Potential to take root in viticulture? An evaluation of mycorrhizal inoculants on the growth and nutrient uptake of young wine grapes planted in live field soil.

AIMS: Incorporating biofertilizers, such as arbuscular mycorrhizal fungal (AM) fungal inoculants, into vineyard management practices may enhance vine growth and reduce environmental impact. Here, we evaluate the effects of commercially available and local AM fungal inoculants on the growth, root colonization, and nutrient uptake of wine grapes (Vitis vinifera) when planted in a field soil substrate. METHODS AND RESULTS: In a greenhouse experiment, young wine grapes were planted in a field soil substrate and inoculated with one of three commercially available mycorrhizal inoculant products, or one of two locally collected whole soil inoculants. After 4 months of growth, inoculated vines showed no differences in plant biomass, colonization of roots by AM fungi, or foliar macronutrient concentrations compared to uninoculated field soil substrate. However, vines grown with local inoculants had greater shoot biomass than vines grown with mycorrhizal inoculant products. CONCLUSIONS: Although effects from inoculations with AM fungi varied by inoculant type and source, inoculations may not improve young vine performance in field soils with a resident microbial community.

The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

Grassland ecosystem type drives AM fungal diversity and functional guild distribution in North American grasslands.

Nutrient exchange forms the basis of the ancient symbiotic relationship that occurs between most land plants and arbuscular mycorrhizal (AM) fungi. Plants provide carbon (C) to AM fungi and fungi provide the plant with nutrients such as nitrogen (N) and phosphorous (P). Nutrient addition can alter this symbiotic coupling in key ways, such as reducing AM fungal root colonization and changing the AM fungal community composition. However, environmental parameters that differentiate ecosystems and drive plant distribution patterns (e.g., pH, moisture), are also known to impact AM fungal communities. Identifying the relative contribution of environmental factors impacting AM fungal distribution patterns is important for predicting biogeochemical cycling patterns and plant-microbe relationships across ecosystems. To evaluate the relative impacts of local environmental conditions and long-term nutrient addition on AM fungal abundance and composition across grasslands, we studied experimental plots amended for 10 years with N, P, or N and P fertilizer in different grassland ecosystem types, including tallgrass prairie, montane, shortgrass prairie, and desert grasslands. Contrary to our hypothesis, we found ecosystem type, not nutrient treatment, was the main driver of AM fungal root colonization, diversity, and community composition, even when accounting for site-specific nutrient limitations. We identified several important environmental drivers of grassland ecosystem AM fungal distribution patterns, including aridity, mean annual temperature, root moisture, and soil pH. This work provides empirical evidence for niche partitioning strategies of AM fungal functional guilds and emphasizes the importance of long-term, large scale research projects to provide ecologically relevant context to nutrient addition studies.

What are mycorrhizal traits?

Traits are inherent properties of organisms, but how are they defined for organismal networks such as mycorrhizal symbioses? Mycorrhizal symbioses are complex and diverse belowground symbioses between plants and fungi that have proved challenging to fit into a unified and coherent trait framework. We propose an inclusive mycorrhizal trait framework that classifies traits as morphological, physiological, and phenological features that have functional implications for the symbiosis. We further classify mycorrhizal traits by location - plant, fungus, or the symbiosis - which highlights new questions in trait-based mycorrhizal ecology designed to charge and challenge the scientific community. This new framework is an opportunity for researchers to interrogate their data to identify novel insights and gaps in our understanding of mycorrhizal symbioses.

Microbial Inoculants: Silver Bullet or Microbial Jurassic Park?

The appeal of using microbial inoculants to mediate plant traits and productivity in managed ecosystems has increased over the past decade, because microbes represent an alternative to fertilizers, pesticides, and direct genetic modification of plants. Using microbes bypasses many societal and environmental concerns because microbial products are considered a more sustainable and benign technology. In our desire to harness the power of plant-microbial symbioses, are we ignoring the possibility of precipitating microbial invasions, potentially setting ourselves up for a microbial Jurassic Park? Here, we outline potential negative consequences of microbial invasions and describe a set of practices (Testing, Regulation, Engineering, and Eradication, TREE) based on the four stages of invasion to prevent microbial inoculants from becoming invasive. We aim to stimulate discussion about best practices to proactively prevent microbial invasions.

Variation in hyphal production rather than turnover regulates standing fungal biomass in temperate hardwood forests.

Soil fungi link above- and belowground carbon (C) fluxes through their interactions with plants and contribute to C and nutrient dynamics through the production, turnover, and activity of fungal hyphae. Despite their importance to ecosystem processes, estimates of hyphal production and turnover rates are relatively uncommon, especially in temperate hardwood forests. We sequentially harvested hyphal ingrowth bags to quantify the rates of Dikarya (Ascomycota and Basidiomycota) hyphal production and turnover in three hardwood forests in the Midwestern United States, where plots differed in their abundance of arbuscular (AM)- vs. ectomycorrhizal (ECM)-associated trees. Hyphal production rates increased linearly with the percentage of ECM trees and annual production rates were 66% higher in ECM- than AM-dominated plots. Hyphal turnover rates did not differ across the mycorrhizal gradient (plots varying in their abundance of AM vs. ECM trees), suggesting that the greater fungal biomass in ECM-dominated plots relates to greater fungal production rather than slower fungal turnover. Differences in hyphal production across the gradient aligned with distinctly different fungal communities and activities. As ECM trees increased in dominance, fungi inside ingrowth bags produced more extracellular enzymes involved in degrading nitrogen (N)-bearing relative to C-bearing compounds, suggesting greater fungal (and possibly plant) N demand in ECM-dominated soils. Collectively, our results demonstrate that shifts in temperate tree species composition that result in changes in the dominant type of mycorrhizal association may have strong impacts on Dikarya hyphal production, fungal community composition and extracellular enzyme activity, with important consequences for soil C and N cycling.

Sensitivity to AMF species is greater in late-successional than early-successional native or nonnative grassland plants.

Sensitivity of plant species to individual arbuscular mycorrhizal (AM) fungal species is of primary importance to understanding the role of AM fungal diversity and composition in plant ecology. Currently, we do not have a predictive framework for understanding which plant species are sensitive to different AM fungal species. In two greenhouse studies, we tested for differences in plant sensitivity to different AM fungal species and mycorrhizal responsiveness across 17 grassland plant species of North America that varied in successional stage, native status, and plant family by growing plants with different AM fungal treatments including eight single AM fungal isolates, diverse mixtures of AM fungi, and non-inoculated controls. We found that late successional grassland plant species were highly responsive to AM fungi and exhibited stronger sensitivity in their response to individual AM fungal taxa compared to nonnative or early successional native grassland plant species. We confirmed these results using a meta-analysis that included 13 experiments, 37 plant species, and 40 fungal isolates (from nine publications and two greenhouse experiments presented herein). Mycorrhizal responsiveness and sensitivity of response (i.e., variation in plant biomass response to different AM fungal taxa) did not differ by the source of fungal inocula (i.e., local or not local) or plant family. Sensitivity of plant response to AM fungal species was consistently correlated with the average mycorrhizal response of that plant species. This study identifies that AM fungal identity is more important to the growth of late successional plant species than early successional or nonnative plant species, thereby predicting that AM fungal composition will be more important to plant community dynamics in late successional communities than in early successional or invaded plant communities.

Dominant mycorrhizal association of trees alters carbon and nutrient cycling by selecting for microbial groups with distinct enzyme function.

While it is well established that plants associating with arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi cycle carbon (C) and nutrients in distinct ways, we have a limited understanding of whether varying abundance of ECM and AM plants in a stand can provide integrative proxies for key biogeochemical processes. We explored linkages between the relative abundance of AM and ECM trees and microbial functioning in three hardwood forests in southern Indiana, USA. Across each site's 'mycorrhizal gradient', we measured fungal biomass, fungal : bacterial (F : B) ratios, extracellular enzyme activities, soil carbon : nitrogen ratio, and soil pH over a growing season. We show that the percentage of AM or ECM trees in a plot promotes microbial communities that both reflect and determine the C to nutrient balance in soil. Soils dominated by ECM trees had higher F : B ratios and more standing fungal biomass than AM stands. Enzyme stoichiometry in ECM soils shifted to higher investment in extracellular enzymes needed for nitrogen and phosphorus acquisition than in C-acquisition enzymes, relative to AM soils. Our results suggest that knowledge of mycorrhizal dominance at the stand or landscape scale may provide a unifying framework for linking plant and microbial community dynamics, and predicting their effects on ecological function.

Phosphorus cycling in deciduous forest soil differs between stands dominated by ecto- and arbuscular mycorrhizal trees.

Although much is known about how trees and their associated microbes influence nitrogen cycling in temperate forest soils, less is known about biotic controls over phosphorus (P) cycling. Given that mycorrhizal fungi are instrumental for P acquisition and that the two dominant associations - arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi - possess different strategies for acquiring P, we hypothesized that P cycling would differ in stands dominated by trees associated with AM vs ECM fungi. We quantified soil solution P, microbial biomass P, and sequentially extracted inorganic and organic P pools from May to November in plots dominated by trees forming either AM or ECM associations in south-central Indiana, USA. Overall, fungal communities in AM and ECM plots were functionally different and soils exhibited fundamental differences in P cycling. Organic forms of P were more available in ECM plots than in AM plots. Yet inorganic P decreased and organic P accumulated over the growing season in both ECM and AM plots, resulting in increasingly P-limited microbial biomass. Collectively, our results suggest that P cycling in hardwood forests is strongly influenced by biotic processes in soil and that these are driven by plant-associated fungal communities.

Spatial soil heterogeneity has a greater effect on symbiotic arbuscular mycorrhizal fungal communities and plant growth than genetic modification with Bacillus thuringiensis toxin genes.

Maize, genetically modified with the insect toxin genes of Bacillus thuringiensis (Bt), is widely cultivated, yet its impacts on soil organisms are poorly understood. Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots and may be uniquely sensitive to genetic changes within a plant host. In this field study, the effects of nine different lines of Bt maize and their corresponding non-Bt parental isolines were evaluated on AMF colonization and community diversity in plant roots. Plants were harvested 60 days after sowing, and data were collected on plant growth and per cent AMF colonization of roots. AMF community composition in roots was assessed using 454 pyrosequencing of the 28S rRNA genes, and spatial variation in mycorrhizal communities within replicated experimental field plots was examined. Growth responses, per cent AMF colonization of roots and AMF community diversity in roots did not differ between Bt and non-Bt maize, but root and shoot biomass and per cent colonization by arbuscules varied by maize cultivar. Plot identity had the most significant effect on plant growth, AMF colonization and AMF community composition in roots, indicating spatial heterogeneity in the field. Mycorrhizal fungal communities in maize roots were autocorrelated within approximately 1 m, but at greater distances, AMF community composition of roots differed between plants. Our findings indicate that spatial variation and heterogeneity in the field has a greater effect on the structure of AMF communities than host plant cultivar or modification by Bt toxin genes.

Field evaluation of arbuscular mycorrhizal fungal colonization in Bacillus thuringiensis toxin-expressing (Bt) and non-Bt maize.

The cultivation of genetically engineered Bacillus thuringiensis toxin-expressing (Bt) maize continues to increase worldwide, yet the effects of Bt crops on arbuscular mycorrhizal fungi (AMF) in soil are poorly understood. In this field experiment, we investigated the impact of seven different genotypes of Bt maize and five corresponding non-Bt parental cultivars on AMF and evaluated plant growth responses at three different physiological time points. Plants were harvested 60 days (active growth), 90 days (tasseling and starting to produce ears), and 130 days (maturity) after sowing, and data on plant growth responses and percent AMF colonization of roots at each harvest were collected. Spore abundance and diversity were also evaluated at the beginning and end of the field season to determine whether the cultivation of Bt maize had a negative effect on AMF propagules in the soil. Plant growth and AMF colonization did not differ between Bt and non-Bt maize at any harvest period, but AMF colonization was positively correlated with leaf chlorophyll content at the 130-day harvest. Cultivation of Bt maize had no effect on spore abundance and diversity in Bt versus non-Bt plots over one field season. Plot had the most significant effect on total spore counts, indicating spatial heterogeneity in the field. Although previous greenhouse studies demonstrated that AMF colonization was lower in some Bt maize lines, our field study did not yield the same results, suggesting that the cultivation of Bt maize may not have an impact on AMF in the soil ecosystem under field conditions.

Evidence of reduced arbuscular mycorrhizal fungal colonization in multiple lines of Bt maize.

PREMISE OF THE STUDY: Insect-resistant Bacillus thuringiensis (Bt) maize is widely cultivated, yet few studies have examined the interaction of symbiotic arbuscular mycorrhizal fungi (AMF) with different lines of Bt maize. As obligate symbionts, AMF may be sensitive to genetic changes within a plant host. Previous evaluations of the impact of Bt crops on AMF have been inconsistent, and because most studies were conducted under disparate experimental conditions, the results are difficult to compare. METHODS: We evaluate AMF colonization in nine Bt maize lines, differing in number and type of engineered trait, and five corresponding near-isogenic parental (P) base hybrids in greenhouse microcosms. Plants were grown in 50% local agricultural soil with low levels of fertilization, and AMF colonization was evaluated at 60 and 100 d. Nontarget effects of Bt cultivation on AMF colonization were tested in a subsequently planted crop, Glycine max, which was seeded into soil that had been preconditioned for 60 d with Bt or P maize. KEY RESULTS: We found that Bt maize had lower levels of AMF colonization in their roots than did the non-Bt parental lines. However, reductions in AMF colonization were not related to the expression of a particular Bt protein. There was no difference in AMF colonization in G. max grown in the Bt- or P-preconditioned soil. CONCLUSIONS: These findings are the first demonstration of a reduction in AMF colonization in multiple Bt maize lines grown under the same experimental conditions and contribute to the growing body of knowledge examining the unanticipated effects of Bt crop cultivation on nontarget soil organisms.

The influence of fertilizer level and spore density on arbuscular mycorrhizal colonization of transgenic Bt 11 maize (Zea mays) in experimental microcosms.

Crop plants genetically modified for the expression of Bacillus thuringiensis (Bt) insecticidal toxins have broad appeal for reducing insect damage in agricultural systems, yet questions remain about the impact of Bt plants on symbiotic soil organisms. Here, arbuscular mycorrhizal fungal (AMF) colonization of transgenic maize isoline Bt 11 (expressing Cry1Ab) and its non-Bt parental line (Providence) was evaluated under different fertilizer level and spore density scenarios. In a three-way factorial design, Bt 11 and non-Bt maize were inoculated with 0, 40, or 80 spores of Glomus mosseae and treated weekly with 'No' (0 g L(-1) ), 'Low' (0.23 g L(-1) ), or 'High' (1.87 g L(-1) ) levels of a complete fertilizer and grown for 60 days in a greenhouse. While no difference in AMF colonization was detected between the Bt 11 and Providence maize cultivars in the lower spore/higher fertilizer treatments, microcosm experiments demonstrated a significant reduction in AMF colonization in Bt 11 maize roots in the 80 spore treatments when fertilizer was limited. These results confirm previous work indicating an altered relationship between this Bt 11 maize isoline and AMF and demonstrate that the magnitude of this response is strongly dependent on both nutrient supply and AMF spore inoculation level.