Ecological Diversity of Bacterial Rhizomicrobiome Core during the Growth of Selected Wheat Cultivars.

One of the latest ecological concepts is the occurrence of a biased rhizosphere of microorganisms recruited mostly through interactions among various components of the rhizosphere, including plant roots and the bulk soil microbiome. We compared the diverse attributes of the core microbiome of wheat rhizosphere communities with wheat (W) and legume (L) forecrops determined by three different methods in this study (membership, composition, and functionality). The conclusions of the three methods of microbiome core definition suggest the presence of generalists, i.e., some representative microorganisms from Proteobacteria, Actinobacteria, Hypomicrobiaceae, Bradyrhizobiaceae, Sphingomonas sp., in the wheat rhizomicrobiome. The relative abundance of the core microbiome accounted for 0.1976% (W) and 0.334% (L)-membership method and 6.425% (W) and 4.253% (L)-composition method. Additionally, bacteria of the specialist group, such as Rhodoplanes sp., are functionally important in the rhizomicrobiome core. This small community is strongly connected with other microbes and is essential for maintenance of the sustainability of certain metabolic pathways.

Changes in the mycobiome structure in response to reduced nitrogen fertilization in two cropping systems of maize.

Nitrogen (N) is an essential element for plant productivity; hence, it is abundantly applied to the soil in the form of organic or chemical fertilizers, which consequently have a negative impact on the environment. Therefore, the main objective of our study was to investigate the structure and richness of the soil mycobiome in response to reduced nitrogen fertilization under two cropping systems: plowing (P) and no-till (NT). Moreover, the scope of the study perfectly falls into the EU "From Field to Table" strategy, which recommends a 20 % reduction of nitrogen fertilization of agricultural soils by 2030. In our study, the samples were collected twice during a single growing season: before maize sowing (without fertilization) and after harvesting the crop (four different fertilization rates). The mycobiome structure was identified based on the next generation sequencing (NGS) technique. Overall, our research has proved that the cropping system is important in terms of the formation of the fungal mycobiome structure and relative abundance. In addition, we confirmed that soil properties have a significant impact on fungal communities. We determined that a 20 % lower nitrogen fertilization rate (92.0 kg N ha-1) had a positive effect on the abundance of fungal communities. Moreover, the highest biodiversity at each of the taxonomic levels tested (phylum, class, genus) in the NT system and at the class and genus levels in the P system was also evidenced at the 20 % lower N fertilization rate. We also recommended potential indicators confirming the positive impact of reduced fertilization in two cropping systems: plowing - Epicoccum, Metarhizium, Mycosphaerella, and Paraconiothyrium and no-till - Peziza, Podospora, Metarhizium, Trechispora, and Umbelopsis.

Functional and Seasonal Changes in the Structure of Microbiome Inhabiting Bottom Sediments of a Pond Intended for Ecological King Carp Farming.

The main goal of the study was to determine changes in the bacterial structure in bottom sediments occurring over the seasons of the year and to estimate microbial metabolic activity. Bottom sediments were collected four times in the year (spring, summer, autumn, and winter) from 10 different measurement points in Cardinal Pond (Slesin, NW Poland). The Next-Generation Sequencing (MiSeq Illumina) and Community-Level Physiological Profiling techniques were used for identification of the bacterial diversity structure and bacterial metabolic and functional activities over the four seasons. It was evident that Proteobacteria, Acidobacteria, and Bacteroidetes were the dominant phyla, while representatives of Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria predominated at the class level in the bottom sediments. An impact of the season on biodiversity and metabolic activity was revealed with the emphasis that the environmental conditions in summer modified the studied parameters most strongly. Carboxylic and acetic acids and carbohydrates were metabolized most frequently, whereas aerobic respiration I with the use of cytochrome C was the main pathway used by the microbiome of the studied bottom sediments.

Phenotype Switching in Metal-Tolerant Bacteria Isolated from a Hyperaccumulator Plant.

As an adaptation to unfavorable conditions, microorganisms may represent different phenotypes. Azolla filiculoides L. is a hyperaccumulator of pollutants, but the functions of its microbiome have not been well recognized to date. We aimed to reveal the potential of the microbiome for degradation of organic compounds, as well as its potential to promote plant growth in the presence of heavy metals. We applied the BiologTM Phenotypic Microarrays platform to study the potential of the microbiome for the degradation of 96 carbon compounds and stress factors and assayed the hydrolytic potential and auxin production by the microorganisms in the presence of Pb, Cd, Cr (VI), Ni, Ag, and Au. We found various phenotype changes depending on the stress factor, suggesting a possible dual function of the studied microorganisms, i.e., in bioremediation and as a biofertilizer for plant growth promotion. Delftia sp., Staphylococcus sp. and Microbacterium sp. exhibited high efficacy in metabolizing organic compounds. Delftia sp., Achromobacter sp. and Agrobacterium sp. were efficient in enzymatic responses and were characterized by metal tolerant. Since each strain exhibited individual phenotype changes due to the studied stresses, they may all be beneficial as both biofertilizers and bioremediation agents, especially when combined in one biopreparation.

Bacterial Endophytes of Spring Wheat Grains and the Potential to Acquire Fe, Cu, and Zn under Their Low Soil Bioavailability.

Wheat grains are usually low in essential micronutrients. In resolving the problem of grain micronutritional quality, microbe-based technologies, including bacterial endophytes, seem to be promising. Thus, we aimed to (1) isolate and identify grain endophytic bacteria from selected spring wheat varieties (bread Oksamyt myronivs'kyi, Struna myronivs'ka, Dubravka, and emmer Holikovs'ka), which were all grown in field conditions with low bioavailability of microelements, and (2) evaluate the relationship between endophytes' abilities to synthesize auxins and the concentration of Fe, Zn, and Cu in grains. The calculated biological accumulation factor (BAF) allowed for comparing the varietal ability to uptake and transport micronutrients to the grains. For the first time, bacterial endophytes were isolated from grains of emmer wheat T. turgidum subsp. dicoccum. Generally, the 12 different isolates identified in the four varieties belonged to the genera Staphylococcus, Pantoea, Sphingobium, Bacillus, Kosakonia, and Micrococcus (NCBI accession numbers: MT302194-MT302204, MT312840). All the studied strains were able to synthesize the indole-related compounds (IRCs; max: 16.57 µg∙mL-1) detected using the Salkowski reagent. The IRCs produced by the bacterial genera Pantoea spp. and Bacillus spp. isolated from high-yielding Oksamyt myronivs'kyi and Holikovs'ka grains may be considered as one of the determinants of the yield of wheat and its nutritional characteristics.

A Comprehensive Analysis Using Colorimetry, Liquid Chromatography-Tandem Mass Spectrometry and Bioassays for the Assessment of Indole Related Compounds Produced by Endophytes of Selected Wheat Cultivars.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS), colorimetry, and bioassays were employed for the evaluation of the ability of endophytic bacterial strains to synthesize indole-related compounds (IRCs) and in particular indole-3-acetic acid (IAA). A total of 54 endophytic strains belonging to seven bacterial genera isolated from tissues of common and spelt wheat cultivars were studied. The endophytic bacteria isolated from different tissues of the tested wheat types were capable of IRCs production, including IAA, which constituted from 1.75% to 52.68% of all IRCs, in in vitro conditions via the tryptophan dependent pathway. The selected post-culture medium was also examined using a plant bioassay. Substantial growth of wheat coleoptile segments treated with the bacterial post-culture medium was observed in several cases. Our data suggest that the studied endophytic bacteria produce auxin-type compounds to support plant development. Summarizing, our approach to use three complementary methods for estimation of IRCs in different endophytic strains provides a comprehensive picture of their effect on wheat growth.

Fungal Indicators of Sensitivity and Resistance to Long-Term Maize Monoculture: A Culture-Independent Approach.

Although fungi are regarded as very important components of soils, the knowledge of their community in agricultural (monocultural) soils is still limited. This indicates that soil fungal communities are investigated less intensively than bacteria. Therefore, the main goal of this paper was to evaluate the fungal mycobiome structure in monoculture soils in a culture-independent approach. Firstly, the study was conducted to identify the core mycobiome composition and its variability at different stages of the maize growing season (spring, summer, and autumn). Secondly, we identified and recommended fungal indicators of both sensitivity and resistance to long-term maize monoculture. Two neighboring fields from the Potulicka Foundation area were selected for the study: K20 sown with a Gorzow mixture (intercropping mixture) to improve soil quality after a maize monoculture in 2020 and K21, where long-term (over 30 years) monoculture cultivation was continued. The basic chemical features [acidity, redox potential, total organic carbon (TOC), and moisture] of soils were determined, fungal genetic diversity was assessed by ITS next generation sequencing (NGS) analyses, and biodiversity indices were calculated. The results of the NGS technique facilitated recognition and classification of the fungal mycobiome to the taxonomic genus level and changes in the fungal structure in the three periods (spring, summer, and autumn) were assessed. It was evidenced that the mycobiome composition was dependent on both the seasons and the agricultural practices. It was also found that even a 1-year break in the monoculture in favor of an intercropping mixture improved soil properties thus contributing to higher biodiversity. Mortierella was recommended as a potential indicator of sensitivity to long-term maize cultivation, whereas Solicoccozyma and Exophiala were proposed as indicators of resistance to long-term maize cultivation. We proved that the precision farming principles applied on the Potulicka Foundation farm had a very positive effect on fungal biodiversity, which was high even in the long-term maize monoculture field. Therefore, the monoculture cultivation carried out in this way does not induce biological degradation of monoculture soils but preserves their good biological quality.

New Insight into the Composition of Wheat Seed Microbiota.

Endophytes are associated with host plants throughout their life history from seed germination to fruit development. One of the most important plant organs colonized by endophytic microbiota is the seed. The aim of this study was to determine the structure of the seed core microbiome inhabiting the endosperms and embryos of eight wheat cultivars with the use of a culture-independent technique. The seeds of Triticum aestivum L. cv. Hondia, Wilejka, STH, Opcja, Tybalt, Euforia and Triticum spelta L. cv. Rokosz and Schwabencorn (producer: Plant Breeding Strzelce Sp. z o.o. Group IHAR) were studied. Rokosz and Hondia were cultured in vitro and in vivo to identify obligatory bacterial endophytes. A restrictive analysis of reads originating from the in vitro plants has demonstrated that the bacterial genera Paenibacillus and Propionibacterium inhabiting Rokosz and Hondia plants have a status of obligatory microorganisms. Greater biodiversity of seed-borne endophytes was found in the seed endosperms than in the embryos. The multiple comparison analysis of the OTU abundance indicated that the seed part significantly influenced the relative abundance. The seed-born microbiome is not statistically significantly dependent on the wheat cultivars; however, it cannot be claimed that every wheat seed is the same.

Azolla filiculoides L. as a source of metal-tolerant microorganisms.

The metal hyperaccumulator Azolla filiculoides is accompanied by a microbiome potentially supporting plant during exposition to heavy metals. We hypothesized that the microbiome exposition to selected heavy metals will reveal metal tolerant strains. We used Next Generation Sequencing technique to identify possible metal tolerant strains isolated from the metal-treated plant (Pb, Cd, Cr(VI), Ni, Au, Ag). The main dominants were Cyanobacteria and Proteobacteria constituting together more than 97% of all reads. Metal treatment led to changes in the composition of the microbiome and showed significantly higher richness in the Pb-, Cd- and Cr-treated plant in comparison with other (95-105 versus 36-44). In these treatments the share of subdominant Actinobacteria (0.4-0.8%), Firmicutes (0.5-0.9%) and Bacteroidetes (0.2-0.9%) were higher than in non-treated plant (respectively: 0.02, 0.2 and 0.001%) and Ni-, Au- and Ag-treatments (respectively: <0.4%, <0.2% and up to 0.2%). The exception was Au-treatment displaying the abundance 1.86% of Bacteroidetes. In addition, possible metal tolerant genera, namely: Acinetobacter, Asticcacaulis, Anabaena, Bacillus, Brevundimonas, Burkholderia, Dyella, Methyloversatilis, Rhizobium and Staphylococcus, which form the core microbiome, were recognized by combining their abundance in all samples with literature data. Additionally, the presence of known metal tolerant genera was confirmed: Mucilaginibacter, Pseudomonas, Mycobacterium, Corynebacterium, Stenotrophomonas, Clostridium, Micrococcus, Achromobacter, Geobacter, Flavobacterium, Arthrobacter and Delftia. We have evidenced that A. filiculoides possess a microbiome whose representatives belong to metal-resistant species which makes the fern the source of biotechnologically useful microorganisms for remediation processes.

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. 'Hondia') and the first report of microbiota in Triticum spelta L. (cv. 'Rokosz').

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. 'Hondia') and Triticum spelta L. (cv. 'Rokosz'), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1). It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the 'Hondia' cv., whereas Pedobacter and Duganella constituted the core microbiome in the 'Rokosz' cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.

Changes in the Substrate Source Reveal Novel Interactions in the Sediment-Derived Methanogenic Microbial Community.

Methanogenesis occurs in many natural environments and is used in biotechnology for biogas production. The efficiency of methane production depends on the microbiome structure that determines interspecies electron transfer. In this research, the microbial community retrieved from mining subsidence reservoir sediment was used to establish enrichment cultures on media containing different carbon sources (tryptone, yeast extract, acetate, CO2/H2). The microbiome composition and methane production rate of the cultures were screened as a function of the substrate and transition stage. The relationships between the microorganisms involved in methane formation were the major focus of this study. Methanogenic consortia were identified by next generation sequencing (NGS) and functional genes connected with organic matter transformation were predicted using the PICRUSt approach and annotated in the KEGG. The methane production rate (exceeding 12.8 mg CH4 L-1 d-1) was highest in the culture grown with tryptone, yeast extract, and CO2/H2. The analysis of communities that developed on various carbon sources casts new light on the ecophysiology of the recently described bacterial phylum Caldiserica and methanogenic Archaea representing the genera Methanomassiliicoccus and Methanothrix. Furthermore, it is hypothesized that representatives of Caldiserica may support hydrogenotrophic methanogenesis.

Methanotrophic Bacterial Biomass as Potential Mineral Feed Ingredients for Animals.

Microorganisms play an important role in animal nutrition, as they can be used as a source of food or feed. The aim of the study was to determine the nutritional elements and fatty acids contained in the biomass of methanotrophic bacteria. Four bacterial consortia composed of Methylocystis and Methylosinus originating from Sphagnum flexuosum (Sp1), S. magellanicum (Sp2), S. fallax II (Sp3), S. magellanicum IV (Sp4), and one composed of Methylocaldum, Methylosinus, and Methylocystis that originated from coalbed rock (Sk108) were studied. Nutritional elements were determined using the flame atomic absorption spectroscopy technique after a biomass mineralization stage, whereas the fatty acid content was analyzed with the GC technique. Additionally, the growth of biomass and dynamics of methane consumption were monitored. It was found that the methanotrophic biomass contained high concentrations of K, Mg, and Fe, i.e., approx. 9.6-19.1, 2.2-7.6, and 2.4-6.6 g kg-1, respectively. Consequently, the biomass can be viewed as an appropriate feed and/or feed additive for supplementation with macroelements and certain microelements. Moreover, all consortia demonstrated higher content of unsaturated acids than saturated ones. Thus, methanotrophic bacteria seem to be a good solution, in natural supplementation of animal diets.

Enrichment culture and identification of endophytic methanotrophs isolated from peatland plants.

Aerobic methane-oxidizing bacteria (MOB) are an environmentally significant group of microorganisms due to their role in the global carbon cycle. Research conducted over the past few decades has increased the interest in discovering novel genera of methane-degrading bacteria, which efficiently utilize methane and decrease the global warming effect. Moreover, methanotrophs have more promising applications in environmental bioengineering, biotechnology, and pharmacy. The investigations were undertaken to recognize the variety of endophytic methanotrophic bacteria associated with Carex nigra, Vaccinium oxycoccus, and Eriophorum vaginatum originating from Moszne peatland (East Poland). Methanotrophic bacteria were isolated from plants by adding sterile fragments of different parts of plants (roots and stems) to agar mineral medium (nitrate mineral salts (NMS)) and incubated at different methane values (1-20% CH4). Single colonies were streaked on new NMS agar media and, after incubation, transferred to liquid NMS medium. Bacterial growth dynamics in the culture solution was studied by optical density-OD600 and methane consumption. Changes in the methane concentration during incubation were controlled by the gas chromatography technique. Characterization of methanotrophs was made by fluorescence in situ hybridization (FISH) with Mg705 and Mg84 for type I methanotrophs and Ma450 for type II methanotrophs. Identification of endophytes was performed after 16S ribosomal RNA (rRNA) and mmoX gene amplification. Our study confirmed the presence of both types of methanotrophic bacteria (types I and II) with the predominance of type I methanotrophs. Among cultivable methanotrophs, there were different strains of the genus Methylomonas and Methylosinus. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged from 0.463 ± 0.067 to 5.928 ± 0.169 µmol/L CH4/mL/day.

Metagenomic Analysis of Some Potential Nitrogen-Fixing Bacteria in Arable Soils at Different Formation Processes.

The main goal of the study was to determine the diversity of the potential nitrogen-fixing (PNF) bacteria inhabiting agricultural (A) soils versus wastelands serving as controls (C). The soils were classified into three groups based on the formation process: autogenic soils (Albic Luvisols, Brunic Arenosols, Haplic Phaeozem) formed on loess material, hydrogenic soils (Mollic Gleysols, Eutric Fluvisol, Eutric Histosol) formed under the effect of stagnant water and lithogenic soils (Rendzina Leptosols) formed on limestone. In order to determine the preferable conditions for PNF bacteria, the relationships between the soil chemical features and bacterial operational taxonomic units (OTUs) were tested. Additionally, the nitrogen content and fertilisation requirement of the lithogenic (LG), autogenic (AG) and hydrogenic (HG) soils were discussed. The composition of the bacterial communities was analysed with the next-generation sequencing (NGS) by the Ion Torrent™ technology. The sequences were clustered into OTU based on a 99 % similarity threshold. The arable soils tested were distinctly dominated by ß-Proteobacteria representatives of PNF bacteria belonging to the genus Burkholderia. Bacteria from the α-Proteobacteria class and Devosia genus were subdominants. A free-living Cyanobacteria population dominated in A rather than in C soils. We have found that both soil agricultural management and soil formation processes are the most conducive factors for PNF bacteria, as a majority of these microorganisms inhabit the AG group of soils, whilst the LG soils with the lowest abundance of PNF bacteria revealed the need for additional mineral fertilisation. Our studies have also indicated that there are close relationships between soil classification with respect to soil formation processes and PNF bacteria preference for occupation of soil niches.

Biological Activity of Autochthonic Bacterial Community in Oil-Contaminated Soil.

Soil microbial communities play an important role in the biodegradation of different petroleum derivates, including hydrocarbons. Also other biological factors such as enzyme and respiration activities and microbial abundance are sensitive to contamination with petroleum derivates. The aim of this study was to evaluate the response of autochthonic microbial community and biological parameters (respiration, dehydrogenase and catalase activities, total microorganisms count) on contamination with car fuels and engine oils. The surface layer (0-20 cm) of Mollic Gleysol was used for the experiment. In laboratory conditions, soil was contaminated with the following petroleum substances: car fuels (petrol, diesel) and car engine oils (new and waste-after 10,000 km). The results demonstrated that, among the investigated hydrocarbon substances, petrol addition seemed to be the most toxic for the microbial activity of the investigated soil. The toxicity of the used hydrocarbon substances to microorganisms might be summarized as follows: diesel > new oil > waste oil > petrol. Species belonging to the genera Micrococcus and Rhodococcus were noted as the major autochthonic bacteria being present in soil contaminated with new automobile oil, whereas species of the genera Bacillus sp. and Paenibacillus sp. were identified in the combination treated with waste oil.

Cultivation and detection of endophytic aerobic methanotrophs isolated from Sphagnum species as a perspective for environmental biotechnology.

Enriched cultures of microorganisms are an essential step in the production of inoculum of these organisms for biotechnology and bioengineering. The potential application of methanotrophic microorganisms for removal of methane produced from landfills and coal mines as well as biodegradation of toxic compounds has been widely studied. Therefore, searching for new sources of methanotrophs can contribute to increasing the possibilities of biotechnology and bioengineering. Enrichment cultures of endophytic methanotrophs from Sphagnum sp. were initiated in NMS medium, a most widely used medium for cultivation of methanotrophic bacteria from various environments proposed in 1970 by Whittenbury. Incubation was carried out at 10, 20, 30, and 37°C with vigorous shaking on a shaker (180 rpm). The source of carbon and energy for endophytes were methane at the concentration range between 1-20%. It appeared that the consortium of endophytic bacteria grew only at the temperature of 20 and 30°C. During the culture of endophytes, the measurements of gas concentration showed a steady loss of methane and oxygen, as well as accumulation of carbon dioxide as a CH4 oxidation product. The use of FISH has made characterization of endophytic consortia possible. It turned out that the population of endophytes consists of type I and II methanotrophs as well as associated non-methanotrophic bacteria. Furthermore, we determined the potential of the examined bacteria for methane oxidation, which ranged up to 4,7 µMCH4 per ml of the population of endophytes per day.