Dark septate endophyte inoculation enhances antioxidant activity in Astragalus membranaceus var. mongholicus under heat stress.

The influence of dark septate endophytic (DSE) on the antioxidant activity of Astragalus membranaceus var. mongholicus under heat stress was investigated. A. membranaceus plants, with or without DSE inoculation, were grown at 28°C for 8 weeks in a greenhouse and subsequently subjected to heat stress conditions (42°C) in an artificial climate chamber. DSE inoculation significantly decreased the malondialdehyde (MDA) content during the initial three days of heat stress. The activities of superoxide dismutase (SOD) and peroxidase (POD) of A. membranaceus leaves were significantly enhanced by DSE inoculation under heat stress, with SOD activities being 63-81% higher than in other treatments. The glutathione (GSH) and putrescine (Put) contents accumulated significantly on the third day under heat stress with DSE inoculation. Additionally, the contents of soluble sugars and proline (Pro) exhibited significant increases on the seventh day of heat stress and were 33-55% and 81-83% higher than in other treatments, respectively. Three-way ANOVA shows that DSE inoculation under heat stress exerted a significant impact on MDA. Multivariate linear regression and structural equality modelling (SEM) further show that the interaction among these antioxidants significantly decreased MDA content and maintained the normal function of cell membranes. In conclusion, DSE inoculation enhanced the heat tolerance of A. membranaceus by boosting its antioxidant capacity and reducing MDA production. This study highlights the potential of utilizing DSE as a strategy to enhance plant heat tolerance.

Growth of alfalfa in the presence of metabolites from a dark septate endophyte strain Alternaria sp. 17463 cultured with a nonionic surfactant and emulsifier.

AIM: Dark septate endophytes (DSE) were widely used in the agriculture and ecological restoration. The objective of this work was to assess the effect of culture media nonionic surfactant and emulsifier on the biomass and metabolites of DSE strain Alternaria sp. 17463. METHODS AND RESULTS: Changes in the composition of DSE metabolites following the addition of Tween 80 during liquid culture of a DSE fungus were analyzed and used in growth tests of alfalfa.Shaking flask fermentation was carried out and the surfactant was fed to the fungus during the fermentation. The residual sugar content and pH declined significantly in the medium and the biomass of DSE increased by 7.27% over controls with no surfactant. Metabolomic analysis showed that adding the surfactant significantly increased the content of 63 metabolites (P < 0.05). These include lipids and lipid-like molecules, organooxygen compounds, amino acids and organic acids, and flavonoids. Enrichment analysis of metabolic pathways indicates that surfactant addition promoted carbohydrate metabolism and amino acid synthesis. A plant hydroponic experiment indicated that these changes in metabolites altered the root structure of alfalfa seedlings. They also promoted significant increases in root length and root surface area, and increased alfalfa total biomass by 50.2%. CONCLUSIONS: The addition of the surfactant promoted sugar utilization by the DSE fungus and increased the synthesis of lipids and amino acids, resulting in the ability of the fungal metabolites to change root structure and promote plant growth.

Effect of Coal Mining on Soil Microorganisms from Stipa krylovii Rhizosphere in Typical Grassland.

The environmental changes caused by coal mining activities caused disturbances to the plant, soil, and microbial health in the mining area. Arbuscular mycorrhizal fungi (AMF) play an important role in the ecological restoration of mining areas. However, it is less understood how soil fungal communities with multiple functional groups respond to coal mining, and the quantitative impact and risk of mining disturbance. Therefore, in this study, the effect of coal mining on soil microorganisms' composition and diversity were analyzed near the edge of an opencast coal-mine dump in the Shengli mining area, Xilingol League, Inner Mongolia. The response strategy of soil fungi to coal mining and the stability of arbuscular mycorrhizal fungi (AMF) in the soil fungal community were determined. Our results showed that coal mining affected AMF and soil fungi in areas within 900 m from the coal mine. The abundance of endophytes increased with the distance between sampling sites and the mine dump, whereas the abundance of saprotroph decreased with the distance between sampling sites and the mine dump. Saprotroph was the dominant functional flora near the mining area. The nodes percentage of Septoglomus and Claroideoglomus and AMF phylogenetic diversity near the mining area were highest. AMF responded to the mining disturbance via the variety and evolution strategy of flora. Furthermore, AMF and soil fungal communities were significantly correlated with edaphic properties and parameters. Soil available phosphorus (AP) was the main influencer of soil AMF and fungal communities. These findings evaluated the risk range of coal mining on AMF and soil fungal communities and elucidated the microbial response strategy to mining disturbance.

AMF Inoculum Enhances Crop Yields of Zea mays L. 'Chenghai No. 618' and Glycine max L. 'Zhonghuang No. 17' without Disturbing Native Fugal Communities in Coal Mine Dump.

For the agricultural development of dumps, increase in land use efficiency and protection of food security, to verify the safety, efficacy and sustainability of field-applied arbuscular mycorrhizal fungi (AMF) inoculum, and to exclude the risk of potential biological invasion, in this study, we determined the effect of AMF inoculation and intercropping patterns (maize-soybean) on the temporal dynamics of soil parameters, native AMF communities and crop yields. AMF communities were analyzed using Illumina MiSeq. A total of 448 AMF operational taxonomic units (OTUs) belonging to six genera and nine families were identified. AMF inoculation treatment significantly improved the yield of intercropping maize and increased the content of available phosphorus. AMF diversity was significantly influenced by cropping pattern and growth stage, but not by the inoculation treatment. Inoculation altered the AMF community composition in the early growth stage and facilitated a more complex AMF network in the early and late growth stages. These results indicate that AMF inoculation affects native AMF only in the early stage, and its impact on yield may be the consequence of cumulative effects due to the advantages of plant growth and nutrient uptake in the early stage.

Growth and metabolism of dark septate endophytes and their stimulatory effects on plant growth.

Dark septate endophytes (DSE) colonize plant roots extensively and increase host plant growth and nutrition. However, the development of DSE-produced metabolites as plant biostimulants has been largely ignored. DSE growth curves and extracellular metabolite components were analyzed and the growth-promoting effects of DSE extracellular metabolites on alfalfa (Medicago sativa L.) grown for 4, 8 12, 16 and 20 days were evaluated. The growth curve of the DSE strain Alternaria sp. shows days 0-8 in the growth phase, days 8-16 in the stable phase, and days 16-20 in the senescent phase. The extracellular metabolite components of DSE were significantly different at different growth stages. The biomass of alfalfa was increased significantly by DSE extracellular metabolites (P < 0.05). Biomass of alfalfa inoculated with DSE extracellular metabolites more than doubled after growth for 8 days and nutrient availability also increased significantly compared with the uninoculated control. Six DSE extracellular metabolites, calycosin 7-galactoside, 1-[(5-amino-5-carboxypentyl)amino]-1-deoxyfructose, N2-fructopyranosylarginine, 2-(4-methyl-5-thiazolyl)ethyl hexanoate, kenposide B, and medinoside E, were significantly positively correlated with alfalfa biomass (P < 0.01). This study combines the DSE extracellular metabolites with plant and soil traits to provide a theoretical basis for the use of DSE metabolites in the product development of plant biostimulants.

Arbuscular mycorrhizal colonization increases plant above-belowground feedback in a northwest Chinese coal mining-degraded soil by increasing photosynthetic carbon assimilation and allocation to maize.

A three-compartment culture system was used to study the mechanism by which the AM fungus Funneliformis mosseae influences host plant growth and soil organic carbon (SOC) content in a northwest China coal mining area. A 13CO2 pulse tracing technique was used to trace the allocation of maize photosynthetic C in shoots, roots, AM fungus, and soil. Carbon accumulation and allocation in mycorrhizal (inoculated with Funneliformis mosseae) and non-mycorrhizal treatments were detected. AM fungal inoculation significantly increased the 13C concentration and content in both above- and below-ground plant parts and also significantly enhanced anti-aging ability by increasing soluble sugars and catalase activity (CAT) in maize leaves while reducing foliar malondialdehyde content (MDA) and leaf temperature and promoted plant growth. AM fungi also increased P uptake to promote maize growth. Soil organic carbon (SOC), glomalin, microbial biomass carbon (MBC), and nitrogen (MBN) contents increased significantly after inoculation. A mutually beneficial system was established involving maize, the AM fungus and the microbiome, and the AM fungus became an important regulator of C flux between the above- and below-ground parts of the system. Inoculation with the AM fungus promoted plant growth, C fixation and allocation belowground to enhance soil quality. A positive above-belowground feedback appeared to be established.

Soil Fungal Community in Grazed Inner Mongolian Grassland Adjacent to Coal-Mining Activity.

Coal mining results in reduced soil quality and makes environments less stable. Soil fungi are suitable indicators of soil quality for monitoring purposes. Here, the objective was therefore to investigate the effects of grazing and mining on the composition of the soil fungal community at the periphery of an opencast coal-mine dump in the Shengli mining area, Xilingol League, Inner Mongolia. A total of 2,110 fungal operational taxonomic units were identified and subdivided into 81 orders and nine categories, based on trophic modes. The sensitive factor to mining was soil pH, and that to grazing were soil nitrate-nitrogen and alkaline phosphatase activity. According to the Pearson correlation and Mantel test, we propose interactions between grazing and coal-mining exist a co-effect and could regulate edaphic variables to alter the behavior of soil fungal community. Moreover, compared with coal-mining, grazing has a greater impact on it. The results provide a basis to further clarify soil fungal ecological functions, and may also contribute to the practice of soil remediation and environmental management in coal-mining areas.

Combined inoculation with dark septate endophytes and arbuscular mycorrhizal fungi: synergistic or competitive growth effects on maize?

BACKGROUND: Effects on maize were assessed of dual inoculation with arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) isolated from other plant species. METHODS: Suspensions of DSE isolated from Stipa krylovii were prepared at different densities (2, 4, and 8 × 105 CFU mL- 1) and inoculated separately (AMF or DSE) or together (AMF + DSE), to explore their effects on maize growth. RESULTS: Inoculation with AMF or medium and high densities of DSE and combined inoculation (AMF + DSE) increased plant above-ground growth and altered root morphology. Differences in plant growth were attributable to differences in DSE density, with negative DSE inoculation responsiveness at low density. AMF promoted plant above-ground growth more than DSE and the high density of DSE promoted root development more than AMF. Combined inoculation might lead to synergistic growth effects on maize at low density of DSE and competitive effects at medium and high DSE densities. CONCLUSIONS: AMF and DSE co-colonized maize roots and they had positive effects on the host plants depending on DSE density. These findings indicate the optimum maize growth-promoting combination of AMF and DSE density and provide a foundation for further exploration of potentially synergistic mechanisms between AMF and DSE in physiological and ecological effects on host plants.

Changes in peanut canopy structure and photosynthetic characteristics induced by an arbuscular mycorrhizal fungus in a nutrient-poor environment.

A well-developed canopy structure can increase the biomass accumulation and yield of crops. Peanut seeds were sown in a soil inoculated with an arbuscular mycorrhizal fungus (AMF) and uninoculated controls were also sown. Canopy structure was monitored using a 3-D laser scanner and photosynthetic characteristics with an LI-6400 XT photosynthesis system after 30, 45 and 70 days of growth to explore the effects of the AMF on growth, canopy structure and photosynthetic characteristics and yield. The AMF colonized the roots and AMF inoculation significantly increased the height, canopy width and total leaf area of the host plants and improved canopy structure. AMF reduced the tiller angle of the upper and middle canopy layers, increased that of the lower layer, reduced the leaf inclination of the upper, middle and lower layers, and increased the average leaf area and leaf area index after 45 days of growth, producing a well-developed and hierarchical canopy. Moreover, AMF inoculation increased the net photosynthetic rate in the upper, middle and lower layers. Plant height, canopy width, and total leaf area were positively correlated with net photosynthetic rate, and the inclination angle and tiller angle of the upper leaves were negatively correlated with net photosynthetic rate. Overall, the results demonstrate the effects of AMF inoculation on plant canopy structure and net photosynthetic rate.

Shifts in composition and function of soil fungal communities and edaphic properties during the reclamation chronosequence of an open-cast coal mining dump.

The diversity, composition and ecological guilds of soil fungal communities in relation to revegetation were assessed during an open-cast mining dump reclamation chronosequence of the soil <1, 5, 10, 15 and 20 years after the start of reclamation. Soil pH and electrical conductivity, total nitrogen (TN), soil organic carbon (SOC), available potassium (AK), and available phosphorus (AP) contents, and soil phosphatase (Pha), urease (U) and invertase (INV) activities were measured. Using high-throughput sequence analysis on internal transcribed spacer (ITS) sequences, 1059 soil fungal operational taxonomic units (OTUs) were identified belonging to 64 orders and these were further categorized by ecological guild. Soil fungal diversity indices were significantly different between the early (<1 year) and later reclamation communities. Nonmetric multidimensional scaling (NMDS) analysis indicates that the composition and ecological guilds of soil fungal communities were significantly different early in the process and at the end of reclamation (P < 0.05). Co-occurrence network and structural equation model analyses show that soil fungal community structure and ecological guilds were correlated with edaphic properties and had an indirect effect on soil available nutrients through direct action on soil enzymes. Overall, the data suggest that soil fungal community composition and function within an open-cast coal mining dump reclamation chronosequence changed during the period following artificial re-vegetation, with interactions between edaphic properties and soil fungal communities associated with these changes.

Grazing and mining influence the population of arbuscular mycorrhizal fungi around the opencast coal mine pit.

The influence of grazing and mining on arbuscular mycorrhizal fungi (AMF) species in grassland were studied around the opencast coal mine pit. The two sampling transects had been designed which was located in the grazing area and in the non-grazing area in the grassland on the north side of the opencast coal mining area. Besides, two control points with the direct distance of 8 km to the opencast coal mining area have been designed. The two points are located in the inside grassland station and outside the grassland station. We collected 3 replicate rhizosphere soil in every point which is from the two transects. We analyzed the genetic diversity of AMF, measured the chemical properties and enzyme of rhizosphere soil of Cleistogenes squarrosa, and explored their correlations using redundancy analysis. And we identified the factors affecting species diversity of AMF around the mining area. Our results showed that grazing on the grassland around the opencast coal mine will aggravate soil degradation and reduce the species richness and diversity of arbuscular mycorrhizal fungi in rhizosphere soil, which can be a theoretical basis for the scientific management of the grassland around the opencast mining area.

Arbuscular mycorrhizal fungal diversity in soils underlying moss biocrusts in coal mining subsidence areas.

The potentially symbiotic mycorrhizal associations dominated by arbuscular mycorrhizal (AM) fungi have become a new topic in bioremediation research in response to global change. Biological soil crusts (biocrusts) play an important role in arid and semi-arid ecosystems. However, AM fungal diversity in the soils underlying moss biocrusts in coal mining subsidence areas remains poorly understood. Here, samples of the soil underlying moss biocrusts in an area inoculated with an AM fungus (AM-BS) and an uninoculated area (CK-BS) plus soil samples from an uninoculated bare area (CK-NBS) were collected from the subsidence area of Shendong Daliuta mine at Yulin, northwest China. AM fungal community diversity indices were maximum in AM-BS, intermediate in CK-BS, and minimum in CK-NBS (P < 0.05). In addition, redundancy analysis (RDA) indicates that the importance of moss biocrust to soil properties followed the sequence: soil water content (SWC) > glomalin-related soil protein (TG) > available phosphorus (Olsen-P) > soil organic matter (SOM) > easily extractable glomalin-related soil protein (EEG) > pH > available nitrogen (alkali-N). SWC, alkali-N, Olsen-P, and SOM were significantly related to the abundance of Glomus and Claroideoglomus, and TG, EEG, and pH were positively related to Diversisipora. In summary, inoculation with the exotic AM fungus and moss biocrust cover created a eutrophic microhabitat for AM fungi in the soils underlying moss biocrusts in the coal mining subsidence area.

Author Correction: Arbuscular mycorrhizal fungi promote the growth of plants in the mining associated clay.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Arbuscular Mycorrhizal Fungi Enhance Sea Buckthorn Growth in Coal Mining Subsidence Areas in Northwest China.

Land subsidence induced by underground coal mining leads to severe ecological and environmental problems. Arbuscular mycorrhizal fungi (AMF) have the potential to improve plant growth and soil properties. We aimed to assess the effects of AMF on the growth and soil properties of sea buckthorn under field conditions at different reclamation times. Inoculation with AMF significantly promoted the survival rate of sea buckthorn over a 50-month period, while also increasing plant height after 14, 26, and 50 months. Crown width after 14 months and ground diameter after 50 months of inoculation treatment were significantly higher than in the uninoculated treatment. AMF inoculation significantly improved plant mycorrhizal colonization rate and promoted an increase in mycelial density in the rhizosphere soil. The pH and electrical conductivity of rhizosphere soil also increased after inoculation. Moreover, after 26 and 50 months the soil organic matter in the inoculation treatment was significantly higher than in the control. The number of inoculated soil rhizosphere microorganisms, as well as acid phosphatase activity, also increased. AMF inoculation may play an active role in promoting plant growth and improving soil quality in the long term and is conducive to the rapid ecological restoration of damaged mining areas.

Arbuscular mycorrhizal fungi promote the growth of plants in the mining associated clay.

It is urgent to restore the ecological function in open-pit mining areas on grassland in Eastern China. The open-pit mines have abundant of mining associated clay, which is desirable for using as a soil source for ecological restoration. The mining associated clay in Hulunbuir district, Inner Mongolia was selected and mixed with a sandy soil at a ratio of 1:1 (S_C soil). Also, effects of arbuscular mycorrhizal fungi (AMF) inoculation on soil functions were studied. The aboveground and underground biomass of maize in S_C soil was 1.49 and 2.41 times higher than that of clay soil, respectively. In the topsoil and S_C soil, the growth hormone (IAA) and cytokinin (CTK) levels of maize were higher than that of clay, while abscission acid (ABA) levels were lower. The inoculation with AMF could significantly improve the biomass of maize and enhance the stress resistance of plants. Through structural equation model (SEM) analyses, it was found that the soil type and AMF inoculation had the most direct impact on maize growth and biomass content. These finds extend our knowledge regarding a low-cost method for physical and biological improvement of mining associated clay, and to provide theoretical support for large-scale application in the future.

An arbuscular mycorrhizal fungus ameliorates plant growth and hormones after moderate root damage due to simulated coal mining subsidence: a microcosm study.

Arbuscular mycorrhizal fungi (AMF) are obligate plant root symbionts delivering a range of benefits to the host plant such as improved nutrient acquisition and resistance to pathogens and abiotic stress. However, whether they can enhance the function of plant root systems damaged due to subsidence caused by excessive coal mining has not been well explored. In the present study, we investigated the effects of AMF using Funneliformis mosseae (FM) as the test fungus on maize (Zea mays L.) growth and hormone levels under different levels of root damage stress by simulating mining subsidence. The results show that plants treated with FM had more shoots, roots, mycorrhizal colonization and higher hyphal density than those without FM under the same simulated mining-induced subsidence conditions. In addition, plants treated with FM also possessed higher N, P, K, Ca, and Mg contents in the shoots and the roots and higher indole-3-acetic acid, gibberellin (GA), and cytokinin (CTK) contents in the roots, indicating that the mycorrhizal association promoted plant biomass and nutrient uptake. FM treatment was no longer beneficial when root damage due to mining-induced subsidence affected more than half of the roots. Soil SOC, AK, and TG were identified as key factors affecting GA, CTK, IAA, and ABA, and AMF can alter plant hormones directly via the hyphae and indirectly by altering soil physicochemical properties under root damage stress. Overall, our results provide baseline data for assessing the biological reclamation effects of AMF on coal mining-induced subsidence.

Arbuscular mycorrhizal fungi alleviate root damage stress induced by simulated coal mining subsidence ground fissures.

Coal mining results in surface subsidence and induces the development of ground fissures that damage surrounding plant roots. Very few studies have explored the stress of root damage caused by ground fissures and whether arbuscular mycorrhizal fungi (AMF) can relieve root damage stress induced by ground fissures. In the present study we simulated ground fissure induced root damage, examined the resultant changes in endogenous hormones, root system morphology, leaf area, leaf chlorophyll content, nutrient content and biomass of maize, and examined the ameliorative effects of AMF on maize with root damage. Ground fissures led to significantly higher levels of endogenous abscisic acid (ABA) but significantly reduced levels of indole-3-acetic acid (IAA), gibberellins (GA) and cytokinin (CTK). In addition, ground fissures led to significantly reduced root biomass, total root length, root tip number, total root volume, plant nutrient content, leaf chlorophyll content and leaf area. The shoot biomass of root damaged maize decreased significantly by 46%. By contrast, AMF increased IAA and CTK levels in maize roots, reduced ABA levels, improved the hormone balance of damaged plants, increased total root length, root tip number, total root volume, leaf area and leaf chlorophyll content, increased nutrient content and increased shoot biomass by 34%. Overall, by simulating coal mining subsidence ground fissures, the study investigated the effects of root damage stress on plant biomass, found that AMF can alleviate the mechanical damages to the root system, and provided a theoretical basis for microbial remediation in areas subject to subsidence due to coal mining.

Arbuscular mycorrhizal fungi enhance soil carbon sequestration in the coalfields, northwest China.

Carbon storage is affected by photosynthesis (Pn) and soil respiration (Rs), which have been studied extensively in natural and agricultural systems. However, the effects of Pn and Rs on carbon storages in the presence of arbuscular mycorrhizal fungi (AMF) in coalfields remain unclear. A field experiment was established in 2014 in Shendong coal mining subsidence area. The treatments comprised two inoculation levels (inoculated with or without 100 g AMF inoculums per seedlings) and four plant species [wild cherry (Prunus discadenia Koebne L.), cerasus humilis (Prunus dictyneura Diels L.), shiny leaf Yellow horn (Xanthoceras sorbifolium Bunge L.) and apricot (Armeniaca sibirica L.)]. AMF increased Pn of four species ranging from 15.3% to 33.1% and carbon storage, averaged by 17.2% compared to controls. Soil organic carbon (OC), easily extractable glomalin-relation soil protein (EE-GRSP), and total glomalin-relation soil protein (T-GRSP) were significantly increased by AMF treatment. The effect of AMF on the sensitivity of Rs depended on soil temperature. The results highlighted the exponential models to explain the responses of Rs to soil temperature, and for the first time quantified AMF caused carbon sequestration and Rs. Thus, to our knowledge, AMF is beneficial to ecosystems through facilitating carbon conservation in coalfield soils.

[Effects of the arbuscular mycorrhizal fungi on environmental phytoremediation in coal mine areas].

To resolve the key environmental problems in coal mine areas of environmental phytoremediation, symbiosis of arbuscular mycorrhizal fungi (AMF) and Amorpha fruticosa was investigated. Effects of AMF on the root growth of Amorpha fruticosa and degenerated soil in coal mining subsidence area were studied. Results showed that after 5 months inoculation, AMF improved the shoot and root growth of Amorpha fruticosa. After inoculation with arbuscular mycorrhiza (AM) for 5 months, the inoculation significantly increased root colonization of Amorpha fruticosa. Total glomalin and easily extractable glomalin were increased significantly in the incubated soil. The content of phosphorus and organic matter were increased in the rhizosphere soil. Population of microorganism increased obviously. All the above results show that their ecological effects are significantly improved. AM would promote rhizosphere soil that will help the sustainability of ecological systems in mining area. It is really of great significance to keep the ecological system stability.