A glance into the black box: Novel species-specific quantitative real-time PCR assays to disentangle aquatic hyphomycete community composition

Baudy, Patrick; Zubrod, Jochen P.; Röder, Nina; Baschien, Christiane; Feckler, Alexander; Schulz, Ralf; Bundschuh, Mirco

doi:10.1016/j.funeco.2019.08.002

Cited by 20 publications

(23 citation statements)

References 51 publications

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“…The R 2 -values, ranging from 0.984 to 1.000, indicate a linear correspondence between the logarithmic genome copy number and their Cq-values. The efficiency (E), as one of the most important indicators of the performance of a qPCR assay, was between 86.4 and 104.7% in simplex, and between 90.6 and 103.4% in multiplex assays, which proves a good performance of the primer-probe sets (Baudy et al, 2019;Lochman et al, 2019). In all four assays, PCR probes appeared to be very specific and no loss of activity was observed upon multiplexing.…”

Section: Performance Of Simplex and Multiplexed Qpcr Assaysmentioning

confidence: 80%

“…High costs for labeling of each end of the probes with different dyes is in comparison to the use of intercalating dyes is compensated by higher selectivity and a reduced process time in multiplexed assays. Multiplex TaqMan qPCR recently appeared to be a very useful tool for the identification and quantification of different microbial communities (Farkas et al, 2017;Baudy et al, 2019;Enora et al, 2019;Lochman et al, 2019). Accordingly, this study aims to establish a multiplex qPCR approach for the simultaneous detection and quantification of eleven frequently reported bacteria and yeast species of milk kefirs.…”

Section: Introductionmentioning

confidence: 99%

“…http://scienceprimer.com/copy-number-calculator-for-realtime-pcr 3 www.ncbi.nlm.nih.gov concentration (copies reaction −1 ) corresponding to the LOD(Baudy et al, 2019).…”

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confidence: 99%

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Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

et al. 2020

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Kefir grains are complex microbial systems of several groups of microorganisms. The identification and quantification of the microbial composition of milk kefirs was described in several studies, which provided an insight into the microbial consortia in this complex ecosystem. Nevertheless, the current methods for identification and quantification are not appropriate for deeper studies on kefir consortia, e.g., population dynamics and microbial interactions in kefir grains. This requires another sensitive and reliable quantitative method. Therefore, this study aims to develop multiplexed qPCR assays to specifically detect and quantify, as an example, several microorganisms of the milk kefir microbial community. Primer-probe sets, which target species-specific genes in six bacteria and five yeasts, were designed, and their sensitivity and specificity to the target species was analyzed in simplex as well as four multiplex qPCR assays. The self-designed multiplex assays were applied for the detection of target bacteria and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all target microorganisms in simplex and multiplex qPCR was achieved by good linearity, efficiency, repeatability and reproducibility in all assays. When the designed assays were applied on six kefirs, all target microorganisms were detected in different samples, but not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens and Lb. kefiri were present in all six kefirs studied, but were associated with different other yeasts and bacteria. Especially on the yeast community a significant diversity was observed. In general, multiplex TaqMan qPCR as developed here was proven to have high potential for specific identification of target microorganisms in kefir samples and for the first time, eleven target bacteria and yeasts of kefir microbiota were rapidly detected and quantified. This study, thus, provides a fast and reliable protocol for future studies on kefir and other similar microbial ecosystems.

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Section: Performance Of Simplex and Multiplexed Qpcr Assaysmentioning

confidence: 80%

Section: Introductionmentioning

confidence: 99%

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Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

et al. 2020

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“…This selection covers ascomycete species frequently detected in aquatic decomposer communities of the temperate latitudes and exhibits a gradient of sensitivities to anthropogenic stress (e.g., Fernández et al 2016, Solé et al 2008). The strains were isolated from German streams and deposited in the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures, Germany;Baudy et al 2019). In the laboratory, isolated strains were exclusively handled under sterile conditions.…”

Section: Microorganisms and Leaf Substratementioning

confidence: 99%

“…where w i and w f refer to the initial and final dry weight of 20 and 15 leaf disks, respectively, and 0.924 and 1.333 are empirical factors controlling for leaf mass losses due to the autoclaving procedure and the destructive sampling of five leaf disks, respectively. Ergosterol and DNA concentrations were expressed as µg/g leaf dry weight and were calculated as described in Baudy et al (2019). Fungal growth rates were calculated from ergosterol concentrations in bacteria-free fungal monocultures and were expressed as µg ergosterol/g leaf dry weight/d.…”

Section: General Calculationsmentioning

confidence: 99%

Fungal–fungal and fungal–bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity

Baudy

Zubrod

Konschak

et al. 2021

Ecology

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Fungi produce a variety of extracellular enzymes making recalcitrant substrates bioavailable. Thus, fungi are central for decomposition of dead organic matter such as leaf litter. Despite their ecological importance, our understanding of relationships between fungal species diversity and ecosystem functioning is limited, especially with regard to aquatic habitats. Moreover, fungal interactions with other groups of microorganisms such as bacteria are rarely investigated. This lack of information may be attributed to methodological limitations in tracking the biomass of individual fungal species in communities, impeding a detailed assessment of deviations from the overall performance expected from the sum of individual species' performances, so-called net diversity effects (NDEs). We used fungal species-specific biomolecular tools to target fungal-fungal and fungal-bacterial interactions on submerged leaves using four cosmopolitan aquatic fungal species and a stream microbial community dominated by bacteria. In microcosms, we experimentally manipulated fungal diversity and bacterial absence/presence and assessed functional performances and fungal community composition after 14 days of incubation. Fungal community data was used to evaluate NDEs on leaf colonization. The individual fungal species were functionally distinct and fungal cultures were on average more efficient than the bacterial culture. In absence of bacteria, NDEs correlated with growth rate (negatively) and genetic divergence (positively), but were predominantly negative, suggesting that higher fungal diversity led to a lower colonization success (niche overlap). In both absence and presence of bacteria, the overall functional performances of the communities were largely defined by their composition (i.e., no interactions at the functional level). In presence of bacteria, NDEs correlated with genetic divergence (positively) and were largely positive, suggesting higher fungal diversity stimulated colonization (niche complementarity). This stimulation may be driven by a bacteria-induced inhibition of fungal growth, alleviating competition among fungi. Resulting feedback loops eventually promote fungal coexistence and synergistic interactions. Nonetheless, overall functional performances are reduced compared to bacteria-free cultures. These findings highlight the necessity to conduct future studies, investigating biodiversity-ecosystem functioning relationships using artificial systems, without exclusion of key organisms naturally co-occurring in the compartment of interest. Otherwise, study outcomes might not reflect true ecological relationships and ultimately misguide conservation strategies.

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As above, so below? Effects of fungicides on microbial organic matter decomposition are stronger in the hyporheic than in the benthic zone

Bollinger

Zubrod

Konschak

et al. 2021

Limnology & Oceanography

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Microbial organic matter decomposition is a critical ecosystem function, which can be negatively affected by chemicals. Although the majority of organic matter is stored in sediments, the impact of chemicals has exclusively been studied in benthic systems. To address this knowledge gap, we assessed the impact of a fungicide mixture at three concentrations on the decomposition of black alder leaves in the benthic and hyporheic zone. We targeted two sediment treatments characterized by fine and coarse grain sizes (1–2 vs. 2–4 mm). Besides microbial communities' functioning (i.e., decomposition), we determined their structure through microbial biomass estimates and community composition. In absence of fungicides, leaf decomposition, microbial biomass estimates and fungal sporulation were lower in the hyporheic zone, while the importance of bacteria was elevated. Leaf decomposition was reduced (40%) under fungicide exposure in fine sediment with an effect size more than twice as high as in the benthic zone (15%). These differences are likely triggered by the lower hydraulic conductivity in the hyporheic zone influencing microbial dispersal as well as oxygen and nutrient fluxes. Since insights from the benthic zone are not easily transferable, these results indicate that the hyporheic zone requires a higher recognition with regard to ecotoxicological effects on organic matter decomposition.

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A glance into the black box: Novel species-specific quantitative real-time PCR assays to disentangle aquatic hyphomycete community composition

Cited by 20 publications

References 51 publications

Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

Quantification of Major Bacteria and Yeast Species in Kefir Consortia by Multiplex TaqMan qPCR

Fungal–fungal and fungal–bacterial interactions in aquatic decomposer communities: bacteria promote fungal diversity

As above, so below? Effects of fungicides on microbial organic matter decomposition are stronger in the hyporheic than in the benthic zone

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