Crop rotation significantly influences the composition of soil, rhizosphere, and root microbiota in canola (Brassica napus L.).

BACKGROUND: Crop rotation is an agronomic practice that is known to enhance productivity and yield, and decrease pest and disease pressure. Economic and other factors have increased the frequency of certain crops, including canola, with unknown effects on the below ground microbial communities that impact plant health and performance. This study investigated the effect of 12 years of crop rotation including canola-wheat; canola-pea-barley; and unrotated canola across three geographic sites in Western Canada with diverse soil types and environmental conditions. To provide data on mature, established crop rotation strategies, root exudate profiles, soil nutrient fluxes, and bacterial and fungal microbial community profiles were determined at the flowering stage in the final two (canola) years of the 12-year rotations. RESULTS: After 12 years of rotation, nutrient fluxes were affected in the soil in an inconsistent manner, with K, NO3, Mg, Ca, P, and Fe fluxes variably impacted by rotation depending on the year and site of sampling. As expected, rotation positively influenced yield and oil content, and decreased disease pressure from Leptosphaeria and Alternaria. In two of the three sites, root exudate profiles were significantly influenced by crop rotation. Bacterial soil, root, and rhizosphere communities were less impacted by crop rotation than the fungal communities. Fungal sequences that were associated with specific rotation strategies were identified in the bulk soil, and included known fungal pathogens in the canola-only strategy. Two closely related fungal sequences identified as Olpidium brassicae were extremely abundant at all sites in both years. One of these sequences was observed uniquely at a single site and was significantly associated with monocropped canola; moreover, its abundance correlated negatively with yield in both years. CONCLUSIONS: Long-term canola monoculture affected root exudate profiles and soil nutrient fluxes differently in the three geographic locations. Bacterial communities were less impacted by rotation compared to the fungal communities, which consistently exhibited changes in composition in all ecological niches at all sites, in both years. Fungal sequences identified as O. brassicae were highly abundant at all sites, one of which was strongly associated with canola monoculture. Soil management decisions should include consideration of the effects on the microbial ecosystems associated with the plants in order to inform best management practices.

Brassica napus Bacterial Assembly Processes Vary with Plant Compartment and Growth Stage but Not between Lines.

Holobiont bacterial community assembly processes are an essential element to understanding the plant microbiome. To elucidate these processes, leaf, root, and rhizosphere samples were collected from eight lines of Brassica napus in Saskatchewan over the course of 10 weeks. We then used ecological null modeling to disentangle the community assembly processes over the growing season in each plant part. The root was primarily dominated by stochastic community assembly processes, which is inconsistent with previous studies that suggest of a highly selective root environment. Leaf assembly processes were primarily stochastic as well. In contrast, the rhizosphere was a highly selective environment. The dominant rhizosphere selection process leads to more similar communities. Assembly processes in all plant compartments were dependent on plant growth stage with little line effect on community assembly. The foundations of assembly in the leaf were due to the harsh environment, leading to dominance of stochastic effects, whereas the stochastic effects in the root interior likely arise due to competitive exclusion or priority effects. Engineering canola microbiomes should occur during periods of strong selection assuming strong selection could promote beneficial bacteria. For example, engineering the microbiome to resist pathogens, which are typically aerially born, should focus on the flowering period, whereas microbiomes to enhance yield should likely be engineered postflowering as the rhizosphere is undergoing strong selection. IMPORTANCE In order to harness the microbiome for more sustainable crop production, we must first have a better understanding of microbial community assembly processes that occurring during plant development. This study examines the bacterial community assembly processes of the leaf, root, and rhizosphere of eight different lines of Brassica napus over the growing season. The influence of growth stage and B. napus line were examined in conjunction with the assembly processes. Understanding what influences the assembly processes of crops might allow for more targeted breeding efforts by working with the plant to manipulate the microbiome when it is undergoing the strongest selection pressure.

Assembly and potential transmission of the Lens culinaris seed microbiome.

Soil is an important source of bacteria and fungi for the plant, but seeds can also provide microbial inocula through heritable or stochastic assembly. Seed-associated microbial communities can potentially interact with the host plant through multiple generations. Here, we assessed the impact of two different soil types on the seed microbiome assembly of seven lentil (Lens culinaris) genotypes under environmentally controlled conditions and examined the vertical transmission of bacterial communities from seed to seed across two generations. Bulk soil microbiomes and seed microbiomes were characterized using high-throughput amplicon sequencing of the bacterial 16S rRNA gene. Our results revealed that bacterial communities in the two soils differed significantly and that bacterial communities associated with seeds were significantly impacted by genotype (15%) in one of the soils. Co-occurrence of amplicon sequence variants between generations suggests that members of the genera Cutibacterium, Methylobacterium, Sphingomonas, Streptococcus and Tepidimonas are transmitted and preserved in lentil genotypes irrespective of the soil in which they were grown. Increasing our knowledge of how microbial communities carried by seeds are assembled, transmitted and preserved offers a promising way for future breeding programs to consider microbial communities when selecting for more resilient and productive cultivars.

Crop, genotype, and field environmental conditions shape bacterial and fungal seed epiphytic microbiomes.

Seeds are reproductive structures able to carry and transfer microorganisms that play an important role in plant fitness. Genetic and external factors are reported to be partly responsible for the plant microbiome assemblage, but their contribution in seeds is poorly understood. In this study, wheat, canola, and lentil seeds were analyzed to characterize diversity, structure, and persistence of seed-associated microbial communities. Five lines and 2 generations of each crop were subjected to high-throughput amplicon sequencing of the 16S rRNA and internal transcribed spacer (ITS) regions. Bacterial and fungal communities differed most by crop type (30% and 47% of the variance), while generation explained an additional 10% and 15% of the variance. The offspring (i.e., generation harvested in 2016 at the same location) exhibited a higher number of common amplicon sequence variants (ASVs) and less variability in microbial composition. Additionally, in every sample analyzed, a "core microbiome" was detected consisting of 5 bacterial and 12 fungal ASVs. Our results suggest that crop, genotype, and field environmental conditions contributed to the seed-associated microbial assemblage. These findings not only expand our understanding of the factors influencing the seed microbiome but may also help us to manipulate and exploit the microbiota naturally carried by seeds.

An intensive multilocation temporal dataset of fungal and bacterial communities in the root and rhizosphere of Brassica napus.

The plant microbiome has been recently recognized as a plant phenotype to help in the food security of the future population. However, global plant microbiome datasets are insufficient to be used effectively for breeding this new generation of crop plants. We surveyed the diversity and temporal composition of bacterial and fungal communities in the root and rhizosphere of Brassica napus, the world's second largest oilseed crop, weekly in eight diverse lines at one site and every three weeks in sixteen lines, at three sites in 2016 and 2017 in the Canadian Prairies. We sequenced the bacterial 16S ribosomal RNA gene generating a total of 127.7 million reads and the fungal internal transcribed spacer (ITS) region generating 113.4 million reads. 14,944 unique fungal amplicon sequence variants (ASV) were detected, with an average of 43 ASVs per root and 105 ASVs per rhizosphere sample. We detected 10,882 unique bacterial ASVs with an average of 249 ASVs per sample. Temporal, site-to-site, and line-driven variability were key determinants of microbial community structure. This dataset is a valuable resource to systematically extract information on the belowground microbiome of diverse B. napus lines in different environments, at different times in the growing season, in order to adapt effective varieties for sustainable crop production systems.

An intensive multilocation temporal dataset of fungal communities in the root and rhizosphere of Brassica napus.

The plant microbiome has been recently recognized as a plant phenotype to help in the food security of the future population. However, global plant microbiome datasets are insufficient to be used effectively for breeding this new generation of crop plants. We surveyed the diversity and temporal composition of fungal communities in the root and rhizosphere of Brassica napus, the world's second largest oilseed crop, weekly in eight diverse lines at one site and every three weeks in sixteen lines, at three sites in 2016 and 2017 in the Canadian Prairies. 14,944 unique amplicon sequence variants (ASV) were detected based on the internal transcribed spacer region, with an average of 43 ASVs per root and 105 ASVs per rhizosphere sample. Temporal, site-to-site, and line-driven variability were key determinants of fungal community structure. This dataset is a valuable resource to systematically extract information on the belowground microbiome of diverse B. napus lines in different environments, at different times in the growing season, in order to adapt effective varieties for sustainable crop production systems.

Structural equation modeling of a winnowed soil microbiome identifies how invasive plants re-structure microbial networks.

The development of microbial networks is central to ecosystem functioning and is the hallmark of complex natural systems. Characterizing network development over time and across environmental gradients is hindered by the millions of potential interactions among community members, limiting interpretations of network evolution. We developed a feature selection approach using data winnowing that identifies the most ecologically influential microorganisms within a network undergoing change. Using a combination of graph theory, leave-one-out analysis, and statistical inference, complex microbial communities are winnowed to identify the core organisms responding to external gradients or functionality, and then network development is evaluated against these externalities. In a plant invasion case study, the winnowed microbial network became more influential as the plant invasion progressed as a result of direct plant-microbe links rather than the expected indirect plant-soil-microbe links. This represents the first use of structural equation modeling to predict microbial network evolution, which requires identification of keystone taxa and quantification of the ecological processes underpinning community structure and function patterns.

Core and Differentially Abundant Bacterial Taxa in the Rhizosphere of Field Grown Brassica napus Genotypes: Implications for Canola Breeding.

Modifying the rhizosphere microbiome through targeted plant breeding is key to harnessing positive plant-microbial interrelationships in cropping agroecosystems. Here, we examine the composition of rhizosphere bacterial communities of diverse Brassica napus genotypes to identify: (1) taxa that preferentially associate with genotypes, (2) core bacterial microbiota associated with B. napus, (3) heritable alpha diversity measures at flowering and whole growing season, and (4) correlation between microbial and plant genetic distance among canola genotypes at different growth stages. Our aim is to identify and describe signature microbiota with potential positive benefits that could be integrated in B. napus breeding and management strategies. Rhizosphere soils of 16 diverse genotypes sampled weekly over a 10-week period at single location as well as at three time points at two additional locations were analyzed using 16S rRNA gene amplicon sequencing. The B. napus rhizosphere microbiome was characterized by diverse bacterial communities with 32 named bacterial phyla. The most abundant phyla were Proteobacteria, Actinobacteria, and Acidobacteria. Overall microbial and plant genetic distances were highly correlated (R = 0.65). Alpha diversity heritability estimates were between 0.16 and 0.41 when evaluated across growth stage and between 0.24 and 0.59 at flowering. Compared with a reference B. napus genotype, a total of 81 genera were significantly more abundant and 71 were significantly less abundant in at least one B. napus genotype out of the total 558 bacterial genera. Most differentially abundant genera were Proteobacteria and Actinobacteria followed by Bacteroidetes and Firmicutes. Here, we also show that B. napus genotypes select an overall core bacterial microbiome with growth-stage-related patterns as to how taxa joined the core membership. In addition, we report that sets of B. napus core taxa were consistent across our three sites and 2 years. Both differential abundance and core analysis implicate numerous bacteria that have been reported to have beneficial effects on plant growth including disease suppression, antifungal properties, and plant growth promotion. Using a multi-site year, temporally intensive field sampling approach, we showed that small plant genetic differences cause predictable changes in canola microbiome and are potential target for direct and indirect selection within breeding programs.

Resource Legacies of Organic and Conventional Management Differentiate Soil Microbial Carbon Use.

Long-term contrasts in agricultural management can shift soil resource availability with potential consequences to microbial carbon (C) use efficiency (CUE) and the fate of C in soils. Isothermal calorimetry was combined with 13C-labeled glucose stable isotope probing (SIP) of 16S rRNA genes to test the hypothesis that organically managed soils would support microbial communities with greater thermodynamic efficiency compared to conventional soils due to a legacy of lower resource availability and a resultant shift toward communities supportive of more oligotrophic taxa. Resource availability was greater in conventionally managed soils, with 3.5 times higher available phosphorus, 5% more nitrate, and 36% more dissolved organic C. The two management systems harbored distinct glucose-utilizing populations of Proteobacteria and Actinobacteria, with a higher Proteobacteria:Actinobacteria ratio (2.4 vs. 0.7) in conventional soils. Organically managed soils also harbored notable activity of Firmicutes. Thermodynamic efficiency indices were similar between soils, indicating that glucose was metabolized at similar energetic cost. However, differentially abundant glucose utilizers in organically managed soils were positively correlated with soil organic matter (SOM) priming and negatively correlated to soil nutrient and carbon availability, respiration, and heat production. These correlation patterns were strongly reversed in the conventionally managed soils indicating clear differentiation of microbial functioning related to soil resource availability. Fresh C addition caused proportionally more priming of SOM decomposition (57 vs. 51%) in organically managed soils likely due to mineralization of organic nutrients to satisfy microbial demands during glucose utilization in these more resource deprived soils. The additional heat released from SOM oxidation may explain the similar community level thermodynamic efficiencies between management systems. Restoring fertility to soils with a legacy of nutrient limitation requires a balanced supply of both nutrients and energy to protect stable SOM from microbial degradation. These results highlight the need to consider managing C for the energy it provides to icritical biological processes that underpin soil health.

Endophytic root bacteria associated with the natural vegetation growing at the hydrocarbon-contaminated Bitumount Provincial Historic site.

The Bitumount Provincial Historic site is the location of 2 of the world's first oil-extracting and -refining operations. Despite hydrocarbon levels ranging from 330 to 24 700 mg·(kg soil)-1, plants have been able to recolonize the site through means of natural revegetation. This study was designed to achieve a better understanding of the plant-root-associated bacterial partnerships occurring within naturally revegetated hydrocarbon-contaminated soils. Root endophytic bacterial communities were characterized from representative plant species throughout the site by both high-throughput sequencing and culturing techniques. Population abundance of rhizosphere and root endosphere bacteria was significantly influenced (p < 0.05) by plant species and sampling location. In general, members of the Actinomycetales, Rhizobiales, Pseudomonadales, Burkholderiales, and Sphingomonadales orders were the most commonly identified orders. Community structure of root-associated bacteria was influenced by both plant species and sampling location. Quantitative real-time polymerase chain reaction was used to determine the potential functional diversity of the root endophytic bacteria. The gene copy numbers of 16S rRNA and 2 hydrocarbon-degrading genes (CYP153 and alkB) were significantly affected (p < 0.05) by the interaction of plant species and sampling location. Our findings suggest that some of the bacterial communities detected are known to exhibit plant growth promotion characteristics.

Litter decay controlled by temperature, not soil properties, affecting future soil carbon.

Widespread global changes, including rising atmospheric CO2 concentrations, climate warming and loss of biodiversity, are predicted for this century; all of these will affect terrestrial ecosystem processes like plant litter decomposition. Conversely, increased plant litter decomposition can have potential carbon-cycle feedbacks on atmospheric CO2 levels, climate warming and biodiversity. But predicting litter decomposition is difficult because of many interacting factors related to the chemical, physical and biological properties of soil, as well as to climate and agricultural management practices. We applied 13 C-labelled plant litter to soil at ten sites spanning a 3500-km transect across the agricultural regions of Canada and measured its decomposition over five years. Despite large differences in soil type and climatic conditions, we found that the kinetics of litter decomposition were similar once the effect of temperature had been removed, indicating no measurable effect of soil properties. A two-pool exponential decay model expressing undecomposed carbon simply as a function of thermal time accurately described kinetics of decomposition. (R2  = 0.94; RMSE = 0.0508). Soil properties such as texture, cation exchange capacity, pH and moisture, although very different among sites, had minimal discernible influence on decomposition kinetics. Using this kinetic model under different climate change scenarios, we projected that the time required to decompose 50% of the litter (i.e. the labile fractions) would be reduced by 1-4 months, whereas time required to decompose 90% of the litter (including recalcitrant fractions) would be reduced by 1 year in cooler sites to as much as 2 years in warmer sites. These findings confirm quantitatively the sensitivity of litter decomposition to temperature increases and demonstrate how climate change may constrain future soil carbon storage, an effect apparently not influenced by soil properties.

Predicting phosphorus availability from soil-applied composted and non-composted cattle feedlot manure.

Prediction of phosphorus (P) availability from soil-applied composts and manure is important for agronomic and environmental reasons. This study utilized chemical properties of eight composted and two non-composted beef cattle (Bos taurus) manures to predict cumulative phosphorus uptake (CPU) during a 363-d controlled environment chamber bioassay. Ten growth cycles of canola (Brassica napus L.) were raised in pots containing 2 kg of a Dark Brown Chernozemic clay loam soil (fine-loamy, mixed, Typic Haploboroll) mixed with 0.04 kg of the amendments. Inorganic P fertilizer (KH2PO4) and an unamended control were included for comparison. All treatments received a nutrient solution containing an adequate supply of all essential nutrients, except P, which was supplied by the amendments. Cumulative P uptake was similar for composted (74 mg kg-1 soil) and non-composted manures (60 mg kg-1 soil) and for the latter and the fertilizer (40 mg kg-1 soil). However, the CPU was significantly higher for organic amendments than the control (24 mg kg-1 soil) and for composted manure than the fertilizer. Apparent phosphorus recovery (APR) from composted manure (24%) was significantly lower than that from non-composted manure (33%), but there was no significant difference in APR between the organic amendments and the fertilizer (27%). Partial least squares (PLS) regression indicated that only two parameters [total water-extractable phosphorus (TPH2O) and total phosphorus (TP) concentration of amendments] were adequate to model amendment-derived cumulative phosphorus uptake (ACPU), explaining 81% of the variation in ACPU. These results suggest that P availability from soil-applied composted and non-composted manures can be adequately predicted from a few simple amendment chemical measurements. Accurate prediction of P availability and plant P recovery may help tailor manure and compost applications to plant needs and minimize the buildup of bioavailable P, which can contribute to eutrophication of sensitive aquatic systems.