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Studying the microorganisms in soil remains a challenge as soils are one of the most complex and diverse environments. Compounding these challenges is the lack of culturable representatives in soil, with over 99% of soil microorganisms yet to be cultivated in a laboratory setting. Leveraging next-generation sequencing technologies, which bypass traditional culture-dependent methods, scientists are now able to attain low-cost, high-throughput DNA sequencing that can detect even the rarest microorganisms within samples. The present study rigorously compares amplicon and shotgun sequencing techniques in profiling microbial communities across diverse temperate grassland soil samples, focusing on how different databases, classifiers, and sequencing methods influence the results. Our study underscores the crucial need for a harmonized taxonomic database that could greatly enhance comparability and accuracy in the understanding of soil microbiomes.

Climate models project overall a reduction in rainfall amounts and shifts in the timing of rainfall events in mid‐latitudes and sub‐tropical dry regions, which threatens the productivity and diversity of grasslands. Arbuscular mycorrhizal (AM) fungi may help plants to cope with expected changes but may also be impacted by changing rainfall, either via the direct effects of low soil moisture on survival and function or indirectly via changes in the plant community. In an Australian mesic grassland (former pasture) system, we characterized plant and AM fungal communities every 6 months for nearly 4 years to two altered rainfall regimes: (a) ambient, (b) rainfall reduced by 50% relative to ambient over the entire year and (c) total summer rainfall exclusion. Using Illumina sequencing, we assessed the response of AM fungal communities sampled from contrasting rainfall treatments and evaluated whether variation in AM fungal communities was associated with variation in plant community richness and composition. We found that rainfall reduction influenced the fungal communities, with the nature of the response depending on the type of manipulation, but that consistent results were only observed after more than 2 years of rainfall manipulation. We observed significant co‐associations between plant and AM fungal communities on multiple dates. Predictive co‐correspondence analyses indicated more support for the hypothesis that fungal community composition influenced plant community composition than vice versa. However, we found no evidence that altered rainfall regimes were leading to distinct co‐associations between plants and AM fungi. Overall, our results provide evidence that grassland plant communities are intricately tied to variation in AM fungal communities. However, in this system, plant responses to climate change may not be directly related to impacts of altered rainfall regimes on AM fungal communities. Synthesis. Our study shows that arbuscular mycorrhizal (AM) fungal communities respond to changes in rainfall but that this effect was not immediate. The AM fungal community may influence the composition of the plant community. However, our results suggest that plant responses to altered rainfall regimes at our site may not be resulting via changes in the AM fungal communities. Our study shows that AM fungal communities respond to changes in rainfall but that this effect was not immediate. The AM fungal community may influence the composition of the plant community. However, our results suggest that plant responses to altered rainfall regimes at our site may not be resulting via changes in the AM fungal communities.

Arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota) are obligate symbionts with plants influencing plant health, soil a(biotic) processes, and ecosystem functioning. Despite advancements in molecular techniques, understanding the role of AM fungal communities on a(biotic) processes based on AM fungal taxonomy remains challenging. This review advocates for a standardized trait-based framework to elucidate the life-history traits of AM fungi, focusing on their roles in three dimensions: host plants, soil, and AM fungal ecology. We define morphological, physiological, and genetic key traits, explore their functional roles and propose methodologies for their consistent measurement, enabling cross-study comparisons towards improved predictability of ecological function. We aim for this review to lay the groundwork for establishing a baseline of AM fungal trait responses under varying environmental conditions. Furthermore, we emphasize the need to include underrepresented taxa in research and utilize advances in machine learning and microphotography for data standardization.

Taxonomy of arbuscular mycorrhizal fungi (Glomeromycota) historically has been based mostly on analyses of spore morphology. Molecular evidence has been widely used in phylogeny since the turn of the century and has contributed to the nomenclature of arbuscular mycorrhizal fungi. Considering that some species were described solely from field collected spores which often are degraded, synonymy amongst described species is likely. Type and living cultures of Rhizophagus clarus and Rhizophagus manihotis , and protologue of Glomus zaozhuangianus were analyzed to compare spore wall structure. Sequences of the large subunit (LSU) of the rDNA gene of living isolates of Rhizophagus clarus and Rhizophagus manihotis also were used to test phylogenetic relationships. A comprehensive biogeography of arbuscular mycorrhizal fungi was used to investigate species distribution according to soil and climate factors. Spore wall structure analysis indicates that the three species are morphologically indistinguishable. Spore color, size, and shape all overlap highly among the three species. The spore wall of each is composed of an outer hyaline mucilaginous layer, a rigid hyaline laminated layer conferring a visible “halo” to mature spores, and a third rigid pigmented laminated layer that confers spore color. Phylogenetic analysis shows that living isolates identified as R. manihotis were nested with living isolates of R. clarus , forming a monophyletic clade with 99% bootstrap support. Spores of R. clarus (as amended here) have been recorded in six continents and 31 countries in 10 biogeographical realms. R. clarus was detected most often in soil pH 5.0–6.0, soil P up to 5 mg/dm 3 , and soil organic matter up to 2.5%. Polynomial models indicate that the probability of occurrence of R. clarus is optimized at a temperature of 20 o C and 2,000 mm precipitation.

Abstract The influence of soil phosphorous (P) content on the N-cycling communities and subsequent effects on N2O emissions remains unclear. Two laboratory incubation experiments were conducted on soils collected from a long-term (est. 1995) P-addition field trial sampled in summer 2018 and winter 2019. Incubations were treated with a typical field amendment rate of N as well as a C-amendment to stimulate microbial activity. Throughout both incubations, soil subsamples were collected prior to fertiliser amendment and then throughout the incubations, to quantify the abundance of bacteria (16S rRNA), fungi (ITS) and Thaumarcheota (16S rRNA) as well as functional guilds of genes involved in nitrification (bacterial and archaeal amoA, and comammox) and denitrification (nirS, nirK, nosZ clade I and II) using quantitative PCR (qPCR). We also evaluated the correlations between each gene abundance and the associated N2O emissions depending on P-treatments. Our results show that long-term P-application influenced N-cycling genes abundance differently. Except for comammox, overall nitrifiers’ genes were most abundant in low P while the opposite trend was found for denitrifiers’ genes. C and N-amendments strongly influenced the abundance of most genes with changes observed as soon as 24 h after application. ITS was the only gene correlated to N2O emissions in the low P-soils while microbes were mostly correlated to emissions in high P, suggesting possible changes in the organisms involved in N2O production depending on soil P-content. This study highlights the importance of long-term P addition on shaping the microbial community function which in turn stimulates a direct impact on the subsequent N emissions.

A recent study by Sugiura and coworkers reported the nonsymbiotic growth and spore production of an arbuscular mycorrhizal (AM) fungus, Rhizophagus irregularis, when the fungus received an external supply of certain fatty acids, myristates (C:14). This discovery follows the insight that AM fungi receive fatty acids from their hosts when in symbiosis. If this result holds up and can be repeated under nonsterile conditions and with a broader range of fungi, it has numerous consequences for our understanding of AM fungal ecology, from the level of the fungus, at the plant community level, and to functional consequences in ecosystems. In addition, myristate may open up several avenues from a more applied perspective, including improved fungal culture and supplementation of AM fungi or inoculum in the field. We here map these potential opportunities, and additionally offer thoughts on potential risks of this potentially new technology. Lastly, we discuss the specific research challenges that need to be overcome to come to an understanding of the potential role of myristate in AM ecology.

Climate models predict an overall reduction in rainfall in mid-latitudes and sub-tropical dry regions. In southeast Australian grasslands, changes in rainfall may affect plant productivity and diversity because both are highly responsive to rainfall regimes. Associated soil microorganisms may help plants cope with these changes but also may respond themselves to altered rainfall patterns, directly or indirectly via responses of plants. Arbuscular mycorrhizal (AM) fungi are an important component of the soil microbial community in grasslands. They form symbiotic associations with the majority of plant species and are dependent on the carbon provided by their host. In return, they contribute to plant nutrition and tolerance of environmental stress, including drought. The overarching goal of this work was to study the response of AM fungal communities to altered rainfall regimes. In particular, I evaluated AM fungal responses to changes in rainfall in association with changes in root traits (chapter 2) and in the composition and richness of the plant community (chapter 4), using DNA sequencing techniques. I also used a trait-based approach to understand how precipitation regimes affect the AM fungal community (chapter 3). AM fungal communities may respond to altered rainfall regimes either directly or indirectly via changes in host traits. I studied the response of AM fungal communities associated with roots of four common plant species to experimentally altered rainfall patterns in replicated field plots established within an Australian mesic grassland. I found that altered rainfall affected the composition, but not the richness, of the AM fungal community. Specific root length was observed to correlate with AM fungal richness, while concentrations of phosphorus and calcium in root tissue and the proportion of root length allocated to fine roots were correlated with AM fungal community composition. However, I found no evidence that AM fungal response to rainfall resulted via changes in the host because none of the studied traits were affected by rainfall manipulations. However, the effect of altered rainfall patterns via root traits may occur in more responsive plant species or more water limited environments. In addition, I observed that variability among AM fungal communities was high even in communities from the same treatments, and a large proportion of the variation remained unexplained. The high variability observed in molecular community data may be masking the effects of changes in the environment on AM fungal community assembly, which makes predicting AM fungal responses to climate change difficult. I used a trait-based approach to improve our understanding of AM fungal community assembly because we expect that taxa are filtered into local communities according, in part, to their traits and the roles that these traits play in adaptation to environmental conditions. Spores represent a key life history stage for colonisation and survival within stressful environments, therefore I studied spore traits hypothesized to enhance AM fungal fitness in arid environments. I used microscopy, image analysis and a colorimetric assay to measure spore traits at the community level in samples collected in six sites along gradients of aridity in New South Wales and Queensland, Australia, and in communities exposed to two years of experimentally reduced rainfall in replicated field plots in Richmond, NSW. Overall, I found melanin content were higher in more arid environments. I observed a large range of spore colours at all sites but greater range, with a higher proportion of both dark and light spores, in more arid sites. Average spore density differed depending on arid site but I found no evidence that density varied in relation to the aridity gradient. Average spore size increased in the summer rainfall-exclusion treatment after approximately two years, but was not observed to vary along the aridity gradient. Community responses to climate change can take time to manifest and vary over time, making temporal dynamics important to consider. I therefore studied the response of the AM fungal communities in soil to altered rainfall regimes every six months over a period of almost four years and evaluated whether changes in AM fungal communities were associated with plant community richness and composition. I observed that altered rainfall regimes resulted in distinct AM fungal communities differing in richness and composition three years after rainfall manipulations began. I found that plant and AM fungal communities co-varied but found more support for the hypothesis that fungal community composition influence plant community composition than vice versa. However, I found no evidence that altered rainfall regimes were leading to distinct co-associations between plants and AM fungi, suggesting that plant-fungal co-associations and responses to rainfall are decoupled in this system. Overall, my results provide some evidence that grassland plant community at the studied site may not be responding to altered rainfall regimes via changes in the AM fungal communities. Based on my work in eastern Australia, I demonstrated that changes in rainfall regimes influence AM fungal community richness and composition. Shifts in the AM fungal communities may not be immediate, and the direction in which these communities are expected to respond to altered rainfall regimes will depend on the specifics of future climatic conditions. Although I did find associations between AM fungal communities and the identity of host plants and the composition of plant communities, I did not find evidence supporting my predictions that effects of altered rainfall regimes would occur via changes in the host root traits or via changes in the plant community composition. I identified traits potentially associated with rainfall regimes and results suggest that AM fungal communities found in drier environments may have smaller spores with a higher melanin content, and were characterised by a high frequency of both light and dark spores and a lower frequency of intermediate types. Further research is needed to predict how AM fungal communities will respond to climate change, as well as to study how these changes will influence the structure and functioning of grassland ecosystems (approaches for such studies are detailed in chapter 5).