Mycoviruses Increase the Attractiveness of Fusarium graminearum for Fungivores and Suppress Production of the Mycotoxin Deoxynivalenol.

RNA viruses of the genera Ambivirus, Mitovirus, Sclerotimonavirus, and Partitivirus were found in a single isolate of Fusarium graminearum. The genomes of the mitovirus, sclerotimonavirus, and partitivirus were assigned to previously described viruses, whereas the ambivirus genome putatively represents a new species, named Fusarium graminearum ambivirus 1 (FgAV1). To investigate the effect of mycoviruses on the fungal phenotype, the spontaneous loss of mycoviruses during meiosis and the transmission of mycoviruses into a new strain via anastomosis were used to obtain isogenic F. graminearum strains both with and without mycoviruses. Notable effects observed in mycovirus-harboring strains were (i) the suppression of the synthesis of trichothecene mycotoxins and their precursor trichodiene, (ii) the suppression of the synthesis of the defense compound aurofusarin, (iii) the stimulation of the emission of 2-methyl-1-butanol and 3-methyl-1-butanol, and (iv) the increased attractiveness of fungal mycelia for fungivorous collembolans. The increased attractiveness of mycovirus-infected filamentous fungi to animal predators opens new perspectives on the ecological implications of the infection of fungi with viruses.

The Role of Mycotoxins in Interactions between Fusarium graminearum and F. verticillioides Growing in Saprophytic Cultures and Co-Infecting Maize Plants.

Fusarium graminearum (FG) and Fusarium verticillioides (FV) co-occur in infected plants and plant residues. In maize ears, the growth of FV is stimulated while FG is suppressed. To elucidate the role of mycotoxins in these effects, we used FG mutants with disrupted synthesis of nivalenol (NIV) and deoxynivalenol (DON) and a FV mutant with disrupted synthesis of fumonisins to monitor fungal growth in mixed cultures in vitro and in co-infected plants by real-time PCR. In autoclaved grains as well as in maize ears, the growth of FV was stimulated by FG regardless of the production of DON or NIV by the latter, whereas the growth of FG was suppressed. In autoclaved grains, fumonisin-producing FV suppressed FG more strongly than a fumonisin-nonproducing strain, indicating that fumonisins act as interference competition agents. In co-infected maize ears, FG suppression was independent of fumonisin production by FV, likely due to heterogeneous infection and a lower level of fumonisins in planta. We conclude that (i) fumonisins are agents of interference competition of FV, and (ii) trichothecenes play no role in the interaction between FG and FV. We hypothesize the following: (i) In vitro, FG stimulates the FV growth by secreting hydrolases that mobilize nutrients. In planta, suppression of plant defense by FG may additionally play a role. (ii) The biological function of fumonisin production in planta is to protect kernels shed on the ground by accumulating protective metabolites before competitors become established. Therefore, to decipher the biological function of mycotoxins, the entire life history of mycotoxin producers must be considered.

Characterization of Peroxidase and Laccase Gene Families and In Silico Identification of Potential Genes Involved in Upstream Steps of Lignan Formation in Sesame.

Peroxidases and laccases are oxidative enzymes involved in physiological processes in plants, covering responses to biotic and abiotic stress as well as biosynthesis of health-promoting specialized metabolites. Although they are thought to be involved in the biosynthesis of (+)-pinoresinol, a comprehensive investigation of this class of enzymes has not yet been conducted in the emerging oil crop sesame and no information is available regarding the potential (+)-pinoresinol synthase genes in this crop. In the present study, we conducted a pan-genome-wide identification of peroxidase and laccase genes coupled with transcriptome profiling of diverse sesame varieties. A total of 83 and 48 genes have been identified as coding for sesame peroxidase and laccase genes, respectively. Based on their protein domain and Arabidopsis thaliana genes used as baits, the genes were classified into nine and seven groups of peroxidase and laccase genes, respectively. The expression of the genes was evaluated using dynamic transcriptome sequencing data from six sesame varieties, including one elite cultivar, white vs black seed varieties, and high vs low oil content varieties. Two peroxidase genes (SiPOD52 and SiPOD63) and two laccase genes (SiLAC1 and SiLAC39), well conserved within the sesame pan-genome and exhibiting consistent expression patterns within sesame varieties matching the kinetic of (+)-pinoresinol accumulation in seeds, were identified as potential (+)-pinoresinol synthase genes. Cis-acting elements of the candidate genes revealed their potential involvement in development, hormonal signaling, and response to light and other abiotic triggers. Transcription factor enrichment analysis of promoter regions showed the predominance of MYB binding sequences. The findings from this study pave the way for lignans-oriented engineering of sesame with wide potential applications in food, health and medicinal domains.

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.

Comparative genomics reveals low levels of inter- and intraspecies diversity in the causal agents of dwarf and common bunt of wheat and hint at conspecificity of Tilletia caries and T. laevis.

Tilletia caries and T. laevis, which are the causal agents of common bunt, as well as T. controversa, which causes dwarf bunt of wheat, threaten especially organic wheat farming. The three closely related fungal species differ in their teliospore morphology and partially in their physiology and infection biology. The gene content as well as intraspecies variation in these species and the genetic basis of their separation is unknown. We sequenced the genome of four T. caries, five T. controversa, and two T. laevis and extended this dataset with five publicly available ones. The genomes of the three species displayed microsynteny with up to 94.3% pairwise aligned regions excluding repetitive regions. The majority of functionally characterized genes involved in pathogenicity, life cycle, and infection of corn smut, Ustilago maydis, were found to be absent or poorly conserved in the draft genomes and the biosynthetic pathway for trimethylamine in Tilletia spp. could be different from bacteria. Overall, 75% of the identified protein-coding genes comprising 84% of the total predicted carbohydrate utilizing enzymes, 72.5% putatively secreted proteins, and 47.4% of effector-like proteins were conserved and shared across all 16 isolates. We predicted nine highly identical secondary metabolite biosynthesis gene clusters comprising in total 62 genes in all species and none were species-specific. Less than 0.1% of the protein-coding genes were species-specific and their function remained mostly unknown. Tilletia controversa had the highest intraspecies genetic variation, followed by T. caries and the lowest in T. laevis. Although the genomes of the three species are very similar, employing 241 single copy genes T. controversa was phylogenetically distinct from T. caries and T. laevis, however these two could not be resolved as individual monophyletic groups. This was in line with the genome-wide number of single nucleotide polymorphisms and small insertions and deletions. Despite the conspicuously different teliospore ornamentation of T. caries and T. laevis, a high degree of genomic identity and scarcity of species-specific genes indicate that the two species could be conspecific.

Monomer Release from Dental Resins: The Current Status on Study Setup, Detection and Quantification for In Vitro Testing.

Improvements in mechanical properties and a shift of focus towards esthetic dentistry led to the application of dental resins in various areas of dentistry. However, dental resins are not inert in the oral environment and may release monomers and other substances such as Bisphenol-A (BPA) due to incomplete polymerization and intraoral degradation. Current research shows that various monomers present cytotoxic, genotoxic, proinflammatory, and even mutagenic effects. Of these eluting substances, the elution of BPA in the oral environment is of particular interest due to its role as an endocrine disruptor. For this reason, the release of residual monomers and especially BPA from dental resins has been a cause for public concern. The assessment of patient exposure and potential health risks of dental monomers require a reliable experimental and analytical setup. However, the heterogeneous study design applied in current research hinders biocompatibility testing by impeding comparative analysis of different studies and transfer to the clinical situation. Therefore, this review aims to provide information on each step of a robust experimental and analytical in vitro setup that allows the collection of clinically relevant data and future meta-analytical evaluations.

Fusaric acid detoxification: a strategy of Gliocladium roseum involved in its antagonism against Fusarium verticillioides.

Fungal co-culture has several biotechnological applications including the discovery or the enhanced production of secondary metabolites. It is also a powerful tool aiding to elucidate the involvement of secondary metabolism in fungus-fungus interactions. The aim of this work was to investigate secondary metabolites produced when Fusarium verticillioides is co-cultured with Gliocladium roseum. Secreted metabolites were analyzed by HPLC-MS, and fusaric acid (FA) was quantified by HPLC-DAD. Four FA derivatives were identified only in the F. verticillioides-G. roseum co-culture. Mass spectrometry and one- and two-dimensional NMR spectra indicated that they were 5-butylpyridine-2-carboxylic acid methyl ester (5B2CAM), 4-(5-butylpicolinamido) butanoic acid (45BBA), methyl 4-(5-butylpicolinamido) butanoate (M45BBA), and bis(5-butyl-2-pyridinecarboxylate-N1,O2)-copper (B52P). 45BBA and M45BBA are reported for the first time and were FA biotransformation products generated by G. roseum. The antifungal activity of 5B2CAM, 45BBA, and M45BBA was evaluated in vitro against Botrytis cinerea and Aspergillus niger. They were less fungitoxic than FA, with 45BBA as the least toxic. Our results suggest that the effective antagonism exerted by G. roseum against F. verticillioides is due, at least in part, to its detoxifying ability against FA.

A comparative in vitro study on monomer release from bisphenol A-free and conventional temporary crown and bridge materials.

This study aimed to investigate the release of common monomers from two conventional and two bisphenol A (BPA)-free temporary crown and bridge materials. Cylindrical samples of all materials were prepared (N = 90; five samples for each material and cycle of analysis). All samples were immersed in high-performance liquid chromatography (HPLC)-grade water and incubated for 1 h, 12 h, 24 h, and 7 days in an incubation shaker at 37°C and 112 rpm. Extraction was performed in accordance with ISO 10993-12. Eluted monomers were detected and quantified by HPLC coupled with ultraviolet-visible spectroscopy and mass spectrometry (HPLC-UV/Vis-MS). Analysis of BPA was performed by HPLC coupled with ultraviolet-visible spectroscopy (HPLC-UV/Vis) and positive results were verified by HPLC-tandem mass spectrometry (HPLC-MS/MS). Neither bisphenol A-glycidyl methacrylate (Bis-GMA) nor BPA was quantifiable in any of the crown and bridge samples investigated in the present study. However, all samples contained triethylene glycol dimethacrylate (TEGDMA) and/or urethane dimethacrylate (UDMA) after 24 h of incubation. Statistical analysis showed that significantly more UDMA was released from the BPA-free materials than from the conventional materials. All concentrations of UDMA measured were below the effective cytotoxic concentrations previously reported. However, for a few materials, especially BPA-free temporary crown and bridge materials, the levels of UDMA were above previously reported potentially harmful concentrations for local cells. As BPA-free materials were introduced as being more biocompatible than materials containing BPA, substitution of Bis-GMA with UDMA should be further investigated.

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.

Secondary metabolites of Hülle cells mediate protection of fungal reproductive and overwintering structures against fungivorous animals.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Development of a loop-mediated isothermal amplification assay for the detection of Tilletia controversa based on genome comparison.

Tilletia controversa causing dwarf bunt of wheat is a quarantine pathogen in several countries. Therefore, its specific detection is of great phytosanitary importance. Genomic regions routinely used for phylogenetic inferences lack suitable polymorphisms for the development of species-specific markers. We therefore compared 21 genomes of six Tilletia species to identify DNA regions that were unique and conserved in all T. controversa isolates and had no or limited homology to other Tilletia species. A loop-mediated isothermal amplification (LAMP) assay for T. controversa was developed based on one of these DNA regions. The specificity of the assay was verified using 223 fungal samples comprising 43 fungal species including 11 Tilletia species, in particular 39 specimens of T. controversa, 92 of T. caries and 40 of T. laevis, respectively. The assay specifically amplified genomic DNA of T. controversa from pure cultures and teliospores. Only Tilletia trabutii generated false positive signals. The detection limit of the LAMP assay was 5 pg of genomic DNA per reaction. A test performance study that included five laboratories in Germany resulted in 100% sensitivity and 97.7% specificity of the assay. Genomic regions, specific to common bunt (Tilletia caries and Tilletia laevis together) are also provided.

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.

Lignans of Sesame (Sesamum indicum L.): A Comprehensive Review.

Major lignans of sesame sesamin and sesamolin are benzodioxol--substituted furofurans. Sesamol, sesaminol, its epimers, and episesamin are transformation products found in processed products. Synthetic routes to all lignans are known but only sesamol is synthesized industrially. Biosynthesis of furofuran lignans begins with the dimerization of coniferyl alcohol, followed by the formation of dioxoles, oxidation, and glycosylation. Most genes of the lignan pathway in sesame have been identified but the inheritance of lignan content is poorly understood. Health-promoting properties make lignans attractive components of functional food. Lignans enhance the efficiency of insecticides and possess antifeedant activity, but their biological function in plants remains hypothetical. In this work, extensive literature including historical texts is reviewed, controversial issues are critically examined, and errors perpetuated in literature are corrected. The following aspects are covered: chemical properties and transformations of lignans; analysis, purification, and total synthesis; occurrence in Seseamum indicum and related plants; biosynthesis and genetics; biological activities; health-promoting properties; and biological functions. Finally, the improvement of lignan content in sesame seeds by breeding and biotechnology and the potential of hairy roots for manufacturing lignans in vitro are outlined.

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.

Occurrence, Pathogenicity, and Mycotoxin Production of Fusarium temperatum in Relation to Other Fusarium Species on Maize in Germany.

Fusarium subglutinans is a plant pathogenic fungus infecting cereal grain crops. In 2011, the species was divided in Fusarium temperatumsp. nov. and F. subglutinans sensu stricto. In order to determine the occurrence and significance of F. temperatum and F. subglutinans on maize, a monitoring of maize ears and stalks was carried out in Germany in 2017 and 2018. Species identification was conducted by analysis of the translation elongation factor 1α (TEF-1α) gene. Ninety-four isolates of F. temperatum and eight isolates of F. subglutinans were obtained during two years of monitoring from 60 sampling sites in nine federal states of Germany. Inoculation of maize ears revealed a superior aggressiveness for F. temperatum, followed by Fusarium graminearum, Fusarium verticillioides, and F. subglutinans. On maize stalks, F. graminearum was the most aggressive species while F. temperatum and F. subglutinans caused only small lesions. The optimal temperature for infection of maize ears with F. temperatum was 24 °C and 21 °C for F. subglutinans. All strains of F. temperatum and F. subglutinans were pathogenic on wheat and capable to cause moderate to severe head blight symptoms. The assessment of mycotoxin production of 60 strains of F. temperatum cultivated on rice revealed that all strains produced beauvericin, moniliformin, fusaric acid, and fusaproliferin. The results demonstrate a higher prevalence and aggressiveness of F. temperatum compared to F. subglutinans in German maize cultivation areas.

Protection of Citrus Fruits from Postharvest Infection with Penicillium digitatum and Degradation of Patulin by Biocontrol Yeast Clavispora lusitaniae 146.

Fungal rots are one of the main causes of large economic losses and deterioration in the quality and nutrient composition of fruits during the postharvest stage. The yeast Clavispora lusitaniae 146 has previously been shown to efficiently protect lemons from green mold caused by Penicillium digitatum. In this work, the effect of yeast concentration and exposure time on biocontrol efficiency was assessed; the protection of various citrus fruits against P. digitatum by C. lusitaniae 146 was evaluated; the ability of strain 146 to degrade mycotoxin patulin was tested; and the effect of the treatment on the sensory properties of fruits was determined. An efficient protection of lemons was achieved after minimum exposure to a relatively low yeast cell concentration. Apart from lemons, the yeast prevented green mold in grapefruits, mandarins, oranges, and tangerines, implying that it can be used as a broad-range biocontrol agent in citrus. The ability to degrade patulin indicated that strain 146 may be suitable for the control of further Penicillium species. Yeast treatment did not alter the sensory perception of the aroma of fruits. These results corroborate the potential of C. lusitaniae 146 for the control of postharvest diseases of citrus fruits and indicate its suitability for industrial-scale fruit processing.

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.