, our understanding of microbial community development in soil ecosystems exposed to different inorganic and organic pollutants has been limited to culturable microorganisms because of the techniques available. The discovery that most soil microorganisms are non-culturable but potentially viable and metabolically active accelerated the application of different culture-independent methods for structural diversity assessments of the microbial community. This review examines the results of recent studies on the impact of heavy metals and organic pollutants on the diversity of the microflora obtained with methods based on analyses of signature biomarkers such as nucleic acids and fatty acids. The application of these techniques allowed researchers to pinpoint reduction of microbial diversity in contaminated soil, and significant shifts in the community structure, leading to the dominance of only a few populations (species) and the disappearance of others, some of which were never isolated by conventional methods (e.g. an increase in Acidobacterium or a decrease in terrestrial non-thermophilic Crenarchaeota). Although the new techniques are not free from limitations, they allow the monitoring of the virtual impact of stressors on soil microorganisms and the direction of resuscitation of the microbial community during natural or induced bioremediation, especially when using combined approaches.

Effects of agronomical measures on the microbial diversity of soils as related to the suppression of soil-borne plant pathogens.

The diversity of soil microbial communities can be key to the capacity of soils tosuppress soil-borne plant diseases. As agricultural practice, as well as directedagronomical measures, are known to be able to affect soil microbial diversity, it isplausible that the soil microflora can be geared towards a greater suppressivity ofsoil-borne diseases as a result of the selection of suitable soil management regimes.In the context of a programme aimed at investigating the microbial diversity of soilsunder different agricultural regimes, including permanent grassland versus arableland under agricultural rotation, we assessed how soil microbial diversity is affectedin relation to the suppression of the soil-borne potato pathogen Rhizoctoniasolani AG3. The diversity in the microbial communities over about a growingseason was described by using cultivation-based – plating on different media – and cultivation-independent – soil DNA-based PCR followed by denaturing gradient gel electrophoresis (DGGE) community fingerprinting – methods. The results showed great diversity in the soil microbiota at both the culturable and cultivation-independent detection levels. Using cultivation methods, various differences between treatments with respect to sizes of bacterial and fungal populations were detected, with highest population sizes generally found in rhizospheres. In addition, the evenness of eco-physiologically differing bacterial types was higher in grassland than in arable land under rotation. At the cultivation-independent level, clear differences in the diversities of several microbial groups between permanent grassland and arable land under rotation were apparent. Bio-assays that assessed the growth of R. solani AG3 hyphae through soil indicated a greater growth suppression in grassland than in arable land soils. Similarly, an experiment performed in the glasshouse showed clear differences in both microbial diversities and suppressiveness of R. solani growth in soil, depending on the presence of either maizeor oats as the crop. The significance of these findings for designing soil managementstrategies is discussed.

Multivariate analyses of Burkholderia species in soil: Effect of crop and land use history

ABSTRACT The assessment of Burkholderia diversity in agricultural areas is important considering the potential use of this genus for agronomic and environmental applications. Therefore, the aim of this work was to ascertain how plant species and land use management drive the diversity of the genus Burkholderia. In a greenhouse experiment, different crops, i.e., maize, oat, barley, and grass, were planted in pots containing soils with different land use histories, i.e., maize monoculture, crop rotation, and permanent grassland, for three consecutive growth cycles. The diversity of Burkholderia spp. in the rhizosphere soil was assessed by genus-specific PCR-denaturing gradient gel electrophoresis (DGGE) and analyzed by canonical correspondence analysis (CCA). CCA ordination plots showed that previous land use was the main factor affecting the composition of the Burkholderia community. Although most variation in the Burkholderia community structure was observed between the permanent grassland and agricultural areas, differences between the crop rotation and maize monoculture groups were also observed. Plant species affected Burkholderia community structure to a lesser extent than did land use history. Similarities were observed between Burkholderia populations associated with maize and grass, on the one hand, and between those associated with barley and oat, on the other hand. Additionally, CCA ordination plots demonstrated that these two groups (maize/grass versus barley/oat) had a negative correlation. The identification of bands from the DGGE patterns demonstrated that the species correlated with the environmental variables were mainly affiliated with Burkholderia species that are commonly isolated from soil, in particular Burkholderia glathei, B. caledonica, B. hospita, and B. caribiensis.

Mangrove bacterial diversity and the impact of oil contamination revealed by pyrosequencing: Bacterial proxies for oil pollution

Background Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills. Methodology/Principal Findings A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings. Conclusions/Significance We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest.