Distinct changes in tomato-associated multi-kingdom microbiomes during Meloidogyne incognita parasitism.

BACKGROUND: The interplay between root-knot nematode (RKN) parasitism and the complex web of host-associated microbiota has been recognized as pivotal for effective management of the pest. However, studies assessing this relationship have focussed on the bacterial and fungal communities, neglecting the unicellular eukaryotic members. Here, we employed amplicon sequencing analysis of the bacterial 16S rRNA, fungal ITS and eukaryotic 18S rRNA genes, and comprehensively examined how the microbiome composition, diversity and networking developed with time in the rhizospheres and roots of RKN-inoculated and non-inoculated tomato plants. RESULTS: As expected, infection with the RKN Meloidogyne incognita decreased plant growth. At individual timepoints, we found distinct bacterial, fungal and eukaryote community structures in the RKN-inoculated and non-inoculated rhizospheres and roots, and RKN inoculation affected several taxa in the root-associated microbiome differentially. Correlation analysis revealed several bacterial and fungal and few protist taxa that correlated negatively or positively with M. incognita. Moreover, network analysis using bacterial, fungal and eukaryotic data revealed more dynamic networks with higher robustness to disturbances in the RKN-inoculated than in the non-inoculated rhizospheres/roots. Hub taxa displayed a noticeable successional pattern that coincided with different phases of M. incognita parasitism. We found that fungal hubs had strong negative correlations with bacteria and eukaryotes, while positive correlations characterized hub members within individual kingdoms. CONCLUSION: Our results reveal dynamic tomato-associated microbiomes that develop along different trajectories in plants suffering M. incognita infestation and non-infested plants. Overall, the results identify stronger associations between RKN and bacterial and fungal taxa than between eukaryotic taxa and RKN, suggesting that fungal and bacterial communities could play a larger role in the regulation of RKN. The study identifies several putative RKN-antagonistic bacterial and fungal taxa and confirms the antagonistic potential previously identified in other taxa.

A field study reveals links between hyperaccumulating Sedum plants-associated bacterial communities and Cd/Zn uptake and translocation.

Hyperaccumulating ecotypes of Sedum plants are promising Cd/Zn phytoextractors, with potential for leveraging its rhizospheric or endophytic microbiomes to improve phytoremediation efficiency. However, research of bacteria associated with Sedum at field scale is still lacking. Here, we presented a detailed investigation of the bacterial microbiome of hyperaccumulating Sedum ecotypes (S. alfredii and S. plumbizincicola) and a non-hyperaccumulating S. alfredii ecotype, which grow at different soil environments. Moreover, we evaluated the heavy metal uptake and translocation potential of Sedum plants at different locations. The results showed that both HE ecotypes, contrary to the NHE, were efficient for phytoremediation in mine areas and farmlands. For NHE plants, rhizosphere co-occurrence networks were more complex than the networks of other compartments, while networks of HE plants were more complex in bulk soil and roots. The proportion of positive correlations within co-occurrence networks was higher for the HE plants, suggesting a greater potential for mutualistic interactions. Plant compartment and location predominantly shaped the microbiome assembly, and Proteobacteria, Actinobacteria and Acidobacteria dominated the bacterial communities of Sedum plants. Keystone taxa related to Zn hyperaccumulation are similar to those related to Cd hyperaccumulation, and nine bacterial genera had significantly positive correlation with Cd/Zn hyperaccumulation. Taxa, linked to phytoremediation in both mine and farmland (i.e. Actinospica and Streptomyces from Actinobacteria), could be targets for further investigation of their ability to promote metal phytoremediation of Sedum species.

Land use as a driver for protist community structure in soils under agricultural use across Europe.

Soil biodiversity is threatened by intensification of land use. The consequences of different land use on belowground biodiversity remain insufficiently explored for soil protists. Alongside being abundant and extremely diverse in soil, protists provide many ecosystem services: key players in the microbial loop, turnover of organic matter and stimulation of plant growth-promoting rhizobacteria. However, we lack knowledge of effects of site, land use intensity and management on diversity of soil protists. Here we assessed protist communities in four European arable sites with contrasting land use intensities at each site: Lusignan, France; Moskanjci, Slovenia; Castro Verde, Portugal and Scheyern, Germany as well as two grassland sites: Hainich, Germany and Lancaster, UK. Each site has consistent agricultural management history of low and high land use intensities quantified in terms of land use index (LUI). We employed high-throughput sequencing of environmental DNA, targeting the V4 region of the 18S rRNA gene. By assigning the protist composition to trophic groups, we inspected for effects of management, and other biotic and abiotic variables. While overall protist richness was unaffected by LUI within sites, specific trophic groups such as plant pathogens and saprotrophs were affected. Effects on protist biome across land uses and sites were also observed. LUI sensitive taxa were taxonomically diverse in each plot, and their trophic groups responded in specific patterns to specific practices. The most abundant trophic group was phagotrophs (73%), followed by photoautotrophs (16%), plant pathogens (4%), animal parasites (2%) and saprotrophs (1%). Community compositions and factors affecting the structure of individual trophic groups differed between land uses and management systems. The agricultural management selected for distinct protist populations as well as specific functional traits, and the protist community and diversity were indeed affected by site, LUI and management, which indicates the ecological significance of protists in the soil food web.

Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome.

Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays.

Effects of Bacillus cereus Endospores on Free-Living Protist Growth.

We studied the predator-prey interactions between heterotrophic protists and endospores of Bacillus cereus group bacteria, in order to gain insight on survival and dispersal of B. cereus endospores in the environment. It has been hypothesised that the spore stage protects against digestion by predating protists. Therefore, experiments were carried out to investigate the impact of B. cereus endospores and vegetative cells, as the only food source, on individual amoeboid, flagellated and ciliated protists. The presence of fluorescent-labelled intracellular bacteria confirmed that B. cereus endospores as well as vegetative cells were ingested by protists and appeared intact in the food vacuoles when observed by epifluorescence microscopy. Furthermore, protist growth and bacterial predation were followed by qPCR. Protists were able to grow on vegetative cells as well as endospores of B. cereus, despite the lower cell division rates observed for some protists when feeding on bacterial endospores. Survival and proliferation of ingested bacteria inside protists cells was also observed. Finally, B. cereus spore germination and growth was observed within all protists with higher abundance in the amoeboid protist after antibiotic treatment of the protist surface. These observations support that protists can act as a potential breeding ground for B. cereus endospores.

Metagenomes provide valuable comparative information on soil microeukaryotes.

Despite the critical ecological roles of microeukaryotes in terrestrial ecosystems, most descriptive studies of soil microbes published so far focused only on specific groups. Meanwhile, the fast development of metagenome sequencing leads to considerable data accumulation in public repositories, providing microbiologists with substantial amounts of accessible information. We took advantage of public metagenomes in order to investigate microeukaryote communities in a well characterized grassland soil. The data gathered allowed the evaluation of several factors impacting the community structure, including the DNA extraction method, the database choice and also the annotation procedure. While most studies on soil microeukaryotes are based on sequencing of PCR-amplified taxonomic markers (18S rRNA genes, ITS regions), this work represents, to our knowledge, the first report based solely on metagenomic microeukaryote DNA. Choosing the correct annotation procedure and reference database has proven to be crucial, as it considerably limits the risk of wrong assignments. In addition, a significant and pronounced effect of the DNA extraction method on the taxonomical structure of soil microeukaryotes has been identified. Our analyses suggest that publicly available metagenome data can provide valuable information on soil microeukaryotes for comparative purposes when handled appropriately, complementing the current view provided by ribosomal amplicon sequencing methods.

Comparison of three DNA extraction methods for recovery of soil protist DNA.

The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism.

Nematicidal bacteria associated to pinewood nematode produce extracellular proteases.

Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion.

Diversity of cloacal microbial community in migratory shorebirds that use the Tagus estuary as stopover habitat and their potential to harbor and disperse pathogenic microorganisms.

The diversity of the cloacal microbial community in migratory shorebirds, caught at the Tagus estuary, Portugal, was assessed by cultivation (R2A and Nutrient Agar media) and denaturing gradient gel electrophoresis profiling (DGGE) to provide a better understanding of the birds' potential to harbor and disperse pathogens. Three different bird species belonging to four different populations were studied: common redshank (Tringa totanus), black-winged stilt (Himantopus himantopus) and nominate and Icelandic populations of black-tailed godwit (Limosa limosa). DGGE profiling and partial 16S RNA gene sequences of 240 isolates, and 26 DGGE bands resulting in 58 clones, were analyzed. Most isolates were members of the phylum Firmicutes and Actinobacteria and only a small portion belonged to the Proteobacteria and Deinococcus-Thermus phyla. Potentially pathogenic strains carried by the birds were found such as Helicobacter and Staphylococcus in all bird species, and Clostridium, Mycobacterium, Rhodococcus, Legionella and Corynebacterium in black-winged stilts. Unexpectedly, bacteria from the phylum Deinococcus-Thermus were isolated in shorebirds and were present in all the bird species studied.