Soil Chemistry and Soil History Significantly Structure Oomycete Communities in Brassicaceae Crop Rotations.

Oomycetes are critically important in soil microbial communities, especially for agriculture, where they are responsible for major declines in yields. Unfortunately, oomycetes are vastly understudied compared to bacteria and fungi. As such, our understanding of how oomycete biodiversity and community structure vary through time in the soil remains poor. Soil history established by previous crops is one factor known to structure other soil microbes, but this has not been investigated for its influence on oomycetes. In this study, we established three different soil histories in field trials; the following year, these plots were planted with five different Brassicaceae crops. We hypothesized that the previously established soil histories would structure different oomycete communities, regardless of their current Brassicaceae crop host, in both the roots and rhizosphere. We used a nested internal transcribed spacer amplicon strategy incorporated with MiSeq metabarcoding, where the sequencing data was used to infer amplicon sequence variants of the oomycetes present in each sample. This allowed us to determine the impact of different soil histories on the structure and biodiversity of the oomycete root and rhizosphere communities from the five different Brassicaceae crops. We found that each soil history structured distinct oomycete rhizosphere communities, regardless of different Brassicaceae crop hosts, while soil chemistry structured the oomycete communities more during a dry year. Interestingly, soil history appeared specific to oomycetes but was less influential for bacterial communities previously identified from the same samples. These results advance our understanding of how different agricultural practices and inputs can alter edaphic factors to impact future oomycete communities. Examining how different soil histories endure and impact oomycete biodiversity will help clarify how these important communities may be assembled in agricultural soils. IMPORTANCE Oomycetes cause global plant diseases that result in substantial losses, yet they are highly understudied compared to other microbes, like fungi and bacteria. We wanted to investigate how past soil events, like changing crops in rotation, would impact subsequent oomycete communities. We planted different oilseed crops in three different soil histories and found that each soil history structured a distinct oomycete community regardless of which new oilseed crop was planted, e.g., oomycete communities from last year's lentil plots were still detected the following year regardless of which new oilseed crops we planted. This study demonstrated how different agricultural practices can impact future microbial communities differently. Our results also highlight the need for continued monitoring of oomycete biodiversity and quantification.

Brassicaceae host plants mask the feedback from the previous year's soil history on bacterial communities, except when they experience drought.

Soil history operates through time to influence the structure and biodiversity of soil bacterial communities. Examining how different soil histories endure will help clarify the rules of bacterial community assembly. In this study, we established three different soil histories in field trials; the following year these plots were planted with five different Brassicaceae species. We hypothesized that the previously established soil histories would continue to structure the subsequent Brassicaceae bacterial root and rhizosphere communities. We used a MiSeq 16S rRNA metabarcoding strategy to determine the impact of different soil histories on the structure and biodiversity of the bacterial root and rhizosphere communities from the five different Brassicaceae host plants. We found that the Brassicaceae hosts were consistently significant factors in structuring the bacterial communities. Four host plants (Sinapis alba, Brassica napus, B. juncea, B. carinata) formed similar bacterial communities, regardless of different soil histories. Camelina sativa host plants structured phylogenetically distinct bacterial communities compared to the other hosts, particularly in their roots. Soil history established the previous year was only a significant factor for bacterial community structure when the feedback of the Brassicaceae host plants was weakened, potentially due to limited soil moisture during a dry year. Understanding how soil history is involved in the structure and biodiversity of bacterial communities through time is a limitation in microbial ecology and is required for employing microbiome technologies in improving agricultural systems.

Long-term effects of nitrogen and phosphorus fertilization on soil microbial community structure and function under continuous wheat production.

Soil microorganisms play a critical role in the biosphere, and the influence of cropland fertilization on the evolution of soil as a living entity is being actively documented. In this study, we used a shotgun metagenomics approach to globally expose the effects of 50-year N and P fertilization of wheat on soil microbial community structure and function, and their potential involvement in overall N cycling. Nitrogen (N) fertilization increased alpha diversity in archaea and fungi while reducing it in bacteria. Beta diversity of archaea, bacteria and fungi, as well as soil function, were also mainly driven by N fertilization. The abundance of archaea was negatively impacted by N fertilization while bacterial and fungal abundance was increased. The responses of N metabolism-related genes to fertilization differed in archaea, bacteria and fungi. All archaeal N metabolic processes were decreased by N fertilization, while denitrification, assimilatory nitrate reduction and organic-N metabolism were highly increased by N fertilization in bacteria. Nitrate assimilation was the main contribution of fungi to N cycling. Thaumarchaeota and Halobacteria in archaea; Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria in bacteria; and Sordariomycetes in fungi participated dominantly and widely in soil N metabolic processes.

Expression of N-cycling genes of root microbiomes provides insights for sustaining oilseed crop production.

Agricultural production is dependent on inputs of nitrogen (N) whose cycle relies on soil and crop microbiomes. Crop diversification has increased productivity; however, its impact on the expression of microbial genes involved in N-cycling pathways remains unknown. Here, we assessed N-cycling gene expression patterns in the root and rhizosphere microbiomes of five oilseed crops as influenced by three 2-year crop rotations. The first phase consisted of fallow, lentil or wheat, and the second phase consisted of one of five oilseed crops. Expression of bacterial amoA, nirK and nirS genes showed that the microbiome of Ethiopian mustard had the lowest and that of camelina the highest potential for N loss. A preceding rotation phase of lentil significantly increased the expression of nifH gene by 23% compared with wheat and improved nxrA gene expression by 51% with chemical fallow in the following oilseed crops respectively. Lentil substantially increased biological N2 fixation and reduced denitrification in the following oilseed crops. Our results also revealed that most N-cycling gene transcripts are more abundant in the microbiomes associated with roots than with the rhizosphere. The outcome of our investigation brings a new level of understanding on how crop diversification and rotation sequences are related to N-cycling in annual cropping systems.

Intermittent water stress favors microbial traits that better help wheat under drought.

Microorganisms can improve plant resistance to drought through various mechanisms, such as the production of plant hormones, osmolytes, antioxidants, and exopolysaccharides. It is, however, unclear how previous exposure to water stress affects the functional capacity of the soil microbial community to help plants resist drought. We compared two soils that had either a continuous or intermittent water stress history (WSH) for almost 40 years. We grew wheat in these soils and subjected it to water stress, after which we collected the rhizosphere soil and shotgun sequenced its metagenome. Wheat growing in soil with an intermittent WSH maintained a higher biomass when subjected to water stress. Genes related to indole-acetic acid and osmolyte production were more abundant in the metagenome of the soil with an intermittent WSH as compared to the soil with a continuous WSH. We suggest that an intermittent WSH selects traits beneficial for life under water stress.

Impact of land use on arbuscular mycorrhizal fungal communities in rural Canada.

The influence of land use on soil bio-resources is largely unknown. We examined the communities of arbuscular mycorrhizal (AM) fungi in wheat-growing cropland, natural areas, and seminatural areas along roads. We sampled the Canadian prairie extensively (317 sites) and sampled 20 sites in the Atlantic maritime ecozone for comparison. The proportions of the different AM fungal taxa in the communities found at these sites varied with land use type and ecozones, based on pyrosequencing of 18S rRNA gene (rDNA) amplicons, but the lists of AM fungal taxa obtained from the different land use types and ecozones were very similar. In the prairie, the Glomeraceae family was the most abundant and diverse family of Glomeromycota, followed by the Claroideoglomeraceae, but in the Atlantic maritime ecozone, the Claroideoglomeraceae family was most abundant. In the prairie, species richness and Shannon's diversity index were highest in roadsides, whereas cropland had a higher degree of species richness than roadsides in the Atlantic maritime ecozone. The frequencies of occurrence of the different AM fungal taxa in croplands in the prairie and Atlantic maritime ecozones were highly correlated, but the AM fungal communities in these ecozones had different structures. We conclude that the AM fungal resources of soils are resilient to disturbance and that the richness of AM fungi under cropland management has been maintained, despite evidence of a structural shift imposed by this type of land use. Roadsides in the Canadian prairie are a good repository for the conservation of AM fungal diversity.

Effects of nitrogen fertilization and a commercial arbuscular mycorrhizal fungal inoculant on root rot and agronomic production of pea and lentil crops.

In the Canadian prairies, pulse crops such as field pea (Pisum sativum L.) and lentil (Lens culinaris L.) are economically important and widely grown. However, in recent years, root rot, caused by a variety of fungal and oomycete pathogens, including Aphanomyces euteiches, has become a limiting factor on yield. In this study, we examined the impacts of nitrogen (N) fertilization and a commercial arbuscular mycorrhizal fungal (AMF) inoculant on pea and lentil plant health and agronomic production at three locations in Saskatchewan: Swift Current, Indian Head and Melfort. The AMF inoculation had no impact on root rot severity, and therefore is not considered a reliable method to manage root rot in pea and lentil. In contrast, N fertilization led to reductions in root rot in Swift Current, but not the other two sites. However, N fertilization did reduce nodulation. When both pea and lentil are considered, the abundance of A. euteiches in soil increased from pre-seeding to mid-bloom. A negative correlation between soil pH and disease severity was also observed. The high between-site variability highlights the importance of testing root rot mitigation strategies under multiple soil conditions to develop site-specific recommendations. Use of N fertilizer as a root rot management strategy merits further exploration, including investigation into its interactions with other management strategies, soil properties, and costs and benefits.

A multivariate analysis of variation in genome size and endoreduplication in angiosperms reveals strong phylogenetic signal and association with phenotypic traits.

Genome size (C-value) and endopolyploidy (endoreduplication index, EI) are known to correlate with various morphological and ecological traits, in addition to phylogenetic placement. A phylogenetically controlled multivariate analysis was used to explore the relationships between DNA content and phenotype in angiosperms. Seeds from 41 angiosperm species (17 families) were grown in a common glasshouse experiment. Genome size (2C-value and 1Cx-value) and EI (in four tissues: leaf, stem, root, petal) were determined using flow cytometry. The phylogenetic signal was calculated for each measure of DNA content, and phylogenetic canonical correlation analysis (PCCA) explored how the variation in genome size and EI was correlated with 18 morphological and ecological traits. Phylogenetic signal (λ) was strongest for EI in all tissues, and λ was stronger for the 2C-value than the 1Cx-value. PCCA revealed that EI was correlated with pollen length, stem height, seed mass, dispersal mechanism, arbuscular mycorrhizal association, life history and flowering time, and EI and genome size were both correlated with stem height and life history. PCCA provided an effective way to explore multiple factors of DNA content variation and phenotypic traits in a phylogenetic context. Traits that were correlated significantly with DNA content were linked to plant competitive ability.

DNA content variation in monilophytes and lycophytes: large genomes that are not endopolyploid.

Less than 1% of known monilophytes and lycophytes have a genome size estimate, and substantially less is known about the presence and prevalence of endopolyploid nuclei in these groups. Thirty-one monilophyte species (including three horsetails) and six lycophyte species were collected in Ontario, Canada. Using flow cytometry, genome size and degree of endopolyploidy were estimated for 37 species. Across the five orders covered, 1Cx-values averaged 4.2 pg in the Lycopodiales, 18.1 pg for the Equisetales, 5.06 pg for a single representative of the Ophioglossales, 14.3 pg for the Osmundales, and 7.06 pg for the Polypodiales. There was no indication of endoreduplication in any of the leaf, stem, or root tissue analyzed. This information is essential to our understanding of DNA content evolution in land plants.

The response of wheat and its microbiome to contemporary and historical water stress in a field experiment.

In a field experiment, we evaluated the impact of 37 years of contrasting water stress history on the microbial response in various plant compartments at two distinct developmental stages when four wheat genotypes were exposed to contemporary water stress. Seeds were collected and sampled at the end of the experiment to characterize endophytic and epiphytic microbial communities. Amplicon sequencing data revealed that plant development stage and water stress history were the main factors shaping the microbiome of the major plant parts in response to contemporary water limitation. Our results indicate that seeds can become colonized by divergent microbial communities within a single generation based on the initial pool of microbes as determined by historical contingencies, which was modulated by the contemporary environmental conditions and the plant genotype. Such information is essential to incorporate microbial-based strategies into conventional plant breeding to enhance plant resistance to stress.

Soil Depth Significantly Shifted Microbial Community Structures and Functions in a Semiarid Prairie Agroecosystem.

The North American Great Plains cover a large area of the Nearctic ecozone, and an important part of this biome is semiarid. The sustainable intensification of agriculture that is necessary to produce food for an ever-increasing world population requires knowledge of the taxonomic and functional structure of the soil microbial community. In this study, we investigated the influence of soil depth on the composition and functions of the microbial communities hosted in agricultural soils of a semiarid agroecosystem, using metagenomic profiling, and compared them to changes in soil chemical and physical properties. Shotgun sequencing was used to determine the composition and functions of the soil microbial community of 45 soil samples from three soil depths (0-15 cm, 15-30 cm, and 30-60 cm) under different agricultural land use types (native prairie, seeded prairie, and cropland) in southwest Saskatchewan. Analysis of community composition revealed the declining abundance of phyla Verrucomicrobia, Bacteroidetes, Chlorophyta, Bacillariophyta, and Acidobacteria with soil depth, whereas the abundance of phyla Ascomycota, Nitrospirae, Planctomycetes, and Cyanobacteria increased with soil depth. Soil functional genes related to nucleosides and nucleotides, phosphorus (P) metabolism, cell division and cell cycle, amino acids and derivatives, membrane transport, and fatty acids were particularly abundant at 30-60 cm. In contrast, functional genes related to DNA and RNA metabolism, metabolism of nitrogen, sulfur and carbohydrates, and stress response were more abundant in the top soil depth. The RDA analysis of functional genes and soil physico-chemical properties revealed a positive correlation between phages and soil organic P concentrations. In the rooting zone of this semiarid agroecosystem, soil microbes express variable structural patterns of taxonomic and functional diversity at different soil depths. This study shows that the soil microbial community is structured by soil depth and physicochemical properties, with the middle soil depth being an intermediate transition zone with a higher taxonomic diversity. Our results suggest the co-existence of various microbial phyla adapted to upper and lower soil depths in an intermediate-depth transition zone.

The mycorrhizal status and colonization of 26 tree species growing in urban and rural environments.

Urban environments are highly disturbed and fragmented ecosystems that commonly have lower mycorrhizal fungal species richness and diversity compared to rural or natural ecosystems. In this study, we assessed whether the mycorrhizal status and colonization of trees are influenced by the overall environment (rural vs. urban) they are growing in. Soil cores were collected from the rhizosphere of trees growing in urban and rural environments around southern Ontario. Roots were extracted from the soil cores to determine whether the trees were colonized by arbuscular mycorrhizal fungi, ectomycorrhizal fungi, or both, and to quantify the percent colonization of each type of mycorrhizal fungi. All 26 tree species were colonized by arbuscular mycorrhizal fungi, and seven tree species were dually colonized by arbuscular mycorrhizal and ectomycorrhizal fungi. Overall, arbuscular mycorrhizal and ectomycorrhizal fungal colonization was significantly (p < 0.001) lower in trees growing in urban compared to rural environments. It is not clear what 'urban' factors are responsible for the reduction in mycorrhizal fungal colonization; more research is needed to determine whether inoculating urban trees with mycorrhizal fungi would increase colonization levels and growth of the trees.

Pulse Frequency in Crop Rotations Alters Soil Microbial Community Networks and the Relative Abundance of Fungal Plant Pathogens.

Including pulse crops in cereal-based cropping systems has become a widely accepted and useful agronomic practice to increase crop diversification and biologically fixed nitrogen in agroecosystems. However, there is a lack of knowledge regarding how the intensification of pulses in crop rotations influence soil microbial communities. In this study, we used an amplicon sequencing approach to examine the bulk and rhizosphere soil bacterial and fungal communities from the wheat (Triticum aestivum L.) phase (final year of 4 years rotations) of a long-term pulse intensification field trial in the semi-arid region of the Canadian Prairies. Our results revealed pulse frequency had a minimal impact on microbial α-diversity, but caused a significant shift in the composition of the fungal (rhizosphere and bulk soil) and bacterial (bulk soil) communities. This effect was the most pronounced in the Ascomycete and Bacteroidete communities. Increasing pulse frequency also promoted a higher proportion of fungal pathotrophs in the bulk soil, particularly those putatively identified as plant pathogens. The network analysis revealed that rotations with higher pulse frequency promoted increased competition within the soil microbial networks in the rhizosphere and bulk soil. However, we also detected more negative interactions among the dominant pathotrophic taxa with increased pulse frequency, suggesting higher soil-borne disease potential. These findings highlight the potential drawbacks and reduced sustainability of increasing pulse frequency in crop rotations in semiarid environments.

Relative and Quantitative Rhizosphere Microbiome Profiling Results in Distinct Abundance Patterns.

Next-generation sequencing is one of the most popular and cost-effective ways of characterizing microbiome in multiple samples. However, most of the currently available amplicon sequencing approaches are limited, as they result in relative abundance profiles of microbial taxa, which does not represent actual abundance in the environment. Here, we combined amplicon sequencing (16S rRNA gene for bacteria and ITS region for fungi) with real-time quantitative PCR (qPCR) to characterize the rhizosphere microbiome of wheat. We show that changes in the relative abundance of major microbial phyla do not necessarily follow the same pattern as the estimated quantitative abundance. Most of the bacterial phyla linked with the rhizosphere of plants grown in soil with no history of water stress showed enrichment patterns in their estimated absolute abundance, which was in contradiction with the trends observed in the relative abundance data. However, in the case of the fungal groups (except for Basidiomycota), such an enrichment pattern was not observed and the abundance of fungi remained relatively unchanged under different soil water stress history when estimated absolute abundance was considered. Comparing relative and estimated absolute abundances of dominant bacterial and fungal phyla, as well as their correlation with the functional processes in the rhizosphere, our results suggest that the estimated absolute abundance approach gives a different and more realistic perspective than the relative abundance approach. Such a quantification approach provides complementary information that helps to better understand the rhizosphere microbiomes and their associated ecological functional processes.

Bio-fertilizer and rotten straw amendments alter the rhizosphere bacterial community and increase oat productivity in a saline-alkaline environment.

Saline-alkaline conditions can limit crop productivity and the role of soil microbes in nutrient cycling in arid and semi-arid regions throughout the world. A better understanding of how soil amendments and plant varieties affect rhizosphere microbial communities in saline-alkaline environments is important for the development of sustainable and productive agricultural systems under these challenging conditions. The objective of this study was to determine the effect of organic soil amendments on crop yield, soil physicochemical properties and rhizosphere bacterial communities of two oat cultivars in a saline-alkaline soil. The experiment was conducted in a semi-arid region of Northern China and involved growing two oat cultivars with varying levels of saline-alkaline tolerance under four different amendment treatments: (1) control (no amendments), (2) bio-fertilizer, (3) rotten straw, and (4) combination of bio-fertilizer and rotten straw. The combined bio-fertilizer and rotten straw amendment treatment resulted in the highest oat yields, reduced soil pH, and increased soil salt content for both cultivars. Baiyan2 (tolerant cultivar) had a higher bacterial α-diversity, relative abundance of Proteobacteria and Acidobacteria, and lower relative abundance of Firmicutes compared to Caoyou1 (sensitive cultivar). The rotten straw treatment and combined amendment treatment decreased bacterial α-diversity and the abundance of Proteobacteria, and increased the abundance of Firmicutes, which were positively correlated with soil salt, available nitrogen, phosphorous and potassium for both cultivars. Our study suggested using tolerant oat cultivars with the combined application of rotten straw and bio-fertilizer could be an effective strategy in remediating saline-alkaline soils.

Four decades of soil water stress history together with host genotype constrain the response of the wheat microbiome to soil moisture.

There is little understanding about how soil water stress history and host genotype influence the response of wheat-associated microbiome under short-term decreases in soil moisture. To address this, we investigated how plant breeding history (four wheat genotypes; two with recognized drought resistance and two without) and soil water stress history (same wheat field soil from Saskatchewan with contrasting long-term irrigation) independently or interactively influenced the response of the rhizosphere, root and leaf bacterial and fungal microbiota to short-term decreases in soil water content (SWC). We used amplicon sequencing (16S rRNA gene for bacteria and ITS region for fungi) to characterize the wheat microbiome. Fungal and bacterial communities responses to short-term decreases in SWC were mainly constrained by soil water stress history, with some smaller, but significant influence of plant genotype. One exception was the leaf-associated fungal communities, for which the largest constraint was genotype, resulting in a clear differentiation of the communities based on the genotype's sensitivity to water stress. Our results clearly indicate that soil legacy does not only affect the response to water stress of the microbes inhabiting the soil, but also of the microorganisms more closely associated with the plant tissues, and even of the plant itself.

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results.

With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at -20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.

Soil 16S DNA sequence data and corresponding soil property and wheat yield data from a 72-plot field experiment involving pulses and wheat crops grown in rotations in the semiarid prairie.

The soil bacteria diversity and corresponding environmental data made available here are from a 72-field plot experiment testing the effect of pulse frequency in nine wheat-based rotation systems, in the semiarid prairie. The data include sequences of the V6-V8 regions of bacterial 16S rDNA from soil and root extracts, generated using Roche GS FLX Titanium technology, and associated environmental data, specifically levels of soil organic carbon, total carbon, total nitrogen, total phosphorus, pH, electrical conductivity, and extractible sulfate sulfur, copper, iron, manganese, zinc, potassium, nitrate nitrogen, phosphate phosphorus, calcium, and magnesium in the 0-15 cm soil layer, and mineral nitrogen and phosphate in the 0-120 cm soil layer. The grain yield of wheat in the last (4th) phase of the crop rotation systems is also given. The data can be used in meta-analyses of the effect of pea, lentil and chickpea in wheat-based cropping systems on soil bacterial diversity or for monitoring the evolution of soil bacteria communities in cultivated prairie soils in the context of climate change. Samples were collected between 2012 and 2014.

A water stress-adapted inoculum affects rhizosphere fungi, but not bacteria nor wheat.

Here, we tested if inoculating microbial communities adapted to water stress would increase wheat resistance to water stress. Wheat plants were grown for 4 weeks in high and low diversity soils under well-watered conditions, after which they were subjected to a water stress. After another 2 weeks, the rhizospheres were inoculated with microbial communities extracted from soils with or without a history of water stress. The inoculations did not have significant effects on the plant growth, water content and catalase activity, and on the bacterial communities. However, the inoculation did successfully, though modestly, modify the fungal community, shifting the rhizosphere communities toward the inoculated communities. As hypothesized, these shifts were more pronounced and significant in the low diversity soil, and for the inoculum with a water stress history. Whereas the effects of inoculation were relatively subtle, the water stress resulted in large differences in the wheat phenotype and in both the bacterial and fungal communities. Generally, the microbial changes that followed the water stress were in large part due to shifts in the relative abundance of OTUs that were already present before the stress, rather than to the recruitment of microorganisms from the inoculum or the bulk soil.

Intensified Pulse Rotations Buildup Pea Rhizosphere Pathogens in Cereal and Pulse Based Cropping Systems.

The association of plants and microbial communities is crucial for crop production, and host plants influence the composition of rhizosphere microbiomes. Pulse crops play an important role in the development of sustainable cropping systems, and producers in the Canadian prairies often increase the frequency of pulses in their cropping systems. In this study, we determined the shifts in the fungal community of pea (Pisum sativum L.) rhizosphere, as influenced by the frequency of pulses in rotation, using high throughput sequencing. Six cropping systems containing pea (P), lentil (Lens culinaris Medik., L), hybrid canola (Brassica napus L., C), wheat (Triticum aestivum L., W), and oat (Avena sativa L., O) in different intensities were tested. The fungal communities were assessed at the flowering stage in the fourth and fifth year of the 4-year rotations. Cropping system had a significant impact on the composition of the rhizosphere fungal community, and the effect of crop rotation sequence was greater and explained more of the variation than the effect of previous crops. The rotation with consecutive pulses (WPLP) decreased fungal evenness and increased the proportion of pathotrophs. Fusarium was a dominant and ubiquitous pathotrophic genus. Olpidium virulentus, Botrytis cinerea, Fusarium solani, F. graminearum, and Alternaria eichhorniae were generally more abundant in pulse intensive rotations (WPLP, WLOP, and WPOP), the exception being F. solani which was not promoted by lentil. Reads of O. virulentus and B. cinerea were most abundant in pea preceded by lentil followed by the reads of Mortierella elongata in pea preceded by wheat. Pea consistently had higher grain yield when grown in diversified rotations including wheat, canola/lentil, and oat than rotations with two repeated crops (canola or pea). Cropping system affected the soil physicochemical properties, and soil pH was the main driver of fungal community shift. No evidence of beneficial microorganisms involvement in plant productivity was observed, but the high abundance of pathotrophs in pulse intensified rotations suggests the possibility of pathogen buildup in the soil with increasing pulse frequency. Diversifying rotation sequences minimized disease risk and increased pea production, in this study. Careful selection of plant species appears as a strategy for the management of rhizosphere fungal communities and the maintenance of crop production system's health.