Circulation of Shiga Toxin-Producing Escherichia coli Phylogenetic Group B1 Strains Between Calve Stable Manure and Pasture Land With Grazing Heifers.

Escherichia coli strains carrying Shiga toxins 1 and 2 (stx 1 and stx 2), intimin (eae), and hemolysin (ehxA) production genes were found in grass shoot, rhizosphere soil, and stable manure samples from a small-scale cattle farm located at the center of Netherlands, using cultivation-dependent and -independent microbiological detection techniques. Pasture land with grazing heifers in the first year of sampling in 2014 and without grazing cattle in 2015 was physically separated from the stable that housed rose calves during both years. Manure from the stable was applied to pasture via injection into soil once per year in early spring. Among a variety of 35 phylogenetic distinctly related E. coli strains, one large group consisting of 21 closely resembling E. coli O150:H2 (18), O98:H21 (2), and O84:H2 (1) strains, all belonging to phylogenetic group B1 and carrying all screened virulence traits, was found present on grass shoots (10), rhizosphere soil (3), and stable manure (8) in 2014, but not anymore in 2015 when grazing heifers were absent. Presence and absence of these strains, obtained via enrichments, were confirmed via molecular detection using PCR-NALFIA in all ecosystems in both years. We propose that this group of Shiga toxin-producing E. coli phylogenetic group B1 strains was originally introduced via stable manure injection into the pasture. Upon grazing, these potential pathogens proliferated in the intestinal track systems of the heifers resulting in defecation with higher loads of the STEC strain onto the grass cover. The STEC strain was further smeared over the field via the hooves of the heifers resulting in augmentation of the potential pathogen in the pasture in 2014, whereas in 2015, in the absence of heifers, no augmentation occurred and only a more diverse group of potentially mild virulent E. coli phylogenetic group A and B1 strains, indigenous to pasture plants, remained present. Via this model, it was postulated that human pathogens can circulate between plants and farm animals, using the plant as an alternative ecosystem. These data indicate that grazed pasture must be considered as a potential carrier of human pathogenic E. coli strains and possibly also of other pathogens.

Impact of Bacterial-Fungal Interactions on the Colonization of the Endosphere.

Research on different endophyte taxa and the related scientific disciplines have largely developed separately, and comprehensive community-level studies on bacterial and fungal interactions and their importance are lacking. Here, we discuss the transmission modes of bacteria and fungi and the nature of their interactions in the endosphere at both the molecular and physiological level. Mixed-community biofilms in the endosphere may have a role in protecting endophytes against encountered stresses, such as from plant defense systems. However, transmission from static (in biofilms) to free-living (planktonic) forms may be crucial for the exploration of new habitable spaces in plants. Important features previously recognized as plant-microbe interactions or antagonism in endophyte genomes and metagenomes are proposed to have essential roles in the modulation of endophyte communities.

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

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.

The arable ecosystem as battleground for emergence of new human pathogens.

Disease incidences related to Escherichia coli and Salmonella enterica infections by consumption of (fresh) vegetables, sprouts, and occasionally fruits made clear that these pathogens are not only transmitted to humans via the "classical" routes of meat, eggs, and dairy products, but also can be transmitted to humans via plants or products derived from plants. Nowadays, it is of major concern that these human pathogens, especially the ones belonging to the taxonomical family of Enterobacteriaceae, become adapted to environmental habitats without losing their virulence to humans. Adaptation to the plant environment would lead to longer persistence in plants, increasing their chances on transmission to humans via consumption of plant-derived food. One of the mechanisms of adaptation to the plant environment in human pathogens, proposed in this paper, is horizontal transfer of genes from different microbial communities present in the arable ecosystem, like the ones originating from soil, animal digestive track systems (manure), water and plants themselves. Genes that would confer better adaptation to the phytosphere might be genes involved in plant colonization, stress resistance and nutrient acquisition and utilization. Because human pathogenic enterics often were prone to genetic exchanges via phages and conjugative plasmids, it was postulated that these genetic elements may be hold key responsible for horizontal gene transfers between human pathogens and indigenous microbes in agroproduction systems. In analogy to zoonosis, we coin the term phytonosis for a human pathogen that is transmitted via plants and not exclusively via animals.

Rodent species as natural reservoirs of Borrelia burgdorferi sensu lato in different habitats of Ixodes ricinus in The Netherlands.

Rodents are natural reservoirs for human pathogenic spirochaetes of the Borrelia burgdorferi complex [B. burgdorferi sensu lato (s.l.)], and the pathogens are transmitted by Ixodes ricinus ticks to humans in The Netherlands. B. burgdorferi s.l. infection prevalence in questing ticks, rodents, and ticks feeding on these rodents, all sampled within the same short time span of five days in three different areas in The Netherlands, were compared in order to establish the relationship between ticks, reservoir hosts, and B. burgdorferi s.l. Questing nymphs were found in all 3 areas and numbers differed per area and even per site within areas. Infection prevalence in questing nymphs ranged between 0 and 20%. Apodemus sylvaticus and Myodes glareolus were the dominant rodents captured, and their numbers differed per area. Infection prevalence, determined by ear biopsies, ranged between 0 and 33.3% for both rodent species. Larvae were most frequently found feeding on these rodents, and their Borrelia infection prevalence ranged between 0 and 6.3% (A. sylvaticus) and between 0 and 29.4% (M. glareolus). The burden of nymphs feeding on rodents was low and varied per area with only 2 of 42 nymphs infected. Comparisons made on the basis of infection prevalence indicated that there was no clear relationship between rodents and questing nymphs when sampled within the same short time span. However, a possible relationship was present when questing ticks were sampled over longer periods in time (months) within or near the same areas (range of infection prevalence between 3.7 and 39.4). Confounding factors thus play a role in the interaction between rodents, ticks, and B. burgdorferi s.l., and it is very likely that other reservoir host species are responsible for the observed fluctuations. It is concluded that the local variations in rodent-Borrelia-tick interactions only partially explain the Lyme borreliosis risk in the sites studied and that other ecological determinants, notably vertebrate hosts and vegetation structure, should be incorporated in future studies of Lyme borreliosis risk.

Dynamics of seed-borne rice endophytes on early plant growth stages.

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed.

Bacterial communities associated with Chenopodium album and Stellaria media seeds from arable soils.

The bacterial community compositions in Chenopodium album and Stellaria media seeds recovered from soil (soil weed seedbank), from bulk soil, and from seeds harvested from plants grown in the same soils were compared. It was hypothesized that bacterial communities in soil weed seedbanks are distinct from the ones present in bulk soils. For that purpose, bacterial polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints, made from DNA extracts of different soils and seed fractions, were analyzed by principal component analysis. Bacterial fingerprints from C. album and S. media seeds differed from each other and from soil. Further, it revealed that bacterial fingerprints from soil-recovered and plant-harvested seeds from the same species clustered together. Hence, it was concluded that microbial communities associated with seeds in soil mostly originated from the mother plant and not from soil. In addition, the results indicated that the presence of a weed seedbank in arable soils can increase soil microbial diversity. Thus, a change in species composition or size of the soil weed seedbank, for instance, as a result of a change in crop management, could affect soil microbial diversity. The consequence of increased diversity is yet unknown, but by virtue of identification of dominant bands in PCR-DGGE fingerprints as Lysobacter oryzae (among four other species), it became clear that bacteria potentially antagonizing phytopathogens dominate in C. album seeds in soil. The role of these potential antagonists on weed and crop plant growth was discussed.

Isolation and partial characterization of bacterial strains on low organic carbon medium from soils fertilized with different organic amendments.

A total of 720 bacterial strains were isolated from soils with four different organic amendment regimes on a low organic carbon (low-C) agar medium (10 µg C ml(-1)) traditionally used for isolation of oligotrophs. Organic amendments in combination with field history resulted in differences in dissolved organic carbon contents in these soils. There were negative correlations between total and dissolved organic carbon content and the number of isolates on low-C agar medium, whereas these correlations were absent for bacterial strains isolated from the same soil on high-C agar medium (1,000 µg C ml(-1)). Repeated transfers (up to ten times) of the isolates from low-C agar medium to fresh low- and high-C agar media were done to test for exclusive growth under oligotrophic conditions. The number of isolates exclusively growing under oligotrophic conditions dropped after each subsequent transfer from 241 after the first to 98 after the third transfer step. Identification on the basis of partial 16S rRNA gene sequences revealed that most of the 241 isolates (as well as the subset of 98 isolates) belong to widespread genera such as Streptomyces, Rhizobium, Bradyrhizobium, and Mesorhizobium, and the taxonomic composition of dominant genera changed from the first transfer step to the third. A selected subset of 17 isolates were further identified and characterized for exclusive growth on low-C agar medium. Two isolates continued to grow only on low-C agar medium up to the tenth transfer step and matched most closely with Rhizobium sullae and an uncultured bacterium on the basis of the almost full-length 16S rRNA gene. It was concluded that the vast majority of strains which are isolated on low-C agar media belong to the trophic group of microorganisms adapted to a "broad range" of carbon concentrations, including well-known and widespread bacterial genera. Oligotrophy is a physiological, not a taxonomic property, and can only be identified by cultural means so far. We showed that true oligotrophs that are unable to grow on high carbon media are rare and belong to genera that also contain broad-range and copiotrophic strains.

Endophytic colonization of potato (Solanum tuberosum L.) by a novel competent bacterial endophyte, Pseudomonas putida strain P9, and its effect on associated bacterial communities.

Pseudomonas putida strain P9 is a novel competent endophyte from potato. P9 causes cultivar-dependent suppression of Phytophthora infestans. Colonization of the rhizoplane and endosphere of potato plants by P9 and its rifampin-resistant derivative P9R was studied. The purposes of this work were to follow the fate of P9 inside growing potato plants and to establish its effect on associated microbial communities. The effects of P9 and P9R inoculation were studied in two separate experiments. The roots of transplants of three different cultivars of potato were dipped in suspensions of P9 or P9R cells, and the plants were planted in soil. The fate of both strains was followed by examining colony growth and by performing PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Colonies of both strains were recovered from rhizoplane and endosphere samples of all three cultivars at two growth stages. A conspicuous band, representing P9 and P9R, was found in all Pseudomonas PCR-DGGE fingerprints for treated plants. The numbers of P9R CFU and the P9R-specific band intensities for the different replicate samples were positively correlated, as determined by linear regression analysis. The effects of plant growth stage, genotype, and the presence of P9R on associated microbial communities were examined by multivariate and unweighted-pair group method with arithmetic mean cluster analyses of PCR-DGGE fingerprints. The presence of strain P9R had an effect on bacterial groups identified as Pseudomonas azotoformans, Pseudomonas veronii, and Pseudomonas syringae. In conclusion, strain P9 is an avid colonizer of potato plants, competing with microbial populations indigenous to the potato phytosphere. Bacterization with a biocontrol agent has an important and previously unexplored effect on plant-associated communities.

Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa.

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR-DGGE (polymerase chain reaction--denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting alpha- and beta-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa.