Massimiliano Cardinale

Comparison of Different Primer Sets for Use in Automated Ribosomal Intergenic Spacer Analysis of Complex Bacterial Communities

ABSTRACT ITSF and ITSReub, constituting a new primer set designed for the amplification of the 16S-23S rRNA intergenic transcribed spacers, have been compared with primer sets consisting of 1406F and 23Sr (M. M. Fisher and E. W. Triplett, Appl. Environ. Microbiol. 65:4630-4636, 1999) and S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 (L. Ranjard et al., Appl. Environ. Microbiol. 67:4479-4487, 2001), previously proposed for automated ribosomal intergenic spacer analysis (ARISA) of complex bacterial communities. An agricultural soil and a polluted soil, maize silage, goat milk, a small marble sample from the façade of the Certosa of Pavia (Pavia, Italy), and brine from a deep hypersaline anoxic basin in the Mediterranean Sea were analyzed with the three primer sets. The number of peaks in the ARISA profiles, the range of peak size (width of the profile), and the reproducibility of results were used as indices to evaluate the efficiency of the three primer sets. The overall data showed that ITSF and ITSReub generated the most informative (in term of peak number) and reproducible profiles and yielded a wider range of spacer sizes (134 to 1,387) than the other primer sets, which were limited in detecting long fragments. The minimum amount of DNA template and sensitivity in detection of minor DNA populations were evaluated with artificial mixtures of defined bacterial species. ITSF and ITSReub amplified all the bacteria at DNA template concentrations from 280 to 0.14 ng μl−1, while the other primer sets failed to detect the spacers of one or more bacterial strains. Although the primer set consisting of ITSF and ITSReub and that of S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 showed similar sensitivities for the DNA of Allorhizobium undicula mixed with the DNA of other species, the S-D-Bact-1522-b-S-20 and L-D-Bact-132-a-A-18 primer set failed to detect the DNA of Pseudomonas stutzeri.

The versatility and adaptation of bacteria from the genus Stenotrophomonas

The genus Stenotrophomonas comprises at least eight species. These bacteria are found throughout the environment, particularly in close association with plants. Strains of the most predominant species, Stenotrophomonas maltophilia, have an extraordinary range of activities that include beneficial effects for plant growth and health, the breakdown of natural and man-made pollutants that are central to bioremediation and phytoremediation strategies and the production of biomolecules of economic value, as well as detrimental effects, such as multidrug resistance, in human pathogenic strains. Here, we discuss the versatility of the bacteria in the genus Stenotrophomonas and the insight that comparative genomic analysis of clinical and endophytic isolates of S. maltophilia has brought to our understanding of the adaptation of this genus to various niches.

Species-specific structural and functional diversity of bacterial communities in lichen symbioses

Lichens are generally considered as mutualisms between fungi and green algae or cyanobacteria. These partnerships allow light-exposed and long-living joint structures. The unique organization of lichens provides still unexplored environments for microbial communities. To study lichen-associated bacterial communities, we analyze samples, by a polyphasic approach, from three lichen species (Cladonia arbuscula, Lecanora polytropa and Umbilicaria cylindrica) from alpine environments. Our results indicate that bacteria can form highly structured, biofilm-like assemblages on fungal surfaces and reach considerable abundances of up to 108 cells per gram fresh weight. Fluorescence in situ hybridization reveals the predominance of Alphaproteobacteria. Microbial fingerprints performed by PCR-single-strand conformation polymorphism analysis using universal and group-specific primers show distinct patterns for each lichen species. Characterization of cultivable strains and presence of functional genes in the total fraction suggest the involvement of associated bacteria in nutrient cycling. Ubiquitous nifH genes, which encode the nitrogenase reductase, show a high diversity and are assigned to Alphaproteobacteria and Firmicutes, for example, Paenibacillus. Cultivable strains mainly belonging to the genera Acinetobacter, Bacillus, Burkholderia, Methylobacterium and Paenibacillus show lytic (chitinolytic, glucanolytic, and proteolytic) activities, hormone production (indole-3-acetic acid) as well as phosphate mobilization and antagonistic activity toward other microorganisms. The traditional concept of lichens has to be expanded to consider multiple bacterial partners.

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae.

Structure and function of the symbiosis partners of the lung lichen (Lobaria pulmonaria L. Hoffm.) analyzed by metaproteomics

Environmental proteomics, also referred to as metaproteomics, is an emerging technology to study the structure and function of microbial communities. Here, we applied semi‐quantitative label‐free proteomics using one‐dimensional gel electrophoresis combined with LC‐MS/MS and normalized spectral counting together with fluorescence in situ hybridization and confocal laser scanning microscopy to characterize the metaproteome of the lung lichen symbiosis Lobaria pulmonaria. In addition to the myco‐ and photobiont, L. pulmonaria harbors proteins from a highly diverse prokaryotic community, which is dominated by Proteobacteria and including also Archaea. While fungal proteins are most dominant (75.4% of all assigned spectra), about the same amount of spectra were assigned to prokaryotic proteins (10%) and to the green algal photobiont (9%). While the latter proteins were found to be mainly associated with energy and carbohydrate metabolism, a major proportion of fungal and bacterial proteins appeared to be involved in PTMs and protein turnover and other diverse functions.

Sphagnum mosses harbour highly specific bacterial diversity during their whole lifecycle.

Knowledge about Sphagnum-associated microbial communities, their structure and their origin is important to understand and maintain climate-relevant Sphagnum-dominated bog ecosystems. We studied bacterial communities of two cosmopolitan Sphagnum species, which are well adapted to different abiotic parameters (Sphagnum magellanicum, which are strongly acidic and ombrotrophic, and Sphagnum fallax, which are weakly acidic and mesotrophic), in three Alpine bogs in Austria by a multifaceted approach. Great differences between bacterial fingerprints of both Sphagna were found independently from the site. This remarkable specificity was confirmed by a cloning and a deep sequencing approach. Besides the common Alphaproteobacteria, we found a discriminative spectrum of bacteria; although Gammaproteobacteria dominated S. magellanicum, S. fallax was mainly colonised by Verrucomicrobia and Planctomycetes. Using this information for fluorescent in situ hybridisation analyses, corresponding colonisation patterns for Alphaproteobacteria and Planctomycetes were detected. Bacterial colonies were found in high abundances inside the dead big hyalocytes, but they were always connected with the living chlorocytes. Using multivariate statistical analysis, the abiotic factors nutrient richness and pH were identified to modulate the composition of Sphagnum-specific bacterial communities. Interestingly, we found that the immense bacterial diversity was transferred via the sporophyte to the gametophyte, which can explain the high specificity of Sphagnum-associated bacteria over long distances. In contrast to higher plants, which acquire their bacteria mainly from the environment, mosses as the phylogenetically oldest land plants maintain their bacterial diversity within the whole lifecycle.

Culturable bacteria associated with Antarctic lichens: affiliation and psychrotolerance

Antarctic habitats harbour yet unexplored niches for microbial communities. Among these, lichen symbioses are very long-living and stable microenvironments for bacterial colonization. In this work, we present a first assessment of the culturable fraction of bacteria associated with Antarctic lichens. A phylogenetic analysis based on 16S rRNA gene sequence of 30 bacterial strains isolated from five epilithic lichens belonging to four species (Lecanora fuscobrunnea, Umbilicaria decussata, Usnea antarctica, Xanthoria elegans) shows that these represent the main bacterial lineages Actinobacteria, Firmicutes, Proteobacteria and Deinococcus-Thermus. Within the Actinomycetales, two strains group in the genera Arthrobacter and Knoellia, respectively. Most of the other Actinobacteria form well-supported groups, but could be assigned with certainty only at the family level, and one is in isolated position in the Mycobacteriaceae. The strains in Firmicutes and Proteobacteria belong to the genera Paenibacillus,Bacillus and Pseudomonas, which were already reported from lichen thalli. Some genera such as Burkholderia and Azotobacter, reported in the literature as also associated with lichens, have not been detected in this study. One strain represents the first record of Deinococcus in epilithic lichens; it is related to the species Deinococcus alpinitundrae from Alpine environments and may represent a new species. Further separated and well-supported clades indicate the presence of possibly new entities. Some of the examined strains are related to known psychrophilic bacteria isolated from ice and other extreme environments, others with bacteria distributed worldwide even in temperate climates. Most of the strains tested were able to grow at low temperatures, but tolerated a wider range of temperature. Ecological and evolutionary implications of these lichen-associated bacteria are discussed.

Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas in Egypt

Plant protection via disease-suppressive bacteria in desert farming requires specific biological control agents (BCAs) adapted to the unique arid conditions. We performed an ecological study of below-ground communities in desert farm soil and untreated desert soil, and based on these findings, selected antagonists were hierarchically evaluated. In contrast to the highly specific 16S rRNA fingerprints of bacterial communities in soil and cultivated medicinal plants, internal transcribed spacer profiles of fungal communities were less discriminative and mainly characterised by potential pathogens. Therefore, we focused on in vitro bacterial antagonists against pathogenic fungi. Based on the antifungal potential and genomic diversity, 45 unique strains were selected and characterised in detail. Bacillus/Paenibacillus were most frequently identified from agricultural soil, but antagonists from the surrounding desert soil mainly belonged to Streptomyces. All strains produced antibiotics against the nematode Meloidogyne incognita, and one-third showed additional activity against the bacterial pathogen Ralstonia solanacearum. Altogether, 13 broad-spectrum antagonists with antibacterial, antifungal and nematicidal activity were found. They belong to seven different bacterial species of the genera Bacillus and Streptomyces. These Gram-positive, spore-forming bacteria are promising drought-resistant BCAs and a potential source for antibiotics. Their rhizosphere competence was shown by fluorescence in situ hybridisation combined with laser scanning microscopy.

The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome.

Lettuce belongs to the most commonly raw eaten food worldwide and its microbiome plays an important role for both human and plant health. Yet, little is known about the impact of potentially occurring pathogens and beneficial inoculants of the indigenous microorganisms associated with lettuce. To address this question we studied the impact of the phytopathogenic fungus Rhizoctonia solani and the biological control agent Bacillus amyloliquefaciens FZB42 on the indigenous rhizosphere and phyllosphere community of greenhouse-grown lettuce at two plant stages. The rhizosphere and phyllosphere gammaproteobacterial microbiomes of lettuce plants showed clear differences in their overall and core microbiome composition as well as in corresponding diversity indices. The rhizosphere was dominated by Xanthomonadaceae (48%) and Pseudomonadaceae (37%) with Rhodanobacter, Pseudoxanthomonas, Dokdonella, Luteimonas, Steroidobacter, Thermomonas as core inhabitants, while the dominating taxa associated to phyllosphere were Pseudomonadaceae (54%), Moraxellaceae (16%) and Enterobacteriaceae (25%) with Alkanindiges, Pantoea and a group of Enterobacteriaceae unclassified at genus level. The preferential occurrence of enterics in the phyllosphere was the most significant difference between both habitats. Additional enhancement of enterics on the phyllosphere was observed in bottom rot diseased lettuce plants, while Acinetobacter and Alkanindiges were identified as indicators of healthy plants. Interestingly, the microbial diversity was enhanced by treatment with both the pathogen, and the co-inoculated biological control agent. The highest impact and bacterial diversity was found by Rhizoctonia inoculation, but FZB42 lowered the impact of Rhizoctonia on the microbiome. This study shows that the indigenous microbiome shifts as a consequence to pathogen attack but FZB42 can compensate these effects, which supports their role as biocontrol agent and suggests a novel mode of action.

Bacterial networks and co-occurrence relationships in the lettuce root microbiota

Lettuce is one of the most common raw foods worldwide, but occasionally also involved in pathogen outbreaks. To understand the correlative structure of the bacterial community as a network, we studied root microbiota of eight ancient and modern Lactuca sativa cultivars and the wild ancestor Lactuca serriola by pyrosequencing of 16S rRNA gene amplicon libraries. The lettuce microbiota was dominated by Proteobacteria and Bacteriodetes, as well as abundant Chloroflexi and Actinobacteria. Cultivar specificity comprised 12.5% of the species. Diversity indices were not different between lettuce cultivar groups but higher than in L. serriola, suggesting that domestication lead to bacterial diversification in lettuce root system. Spearman correlations between operational taxonomic units (OTUs) showed that co-occurrence prevailed over co-exclusion, and complementary fluorescence in situ hybridization-confocal laser scanning microscopy (FISH-CLSM) analyses revealed that this pattern results from both potential interactions and habitat sharing. Predominant taxa, such as Pseudomonas, Flavobacterium and Sphingomonadaceae rather suggested interactions, even though these are not necessarily part of significant modules in the co-occurrence networks. Without any need for complex interactions, single organisms are able to invade into this microbial network and to colonize lettuce plants, a fact that can influence the susceptibility to pathogens. The approach to combine co-occurrence analysis and FISH-CLSM allows reliably reconstructing and interpreting microbial interaction networks.