High-resolution melting (HRM) curve analysis as a potential tool for the identification of earthworm species and haplotypes.

Background: Earthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa. Methods: We developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment. Results: The developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue. Conclusions: HRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

Fusarium culmorum Produces NX-2 Toxin Simultaneously with Deoxynivalenol and 3-Acetyl-Deoxynivalenol or Nivalenol

Fusarium culmorum is a major pathogen of grain crops. Infected plants accumulate deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), or nivalenol (NIV), which are mycotoxins of the trichothecene B group. These toxins are also produced by F. graminearum species complex. New trichothecenes structurally similar to trichothecenes B but lacking the carbonyl group on C-8, designated NX toxins, were recently discovered in atypical isolates of F. graminearum from North America. Only these isolates and a few strains of a yet to be characterized Fusarium species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 F. culmorum strains isolated from maize, wheat, and oat in Europe and Asia over a period of 70 years, 18 strains produced NX-2 simultaneously with 3-ADON and DON or NIV. Rice cultures of strains producing 3-ADON accumulated NX-2 in amounts corresponding to 2−8% of 3-ADON (1.2−36 mg/kg). A strain producing NIV accumulated NX-2 and NIV at comparable amounts (13.6 and 10.3 mg/kg, respectively). In F. graminearum, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by TRI1 that is unable to oxidize C-8. In F. culmorum, producers and nonproducers of NX-2 possess identical TRI1; the reason for the production of NX-2 is unknown. Our results indicate that the production of NX-2 simultaneously with trichothecenes B is a common feature of F. culmorum.

Abundance, Diversity, and Function of Soil Microorganisms in Temperate Alley-Cropping Agroforestry Systems: A Review.

Modern temperate alley-cropping systems combine rows of trees with rows of crops (agroforestry), which allows for diverse interspecific interactions such as the complementary and competitive use of resources. The complementary use of resources between trees and crops is considered the main advantage of these multifunctional land use systems over cropland monocultures. Moreover, several studies demonstrated that agroforestry systems are environmentally more sustainable than cropland monocultures. Over two decades of research on soil microorganisms in temperate alley-cropping systems are characterized by a variety of different methodological approaches and study designs to investigate the impact of agroforestry on the soil microbiome. Here, we review the available literature on the abundance, diversity, and functionality of soil microorganisms in temperate alley-cropping systems. Further, we identify current knowledge gaps as well as important experimental factors to consider in future studies. Overall, we found that temperate alley-cropping systems increase soil microbial abundance, diversity, and functions as compared to cropland monocultures, which is expected to contribute to enhanced biological soil fertility in these systems.

Soil bacterial community response to rhizoma peanut incorporation into Florida pastures.

Incorporating legumes is one option for improving pasture fertility, sustainability, and biodiversity. Diazotrophic microorganisms, including rhizobia that form symbioses with legumes, represent a small fraction of the total soil microbial community. Yet, they can offset nitrogen (N) fertilizer inputs through their ability to convert atmospheric N2 into plant-usable N via biological N2 fixation (BNF). This study used amplicon sequencing of 16S rRNA genes to investigate soil bacterial community composition and diversity in grazed 'Argentine' bahiagrass (Paspalum notatum Flügge) pastures where N fertilizer was supplanted with legume-derived N from BNF in some treatments. Treatments consisted of bahiagrass fertilized with (a) mineral N (224 kg N ha-1  yr-1 ), (b) combination mineral N (34 kg N ha-1  yr-1 ) and legume-derived N via cool-season clover (CSC) (Trifolium spp.) mix, or (c) combination mineral N (34 kg N ha-1  yr-1 ) and legume-derived N via CSC mix and strips of Ecoturf rhizoma peanut (Arachis glabrata Benth.). Bradyrhizobium spp. relative abundance was 44% greater in the mixed pasture. Other bacterial genera with BNF or denitrification potentials were greater in pastures with legumes, whereas sequences assigned to genera associated with high litter turnover were greater in bahiagrass pastures receiving only mineral N. Soil bacteria alpha diversity was greater in pastures receiving 34 kg ha-1  yr-1 N fertilizer application and the CSC mix than in pastures with the CSC mix and rhizoma peanut strips. Our results demonstrate soil microbial community shifts that may affect soil C and N cycling in pastures common to the southeastern United States.

The travelling particles: community dynamics of biofilms on microplastics transferred along a salinity gradient.

Microplastics (MP), as novel substrata for microbial colonization within aquatic ecosystems, are a matter of growing concern due to their potential to propagate foreign or invasive species across different environments. MP are known to harbour a diversity of microorganisms, yet little is understood of the dynamics of their biofilms and their capacity to successfully displace these microorganisms across different aquatic ecosystems typically marked by steep salinity gradients. To address this, we performed an in situ sequential incubation experiment to simulate MP transport from riverine to coastal seawaters using synthetic (high-density polyethylene, HDPE and tyre wear, TW) and natural (Wood) substrata. Bacterial communities on incubated particles were compared to each other as well as to those in surrounding waters, and their dynamics along the gradient investigated. All communities differed significantly from each other in their overall structure along the salinity gradient and were shaped by different ecological processes. While HDPE communities were governed by environmental selection, those on TW and Wood were dominated by stochastic events of dispersal and drift. Upon transfer into coastal seawaters, an almost complete turnover was observed among HDPE and TW communities. While synthetic particles displaced a minor proportion of communities across the salinity gradient, some of these comprised putatively pathogenic and resistant taxa. Our findings present an extensive assessment of MP biofilms and their dynamics upon displacement across different aquatic systems, presenting new insights into the role of MP as transport vectors.

Are short-read amplicons suitable for the prediction of microbiome functional potential? A critical perspective.

Taxonomic marker gene analysis allows uncovering taxonomic profiles of microbial communities at low cost, making it omnipresent in microbiome research. There is an ever-expanding set of tools to extract further biological information from this kind of data. In this perspective, we enunciate several concerns regarding the biological validity of predicting functional potential from taxonomic profiles, especially when they are generated by short-read sequencing. The taxonomic resolution of marker genes, intragenomic variability of marker genes, and the compositional nature of microbiome data are discussed. Combining actual measurements of microbiome functions with predicted functional potentials is proposed as a powerful approach to better understand microbiome functioning. In this context, the significance of predicted functional potentials for generating and testing hypotheses is highlighted. We argue that functions of microbiomes predicted from microbiome DNA read count data generated by short-read amplicon sequencing should not serve as the only basis to draw biological inferences.

Early response of soil fungal communities to the conversion of monoculture cropland to a temperate agroforestry system.

BACKGROUND: Alley-cropping systems in the temperate zone are a type of agroforestry in which rows of fast-growing trees are alternated with rows of annual crops. With numerous environmental benefits, temperate agroforestry is considered a promising alternative to conventional agriculture and soil fungi may play a key in maintaining productivity of these systems. Agroforestry systems that are established for more than 10 years have shown to increase the fungal biomass and impact the composition of soil fungal communities. Investigations of soil fungi in younger temperate agroforestry systems are scarce and the temporal dynamic of these changes is not understood. METHODS: Our study was conducted in a young poplar-based alley cropping and adjacent monoculture cropland system in an Arenosol soil in north-west Germany. We investigated the temporal dynamics of fungal populations after the establishment of agroforestry by collecting soil samples half, one, and one and a half years after conversion of cropland to agroforestry. Samples were collected within the agroforestry tree row, at 1, 7, and 24 m distance from the tree row within the crop row, and in an adjacent conventional monoculture cropland. The biomass of soil fungi, Asco-, and Basidiomycota was determined by real-time PCR. Soil fungal community composition and diversity were obtained from amplicon sequencing. RESULTS: Differences in the community composition of soil fungi in the tree row and arable land were detected as early as half a year following the conversion of monoculture cropland to agroforestry. In the tree row, soil fungal communities in the plots strongly diverged with the age of the system. The presence of young trees did not affect the biomass of soil fungi. CONCLUSIONS: The composition of soil fungal communities responded rapidly to the integration of trees into arable land through agroforestry, whereas the fungal biomass was not affected during the first one and a half years after planting the trees. Fungal communities under the trees gradually diversified. Adaptation to spatially heterogeneous belowground biomass of the trees and understory vegetation or stochastic phenomena due to limited exchange among fungal populations may account for this effect; long-term monitoring might help unravelling the cause.

Relative Abundances of Species or Sequence Variants Can Be Misleading: Soil Fungal Communities as an Example.

Plant production systems that are more sustainable than conventional monoculture croplands are the vision of future agriculture. With numerous environmental benefits, agroforestry is among the most promising alternatives. Although soil fungi are key drivers of plant productivity and ecosystem processes, investigations of these microorganisms in temperate agroforestry systems are scarce, leaving our understanding of agricultural systems under agroforestry practice incomplete. Here, we assessed the composition and diversity of the soil fungal community as well as the frequency (relative abundance) of fungal groups in three paired temperate poplar-based alley cropping (agroforestry) and monoculture cropland systems by amplicon sequencing. Analysis of microbiomes using relative abundances of species or sequence variants obtained from amplicon sequencing ignores microbial population size, which results in several problems. For example, species stimulated by environmental parameters may appear unaffected or suppressed in amplicon counts. Therefore, we determined absolute abundances of selected fungal groups as well as total fungal population size by real-time polymerase chain reaction (PCR). Tree rows strongly affected the community composition and increased the population size and species richness of soil fungi. Furthermore, ectomycorrhiza were strongly promoted by the tree rows. We speculate that mycorrhiza improved the nutrient acquisition in unfertilized tree rows, thereby contributing to the total productivity of the system. Comparison of relative and absolute abundances revealed dramatic discrepancies, highlighting that amplicon sequencing alone cannot adequately assess population size and dynamics. The results of our study highlight the necessity of combining frequency data based on amplicon sequencing with absolute quantification.

Tree rows in temperate agroforestry croplands alter the composition of soil bacterial communities.

BACKGROUND: Tree-based intercropping (agroforestry) has been advocated to reduce adverse environmental impacts of conventional arable cropping. Modern agroforestry systems in the temperate zone are alley-cropping systems that combine rows of fast-growing trees with rows of arable crops. Soil microbial communities in these systems have been investigated intensively; however, molecular studies with high taxonomical resolution are scarce. METHODS: Here, we assessed the effect of temperate agroforestry on the abundance, diversity and composition of soil bacterial communities at three paired poplar-based alley cropping and conventional monoculture cropland systems using real-time PCR and Illumina sequencing of bacterial 16S rRNA genes. Two of the three systems grew summer barley (Hordeum vulgare); one system grew maize (Zea mays) in the sampling year. To capture the spatial heterogeneity induced by the tree rows, soil samples in the agroforestry systems were collected along transects spanning from the centre of the tree rows to the centre of the agroforestry crop rows. RESULTS: Tree rows of temperate agroforestry systems increased the abundance of soil bacteria while their alpha diversity remained largely unaffected. The composition of the bacterial communities in tree rows differed from those in arable land (crop rows of the agroforestry systems and conventional monoculture croplands). Several bacterial groups in soil showed strong association with either tree rows or arable land, revealing that the introduction of trees into arable land through agroforestry is accompanied by the introduction of a tree row-associated microbiome. CONCLUSION: The presence of tree row-associated bacteria in agroforestry increases the overall microbial diversity of the system. We speculate that the increase in biodiversity is accompanied by functional diversification. Differences in plant-derived nutrients (root exudates and tree litter) and management practices (fertilization and tillage) likely account for the differences between bacterial communities of tree rows and arable land in agroforestry systems.

Improved normalization of species count data in ecology by scaling with ranked subsampling (SRS): application to microbial communities.

BACKGROUND: Analysis of species count data in ecology often requires normalization to an identical sample size. Rarefying (random subsampling without replacement), which is the current standard method for normalization, has been widely criticized for its poor reproducibility and potential distortion of the community structure. In the context of microbiome count data, researchers explicitly advised against the use of rarefying. Here we introduce a normalization method for species count data called scaling with ranked subsampling (SRS) and demonstrate its suitability for the analysis of microbial communities. METHODS: SRS consists of two steps. In the scaling step, the counts for all species or operational taxonomic units (OTUs) are divided by a scaling factor chosen in such a way that the sum of scaled counts equals the selected total number of counts Cmin. The relative frequencies of all OTUs remain unchanged. In the subsequent ranked subsampling step, non-integer count values are converted into integers by an algorithm that minimizes subsampling error with regard to the population structure (relative frequencies of species or OTUs) while keeping the total number of counts equal Cmin. SRS and rarefying were compared by normalizing a test library representing a soil bacterial community. Common parameters of biodiversity and population structure (Shannon index H', species richness, species composition, and relative abundances of OTUs) were determined for libraries normalized to different size by rarefying as well as SRS with 10,000 replications each. An implementation of SRS in R is available for download (https://doi.org/10.20387/BONARES-2657-1NP3). RESULTS: SRS showed greater reproducibility and preserved OTU frequencies and alpha diversity better than rarefying. The variance in Shannon diversity increased with the reduction of the library size after rarefying but remained zero for SRS. Relative abundances of OTUs strongly varied among libraries generated by rarefying, whereas libraries normalized by SRS showed only negligible variation. Bray-Curtis index of dissimilarity among replicates of the same library normalized by rarefying revealed a large variation in species composition, which reached complete dissimilarity (not a single OTU shared) among some libraries rarefied to a small size. The dissimilarity among replicated libraries normalized by SRS remained negligibly low at each library size. The variance in dissimilarity increased with the decreasing library size after rarefying, whereas it remained either zero or negligibly low after SRS. CONCLUSIONS: Normalization of OTU or species counts by scaling with ranked subsampling preserves the original community structure by minimizing subsampling errors. We therefore propose SRS for the normalization of biological count data.

Improved Protocol for DNA Extraction from Subsoils Using Phosphate Lysis Buffer.

As our understanding of soil biology deepens, there is a growing demand for investigations addressing microbial processes in the earth beneath the topsoil layer, called subsoil. High clay content in subsoils often hinders the recovery of sufficient quantities of DNA as clay particles bind nucleic acids. Here, an efficient and reproducible DNA extraction method for 200 mg dried soil based on sodium dodecyl sulfate (SDS) lysis in the presence of phosphate buffer has been developed. The extraction protocol was optimized by quantifying bacterial 16S and fungal 18S rRNA genes amplified from extracts obtained by different combinations of lysis methods and phosphate buffer washes. The combination of one minute of bead beating, followed by ten min incubation at 65°C in the presence of 1 M phosphate buffer with 0.5% SDS, was found to produce the best results. The optimized protocol was compared with a commonly used cetyltrimethylammonium bromide (CTAB) method, using Phaeozem soil collected from 60 cm depth at a conventional agricultural field and validated on five subsoils. The reproducibility and robustness of the protocol was corroborated by an interlaboratory comparison. The DNA extraction protocol offers a reproducible and cost-effective tool for DNA-based studies of subsoil biology.

High-Resolution Melting (HRM) Curve Assay for the Identification of Eight Fusarium Species Causing Ear Rot in Maize.

Maize plants are often infected with fungal pathogens of the genus Fusarium. Taxonomic characterization of these species by microscopic examination of pure cultures or assignment to mating populations is time-consuming and requires specific expertise. Reliable taxonomic assignment may be strengthened by the analysis of DNA sequences. Species-specific PCR assays are available for most Fusarium pathogens, but the number of species that infect maize increases the labor and costs required for analysis. In this work, a diagnostic assay for major Fusarium pathogens of maize based on the analysis of melting curves of PCR amplicons was established. Short segments of genes RPB2 and TEF-1α, which have been widely used in molecular taxonomy of Fusarium, were amplified with universal primers in a real-time thermocycler and high-resolution melting (HRM) curves of the products were recorded. Among major Fusarium pathogens of maize ears, F. cerealis, F. culmorum, F. graminearum, F. equiseti, F. poae, F. temperatum, F. tricinctum, and F. verticillioides, all species except for the pair F. culmorum/F. graminearum could be distinguished by HRM analysis of a 304 bp segment of the RPB2 gene. The latter two species could be differentiated by HRM analysis of a 247 bp segment of the TEF-1α gene. The assay was validated with DNA extracted from pure cultures of fungal strains, successfully applied to total DNA extracted from infected maize ears and also to fungal mycelium that was added directly to the PCR master mix ("colony PCR"). HRM analysis thus offers a cost-efficient method suitable for the diagnosis of multiple fungal pathogens.

Conversion of monoculture cropland and open grassland to agroforestry alters the abundance of soil bacteria, fungi and soil-N-cycling genes.

Integration of trees in agroforestry systems can increase the system sustainability compared to monocultures. The resulting increase in system complexity is likely to affect soil-N cycling by altering soil microbial community structure and functions. Our study aimed to assess the abundance of genes encoding enzymes involved in soil-N cycling in paired monoculture and agroforestry cropland in a Phaeozem soil, and paired open grassland and agroforestry grassland in Histosol and Anthrosol soils. The soil fungi-to-bacteria ratio was greater in the tree row than in the crop or grass rows of the monoculture cropland and open grassland in all soil types, possibly due to increased input of tree residues and the absence of tillage in the Phaeozem (cropland) soil. In the Phaeozem (cropland) soil, gene abundances of amoA indicated a niche differentiation between archaeal and bacterial ammonia oxidizers that distinctly separated the influence of the tree row from the crop row and monoculture system. Abundances of nitrate (napA and narG), nitrite (nirK and nirS) and nitrous oxide reductase genes (nosZ clade I) were largely influenced by soil type rather than management system. The soil types' effects were associated with their differences in soil organic C, total N and pH. Our findings show that in temperate regions, conversion of monoculture cropland and open grassland to agroforestry systems can alter the abundance of soil bacteria and fungi and soil-N-cycling genes, particularly genes involved in ammonium oxidation.

Soil bacterial and fungal communities of six bahiagrass cultivars.

BACKGROUND: Cultivars of bahiagrass (Paspalum notatum Flüggé) are widely used for pasture in the Southeastern USA. Soil microbial communities are unexplored in bahiagrass and they may be cultivar-dependent, as previously proven for other grass species. Understanding the influence of cultivar selection on soil microbial communities is crucial as microbiome taxa have repeatedly been shown to be directly linked to plant performance. OBJECTIVES: This study aimed to determine whether different bahiagrass cultivars interactively influence soil bacterial and fungal communities. METHODS: Six bahiagrass cultivars ('Argentine', 'Pensacola', 'Sand Mountain', 'Tifton 9', 'TifQuik', and 'UF-Riata') were grown in a randomized complete block design with four replicate plots of 4.6 × 1.8 m per cultivar in a Rhodic Kandiudults soil in Northwest Florida, USA. Three soil subsamples per replicate plot were randomly collected. Soil DNA was extracted and bacterial 16S ribosomal RNA and fungal ribosomal internal transcribed spacer 1 genes were amplified and sequenced with one Illumina Miseq Nano. RESULTS: The soil bacterial and fungal community across bahiagrass cultivars showed similarities with communities recovered from other grassland ecosystems. Few differences in community composition and diversity of soil bacteria among cultivars were detected; none were detected for soil fungi. The relative abundance of sequences assigned to nitrite-oxidizing Nitrospira was greater under 'Sand Mountain' than 'UF-Riata'. Indicator species analysis revealed that several bacterial and fungal indicators associated with either a single cultivar or a combination of cultivars are likely to be plant pathogens or antagonists. CONCLUSIONS: Our results suggest a low impact of plant cultivar choice on the soil bacterial community composition, whereas the soil fungal community was unaffected. Shifts in the relative abundance of Nitrospira members in response to cultivar choice may have implications for soil N dynamics. The cultivars associated with presumptive plant pathogens or antagonists indicates that the ability of bahiagrass to control plant pathogens may be cultivar-dependent, however, physiological studies on plant-microbe interactions are required to confirm this presumption. We therefore suggest that future studies should explore the potential of different bahiagrass cultivars on plant pathogen control, particularly in sod-based crop rotation.

Poplar Rows in Temperate Agroforestry Croplands Promote Bacteria, Fungi, and Denitrification Genes in Soils.

Agroforestry, which is the integration of trees into monoculture cropland, can alter soil properties and nutrient cycling. Temperate agroforestry practices have been shown to affect soil microbial communities as indicated by changes in enzyme activities, substrate-induced respiration, and microbial biomass. Research exploring soil microbial communities in temperate agroforestry with the help of molecular tools which allow for the quantification of microbial taxa and selected genes is scarce. Here, we quantified 13 taxonomic groups of microorganisms and nine genes involved in N cycling (N2 fixation, nitrification, and denitrification) in soils of three paired temperate agroforestry and conventional monoculture croplands using real-time PCR. The agroforestry croplands were poplar-based alley-cropping systems in which samples were collected in the tree rows as well as within the crop rows at three distances from the tree rows. The abundance of Acidobacteria, Actinobacteria, Alpha- and Gammaproteobacteria, Firmicutes, and Verrucomicrobia increased in the vicinity of poplar trees, which may be accounted for by the presence of persistent poplar roots as well as by the input of tree litter. The strongest population increase was observed for Basidiomycota, which was likely related to high soil moisture, the accumulation of tree litter, and the absence of tillage in the tree rows. Soil microorganisms carrying denitrification genes were more abundant in the tree rows than in the crop rows and monoculture systems, suggesting a greater potential for nitrate removal through denitrification, which may reduce nitrate leaching. Since microbial communities are involved in critical soil processes, we expect that the combination of real-time PCR with soil process measurements will greatly enhance insights into the microbial control of important soil functions in agroforestry systems.

Single versus repeated applications of CuO and Ag nanomaterials and their effect on soil microflora.

Nanomaterials enter the terrestrial environment via the repeated application of sludge to soils over many years. The goal of this investigation was to compare the effects of CuO and Ag nanomaterials on soil microorganisms after a single application and after repeated applications ultimately resulting in the same test concentrations. The effect on soil microorganisms was determined using the ammonium oxidation (ISO 15685), enzymatic activity patterns (ISO 22939) and MicroResp™ tests on days 28, 56 and 84. The comparability of single and repeated applications of ion-releasing nanomaterials depended on the test endpoint and duration. No significant differences between single and repeated applications were observed when testing nitrifying microorganisms and exoenzymes, but differences were observed in the substrate-induced respiration test. The three test systems used together provide more comprehensive information about the impact of different nanomaterials on the soil microflora and its diversity.