Pore-scale view of microbial turnover: Combining 14C imaging, μCT and zymography after adding soluble carbon to soil pores of specific sizes

The location of microorganisms and substrates within soil pore networks plays a crucial role in organic carbon (C) processing, and its microbial utilization and turnover, and has direct consequences for C and nutrient cycling. An optimal approach to quantify responses to new C inputs from microorganisms residing in specific pores is the addition of new C to pores of target sizes in undisturbed soil cores. We used the matric potential approach to add ¹⁴C-labelled glucose to small (< 40 μm, root free) or large (60–180 μm, potentially inhabited by roots) pores of undisturbed soil cores. Localization of glucose-derived C via ¹⁴C imaging was related to pore size distributions and connectivity, characterized via X-ray computed microtomography (μCT), and to β-glucosidase activity, characterized via zymography. After 2-week incubations, 1.3 times more glucose was mineralized (¹⁴CO₂) when it was added to the large pores; however, more ¹⁴C remained in microbial biomass when glucose was added to the small pores. Consequently, although utilizing the same amounts of easily available C, the microorganisms localized in the large pores had faster turnover compared to microorganisms in small pores. Stronger associations between β-glucosidase activity and glucose-derived C were observed when glucose was added to the large pores. We conclude that (a) the matric potential approach allows placing, albeit not exactly, of soluble substrates into pores of target diameter range, and (b) microorganisms localized in large pores respond to new C inputs with faster turnover, greater growth and more intensive enzyme production compared to those inhabiting the small pores.     

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.     

Pseudomonads from rabbits and their sensitivity to antibiotics and natural antimicrobials

The sensitivity/resistance of Pseudomonas spp. isolated from rabbits gastrointestinal tract and faeces to antibiotics, enterocins and herbal extracts was tested in this study. The counts of Pseudomonas-like bacteria were higher in faeces (3.23-6.16 log₁₀ CFU/mL/g) than in caecum (1.36-4.08 log₁₀ CFU/mL/g). Nineteen isolates (16 faecal, 3 caecal) were oxidase positive. The strains were allotted by phenotypization to Pseudomonas spp., Brevundimonas diminuta and Brevundimonasvesicularis. High percentage of resistant strains was observed to all antibiotics. The tested strains were more susceptible to natural substances, mainly to plant extracts oregano (95%) and sage extracts (58%). Comparing the antibacterial effect of antibiotics and enterocins against rabbits pseudomonads, enterocins were more effective; the strongest inhibitory activity was determined in the case of partially purified enterocins PPBs EF2019, EK13 and EF55.     

Diversity of defence mechanisms in plant–oomycete interactions: a case study of <Emphasis Type="Italic">Lactuca</Emphasis> spp. and <Emphasis Type="Italic">Bremia lactucae</Emphasis>

Plant pathogenic oomycetes, including biotrophic downy mildews and hemibiotrophs/necrotrophs such as Phytophthora and Pythium, cause enormous economic losses on cultivated crops. Lettuce breeders and growers face the threat of Bremia lactucae, the causal agent of lettuce downy mildew. This pathogen damages leaf tissues and lettuce heads and is also frequent on wild Asteraceae plants. The interactions of Lactuca spp. with B. lactucae (abbr. lettuce–Bremia) display extreme variability, due to a long co-evolutionary history. For this reason, during the last 30 years, the lettuce–Bremia pathosystem has been used as a model for many studies at the population, individual, organ, tissue, cellular, physiological and molecular levels, as well as on genetic variability and the genetics of host–parasite interactions. The first part of this review summarizes recent data on host–parasite specificity, host variability, resistance mechanisms and genetics of lettuce–Bremia interactions. The second part focuses on the development infection structures. Phenotypic expression of infection, behaviour of B. lactucae on leaf surfaces, the process of penetration, development of primary infection structures, hyphae and haustoria are discussed in relation to different resistance mechanisms. In the third part, the components of host resistance and the variability of defence responses are analysed. The role of reactive oxygen species (ROS), antioxidant enzymes, nitric oxide (NO), phenolic compounds, reorganization of cytoskeleton, electrolyte leakage, membrane damage, cell wall disruption, hypersensitive reaction and plant energetics are discussed in relation to defence responses. In general, the extreme variability of interactions between lettuce and Bremia, and their phenotypic expression, results from diversity of the genetic background. Different mechanisms of resistance are conditioned by an orchestra of defence responses at the tissue, cell, and molecular levels. The various events responsible for defence involve a complex interaction of the processes and reactions mentioned above. This review also provides an overview on the timing of pathogen development, host pathological anatomy, cytology and physiology of lettuce–Bremia associations. The significance of these factors on the expression of different resistance mechanisms (non-host and host resistance, race-specific and race non-specific resistance, field resistance) is discussed.     

Diatom derived polyunsaturated aldehydes do not structure the planktonic microbial community in a mesocosm study

Several marine and freshwater diatoms produce polyunsaturated aldehydes (PUA) in wound-activated processes. These metabolites are also released by intact diatom cells during algal blooms. Due to their activity in laboratory experiments, PUA are considered as potential mediators of diatom-bacteria interactions. Here, we tested the hypothesis that PUA mediate such processes in a close-to-field mesocosm experiment. Natural plankton communities enriched with Skeletonema marinoi strains that differ in their PUA production, a plankton control, and a plankton control supplemented with PUA at natural and elevated concentrations were observed. We monitored bacterial and viral abundance as well as bacterial community composition and did not observe any influence of PUA on these parameters even at elevated concentrations. We rather detected an alternation of the bacterial diversity over time and differences between the two S. marinoi strains, indicating unique dynamic bacterial communities in these algal blooms. These results suggest that factors other than PUA are of significance for interactions between diatoms and bacteria.     

Allocation and dynamics of C and N within plant–soil system of ash and beech

Forest management requires a profound understanding of how tree species affect C and N cycles in ecosystems. The large C and N stocks in forest soils complicate research on the effects of tree species on C and N pools. In‐situ ¹³C and ¹⁵N labeling in undisturbed, natural forests enable not only tracing of C and N fluxes, but also reveal insight into the interactions at the plant‐soil‐atmosphere interface. In‐situ dual ¹³C and ¹⁵N pulse labeling of 20 beeches (Fagus sylvatica L.) and 20 ashes (Fraxinus excelsior L.) allowed tracing the fate of assimilated C and N in trees and soils in an unmanaged forest system in the Hainich National Park (Germany). Leaf, stem, root, and soil samples as well as microbial biomass were analyzed to quantify the allocation of ¹³C and ¹⁵N for 60 d after labeling and along spatial gradients in the soil with increasing distance from the stem. For trees of similar heights (≈ 4 m), beech (20%) assimilated twice as much as ash (9%) of the applied ¹³CO₂, but beech and ash incorporated similar ¹⁵N amounts (45%) into leaves. The photosynthates were transported belowground through the phloem more rapidly in beech than in ash. Ash preferentially accumulated ¹⁵N and ¹³C in the roots. In contrast, beech released more of this initially assimilated ¹³C (2.0% relative ¹³C allocation) and ¹⁵N (0.1% relative ¹⁵N allocation) via rhizodeposition into the soil than ash (0.2% relative ¹³C, 0.04% relative ¹⁵N allocation), which was also subsequently recovered in microbial biomass. These results on C and N partitioning contribute to an improved understanding of the effects of European beech and ash on the C and N cycles in deciduous broad‐leaved forest. Differences in C and N allocation patterns between ash and beech are one mechanism of niche differentiation in forests containing both species.     

Epidemiologically non-feasible singleton reactors at the final stage of BoHV1 eradication: Serological evidence of BoHV2 cross-reactivity

A voluntary marker-independent Bovine Herpesvirus 1 (BoHV1) eradication program started in 1986; in 1998 it changed to a compulsory one. Certification of free regions in European member states is based on Article 10 of directive 64/432/EEC. According to this rule Bavaria is listed as free of BoHV1 since October 2011. Surveillance of BoHV1-free dairy cattle farms is currently performed with quarterly bulk-milk testing. Non-negative bulk-milk results must be confirmed by blood tests in cattle older than nine months. An increased regional rate of non-negative bulk-milk samples and the subsequent detection of epidemiologically non-feasible singleton BoHV1-reactors by analysis of blood were observed at the final stage of eradication in southwest Bavaria. Nineteen case farms (734 animals) defined by singleton reactors born at least two years after certification of the farms as BoHV1-free, 23 negative control (NC) farms (NC I: 321 animals) from the same region, 11 NC-farms (NC II: 423 animals) from an already-certified Article 10 region in northeast Bavaria and two BoHV1-infected farms (264 animals) were analysed using BoHV1-, BoHV2- and Feline Herpesvirus 1 (FeHV1)-neutralisation tests (NTs), and three commercially available ELISAs supplied by Idexx Laboratories, B.V., The Netherlands: the CHEKIT? Trachitest 2nd Gen. test for milk or serum (Trachitest), Herdchek? gB- (gB-ELISA) and Herdchek? gE-ELISA (gE-ELISA). Significantly increased levels of BoHV2 antibodies were observed on case farms compared to NC I or II farms. Additionally, reactivity by gB-ELISA and the Trachitest was significantly increased for animals with BoHV2 neutralising antibodies. Singleton BoHV1-reactors tested negative by gE-ELISA even if an elevated cut-off of 0.95±0.05 was applied. At this cut-off, the gE-ELISA was as sensitive and specific as the gB-ELISA. Comparative titration of milk samples from seropositive animals from a BoHV1-infected dairy cattle farm and from singleton BoHV1-reactors performed in CHEKIT? Trachitest 2nd Gen. Milk revealed that the slopes of both groups were distinct; therefore, optimised cut-offs for bulk-milk testing to exclude singleton BoHV1-reactors are proposed.