Can nitrogen supersede host identity in shaping the community composition of foliar endophytic fungi in an alpine meadow ecosystem?

The availability of limiting nutrients plays a crucial role in shaping communities of endophytes. Moreover, whether fungal endophytes are host-specific remains controversial. We hypothesized that in a harsh and nitrogen (N)-deficient area, diversity and community composition of foliar endophytic fungi (FEFs) varied substantially among plots with experimentally elevated levels of macronutrients, and thus, N availability, instead of host species identity, would have a greater influence in structuring fungal communities at different scales. We also expected an important subset of taxa shared among numerous host species and N gradients to form a community-wide core microbiome. We measured the leaf functional traits and community structures of FEFs of three commonly seen species in an alpine meadow nested with a long-term N fertilization experiment. We found that host plant identity was a powerful factor driving the endophytic fungal community in leaves, even in habitats where productivity was strongly limited by nitrogen (p < 0.001). We also found that within the same host, nitrogen was an important driving force for the composition of the endophytic fungi community (p < 0.05). In addition, the leaf carbon content was the most important functional trait that limited the diversity of endophytic fungi (p < 0.001). Finally, we documented a distinct core microbiome shared among our three focal species and N gradients.

Effects of Pedicularis kansuensis Expansion on Plant Community Characteristics and Soil Nutrients in an Alpine Grassland.

Pedicularis kansuensis is an indicator species of grassland degradation. Its population expansion dramatically impacts the production and service function of the grassland ecosystem, but the effects and mechanisms of the expansion are still unclear. In order to understand the ecological effects of P. kansuensis, three P. kansuensis patches of different densities were selected in an alpine grassland, and species diversity indexes, biomasses, soil physicochemical properties, and the mechanism among them were analyzed. The results showed that P. kansuensis expansion increased the richness index, the Shannon−Wiener index significantly, and the aboveground biomass ratio (ABR) of the Weed group (p < 0.05), but reduced the total biomass of the community and the ABR of the Gramineae and Cyperaceae decreased insignificantly (p > 0.05); soil moisture, soil AOC, and NO3−·N decreased significantly (p < 0.05), while soil pH and total soil nutrients did not change significantly, and available phosphorus (AP) decreased at first and then increased (p < 0.05). The structural equation model (SEM) showed that P. kansuensis expansion had a significant positive effect on the community richness index, and a significant negative effect followed on the soil AOC from the increase of the index; the increase of pH had a significant negative effect on the soil AOC, NO3−·N, and AP. It indicated that P. kansuensis expansion resulted in the increase of species richness, the ABR of the Weed group, and the community's water demand, which promoted the over-utilization of soil available nutrients in turn, and finally caused the decline of soil quality. This study elucidated a possible mechanism of poisonous weeds expansion, and provided a scientific and theoretical basis for grassland management.

Responses of Soil Microbial Metabolic Activity and Community Structure to Different Degraded and Restored Grassland Gradients of the Tibetan Plateau.

Climate change and land-use disturbances are supposed to have severely affected the degraded alpine grasslands on the Tibetan Plateau. Artificial grassland establishment has been implemented as a restoration tool against grassland degradation. However, the impact of such degradation and restoration processes on soil microbial communities and soil quality is not clearly understood. Here, we aim to investigate how the dynamics of microbial community and soil quality of alpine grasslands respond to a gradient of degradation and that of restoration, respectively. We conducted a randomised experiment with four degradation stages (light, moderate, heavy, and extreme degradation) and three restoration stages (artificial restoration for 1, 5, and 10 years). We analysed the abundance and diversity of soil bacteria and fungi, and measured soil nutrients, enzymatic activity and microbial biomass. The concentration of soil nitrogen (TN), soil organic matter (OM) in heavy degraded grassland decreased significantly by 37.4 and 45.08% compared with that in light degraded grassland. TN and OM in 10-years restored grassland also increased significantly by 33.10 and 30.42% compared to that in 1-year restored grassland. Four soil enzymatic activity indicators related to microbial biomass decreased with degradation gradient and increased with recovery time (i.e., restoration gradient). Both bacterial and fungal community structure was significantly different among grassland degradation or restoration successional stages. The LEfSe analysis revealed that 29 fungal clades and 9 bacterial clades were susceptible to degraded succession, while16 fungal clades and 5 bacterial clades were susceptible to restoration succession. We conclude that soil quality (TN, OM, and enzymatic activity) deteriorated significantly in heavy degraded alpine grassland. Soil microbial community structure of alpine is profoundly impacted by both degradation and restoration processes, fungal communities are more sensitive to grassland succession than bacterial communities. Artificial grasslands can be used as an effective method of restoring degraded grassland, but the soil functions of artificial grassland, even after 10 years of recovery, cannot be restored to the original state of alpine grassland.

Experimental Warming Has Not Affected the Changes in Soil Organic Carbon During the Growing Season in an Alpine Meadow Ecosystem on the Qinghai-Tibet Plateau.

The effects of climate warming and season on soil organic carbon (SOC) have received widespread attention, but how climate warming affects the seasonal changes of SOC remains unclear. Here, we established a gradient warming experiment to investigate plant attributes and soil physicochemical and microbial properties that were potentially associated with changes in SOC at the beginning (May) and end (August) of the growing season in an alpine meadow ecosystem on the Qinghai-Tibet Plateau. The SOC of August was lower than that of May, and the storage of SOC in August decreased by an average of 18.53 million grams of carbon per hectare. Warming not only failed to alter the content of SOC regardless of the season but also did not affect the change in SOC during the growing season. Among all the variables measured, microbial biomass carbon was highly coupled to the change in SOC. These findings indicate that alpine meadow soil is a source of carbon during the growing season, but climate warming has no significant impact on it. This study highlights that in the regulation of carbon source or pool in alpine meadow ecosystem, more attention should be paid to changes in SOC during the growing season, rather than climate warming.

Slope aspect determines the abundance and composition of nitrogen-cycling microbial communities in an alpine ecosystem.

Slope aspect is an important topographic feature that can influence local environmental conditions. While strong effects of slope aspect on aboveground and belowground communities have been frequently elucidated, how slope aspect affects soil nitrogen (N) cycling microbes remains unclear. Here, we characterized the communities of soil N-cycling microbes on south- and north-facing slopes in an alpine ecosystem, by quantifying (qPCR) and high-throughput sequencing six genes involved in N-fixation (nifH), nitrification (archaeal and bacterial amoA) and denitrification (nirK, nirS and nosZ). We found that the abundance, diversity and community composition of major N-cycling microbes differed dramatically between the two slope aspects, and these variances could be well explained by the aspect-driven differences in environmental conditions, especially soil temperature and moisture. The response patterns of different N-cycling groups to slope aspect were much inconsistent, especially for those with similar functions (i.e. ammonia-oxidizing archaea vs. bacteria, nirK- vs. nirS-reducers), indicating strong niche differentiation between these counterparts. We also observed strong preferences and distinct co-occurrence patterns of N-cycling microbial taxa for the two slope aspects. These findings highlight the importance of slope aspect in determining the abundance, species distribution and community structure of N-cycling microbes, and consequently influencing N-cycling processes and ecosystem functioning.

Revegetation differentially influences microbial trophic groups in a Qinghai-Tibetan alpine steppe ecosystem.

Revegetation accelerates the recovery of degraded lands. Different microbial trophic groups underpin this acceleration from the aspects of soil structure stabilization, nutrient accumulation, and ecosystem functions. However, little is known about how revegetation influences the community and biodiversity of different soil microbial trophic groups. Here, six revegetation treatments with different plantings of plant species were established at an excavation pit in the Qinghai-Tibetan Plateau. Communities of plant, bacteria, and several key soil fungal groups were investigated after 12 years of revegetation. Plant and all microbial trophic group compositions were markedly influenced by revegetation treatments. Total fungal and pathogenic fungal compositions were not significantly predicted by any factor of plant and soil, but arbuscular mycorrhizal fungal composition could be mainly predicted by plant composition and plant P content. Bacterial composition was mainly determined by soil total N, organic carbon concentration, and moisture content; and saprotrophic fungal composition was mainly determined by soil organic carbon. Soil pH was the strongest factor to predict bacterial metabolic functions. Our findings highlight that even the differences of microbial compositions were because of different revegetation treatments, but each trophic microbial composition had different relations with plant and/or soil; especially, the bacterial community and metabolic functions and saprotrophic fungal community were more correlated with soil properties rather than plant community or characteristics per se.

Arbuscular mycorrhizal fungi serve as keystone taxa for revegetation on the Tibetan Plateau.

Revegetation is widely used to enhance degraded topsoil recovery with the enhancements of soil nutrient accumulation and soil structure stabilization. Arbuscular mycorrhizal fungi (AMF) are important for the allocation of carbon into the soil and the formation of soil aggregates. Thus, we hypothesized that AMF could construct more niches for other microbes during revegetation, making AMF keystone taxa of soil. Soil fungal and bacterial communities were investigated under a revegetation experiment and correlation networks between soil fungi and bacteria were constructed. Simultaneously, the plant growth level, soil properties and structure, and soil microbial carbon decomposition abilities were measured. The results revealed that AMF were the most central fungi at the phylum (degree = 3), class (degree = 11), and family (degree = 15) levels. The reads number of AMF were positively correlated with both fungal (R2 = 0.431, P < 0.001) and bacterial (R2 = 0.106, P = 0.044) richness. Higher colonization of AMF in roots and/or more AMF extraradical mycelium and spores in soil indicated a better plant growth, more stable soil aggregates, and a higher carbon decomposition ratio. Our results highlight that AMF are keystone taxa in revegetation, as they play significant roles in enhancing the recovery of the belowground microbiome diversity, soil structure stability, and nutrients cycling. The positive roles of AMF in revegetation support the application of AMF in ecosystem recovery.

Comparison of different PCR primers on detecting arbuscular mycorrhizal communities inside plant roots.

OBJECTIVE: Communities of arbuscular mycorrhizal fungi (AMF) colonizing roots have been increasingly investigated by molecular approaches with AMF-specific PCR primers. However, it is difficult to compare the species diversity and species compositions of AMF communities across various studies due to the PCR primers used differently, and also little is known if significant difference of community compositions is characterized by different primers. We aim to compare the difference of efficiency of four primers for AMF. METHODS: We chose four commonly used AMF-specific primer combinations (NS31-AM1, AMLl-AML2, NS31-AML2 and SSUmCf-LSUmBr), and used 18S rDNA clone libraries to describe the AMF diversity and community. RESULTS: Our results showed that the specificity and coverage varied among the tested primers, different primer combinations would yield distinct patterns of species diversity and composition of AMF community. SSUmCf-LSUmBr had the best specificity and coverage in amplifying AMF sequences, followed by NS31-AML2 and NS31-AM1, and AML1-AML2 showed the lowest specificity towards AMF sequences. CONCLUSION: SSUmCf-LSUmBr is not the optimal primer pair for AMF community study in current stage due to limited reference sequences and large DNA size. As an alternative, NS31-AML2 is more suitable in AMF community study, because its target rDNA region could well match the increasingly used virtual taxonomy database (http://maarjam. botany.ut.ee) and also its suitable DNA size could be efficiently used in high-throughput sequencing.

Relative importance of deterministic and stochastic processes in driving arbuscular mycorrhizal fungal assemblage during the spreading of a toxic plant.

Both deterministic and stochastic processes are expected to drive the assemblages of arbuscular mycorrhizal (AM) fungi, but little is known about the relative importance of these processes during the spreading of toxic plants. Here, the species composition and phylogenetic structure of AM fungal communities colonizing the roots of a toxic plant, Ligularia virgaurea, and its neighborhood plants, were analyzed in patches with different individual densities of L. virgaurea (represents the spreading degree). Community compositions of AM fungi in both root systems were changed significantly by the L. virgaurea spreading, and also these communities fitted the neutral model very well. AM fungal communities in patches with absence and presence of L. virgaurea were phylogenetically random and clustered, respectively, suggesting that the principal ecological process determining AM fungal assemblage shifted from stochastic process to environmental filtering when this toxic plant was present. Our results indicate that deterministic and stochastic processes together determine the assemblage of AM fungi, but the dominant process would be changed by the spreading of toxic plants, and suggest that the spreading of toxic plants in alpine meadow ecosystems might be involving the mycorrhizal symbionts.

Direct and indirect influences of 8 yr of nitrogen and phosphorus fertilization on Glomeromycota in an alpine meadow ecosystem.

We measured the influences of soil fertility and plant community composition on Glomeromycota, and tested the prediction of the functional equilibrium hypothesis that increased availability of soil resources will reduce the abundance of arbuscular mycorrhizal (AM) fungi. Communities of plants and AM fungi were measured in mixed roots and in Elymus nutans roots across an experimental fertilization gradient in an alpine meadow on the Tibetan Plateau. As predicted, fertilization reduced the abundance of Glomeromycota as well as the species richness of plants and AM fungi. The response of the glomeromycotan community was strongly linked to the plant community shift towards dominance by Elymus nutans. A reduction in the extraradical hyphae of AM fungi was associated with both the changes in soil factors and shifts in the plant community composition that were caused by fertilization. Our findings highlight the importance of soil fertility in regulating both plant and glomeromycotan communities, and emphasize that high fertilizer inputs can reduce the biodiversity of plants and AM fungi, and influence the sustainability of ecosystems.

Diverse communities of arbuscular mycorrhizal fungi inhabit sites with very high altitude in Tibet Plateau.

Diversity of arbuscular mycorrhizal fungi (AMF) is well studied in many ecosystems, but little is known about AMF in cold-dominated regions with very high altitude. Here, we examined AMF communities associated with two plant species in the Tibet Plateau. Roots and rhizosphere soils of Dracocephalum heterophyllum (pioneer species) and Astragalus polycladus (late-successional species) were sampled at five sites with altitude from 4500 to 4800 m a.s.l. A total of 21 AMF phylotypes were identified from roots and spores following cloning and sequencing of 18S rRNA gene, including eight new phylotypes and one new family-like clade. More AMF phylotypes colonized root samples of D. heterophyllum (5.4±0.49) than of A. polycladus (1.93±0.25). Vegetation coverage was the most important factor influencing AMF community composition in roots. Globally infrequent phylotype Glo-B2 in Glomus group B was the most dominant in roots, followed by globally frequent phylotype Glo-A2 related to Glomus fasciculatum/intraradices group. Our findings suggest that a diverse AMF flora is present in the Tibet Plateau, comprising both potentially habitat-selective and generalist fungi.

[Effects of fertilization on arbuscular mycorrhizal fungi in Elymus nutans roots].

By using traditional staining and DNA cloning and sequencing techniques, this paper studied the effects of fertilization on the arbuscular mycorrhizal (AM) colonization and AM fungal community in Elymus nutans roots across an 8-year nitrogen (N) and phosphorus (P) fertilization gradient. With the increasing level of fertilization, the AM colonization and arbuscular colonization per unit root length reduced from 67.5% to 7.3% and from 5.2% to 0.1%, respectively. A total of 24 AM fungal phylotypes were detected in E. nutans roots, whilst the mean AM fungal richness reduced from 6 to 2.6 across the fertilization gradient. Different fertilization had significant effects on the AM fungal community composition, and there existed significant correlations between the AM fungal community composition and the soil available P and root N contents. The availability of N and P increased gradually across the fertilization gradient, and had significant negative correlations with both AM colonization and AM fungal richness. It was suggested that high level fertilization would reduce the activity of AM symbionts, and result in the large loss of AM fungal biodiversity.