A Biological Comparison of Three Colletotrichum Species Associated with Alfalfa Anthracnose in Northern China.

Anthracnose caused by various species of Colletotrichum is one of the most prevalent diseases in alfalfa worldwide that not only reduces forage yields but also severely compromises forage quality. A comprehensive survey was conducted in 2020 in the main production regions of northern China. The survey results showed that alfalfa anthracnose is prevalent in northern China, with the disease incidence ranging from 9% to 45% and the disease index from 5 to 17 (maximum possible score: 100). In total, 24 isolates were collected and identified as three Colletotrichum species (C. trifolii, C. truncatum and C. americae-borealis) based on morphological characteristics and phylogenetic analysis (combined sequences ITS, HIS3, ACT and GAPDH). The three species displayed remarkable environmental adaptability, exhibiting a capacity for growth, sporulation and conidial germination in temperatures ranging from 4 to 35 °C and in different nutrient conditions. Pathogenicity assays showed that C. trifolii was more virulent than the other two species, although the growth vigor (in terms of colony diameter, sporulation and conidial germination) of C. truncatum was the greatest.

The effects of grassland degradation on the genetic structure of a small mammal.

Grassland degradation is challenging the health of grassland ecosystems globally and causing biodiversity decline. Previous studies have demonstrated the impact of grassland degradation on the abundance and behavior of small mammals. Little is known about how it affects the genetic structure of gregarious mammals in the wild. This study explores the effects of grassland degradation on the genetic structure of a small burrowing mammal, plateau pika (Ochotona curzoniae). We used nine microsatellite loci to analyze the genetic diversity and genetic differentiation between colonies and genetic relatedness between individuals within the colony. We found that pikas in severely degraded grasslands had a significantly higher genetic diversity within colonies, a higher level of gene flow between colonies, and a lower genetic differentiation between colonies compared to pikas in less degraded grasslands. Individuals within colonies had a significantly lower genetic relatedness in severely degraded grasslands than in less degraded grasslands. This study has provided potential evidence of a significant impact of grassland degradation on the genetic structure of pikas, which has caused a breakdown of their kin-selected colony structure.

Endophytes Alleviate Drought-Derived Oxidative Damage in Achnatherum inebrians Plants Through Increasing Antioxidants and Regulating Host Stress Responses.

Endophytes generally increase antioxidant contents of plants subjected to environmental stresses. However, the mechanisms by which endophytes alter the accumulation of antioxidants in plant tissues are not entirely clear. We hypothesized that, in stress situations, endophytes would simultaneously reduce oxidative damage and increase antioxidant contents of plants and that the accumulation of antioxidants would be a consequence of the endophyte ability to regulate the expression of plant antioxidant genes. We investigated the effects of the fungal endophyte Epichloë gansuensis (C.J. Li & Nan) on oxidative damage, antioxidant contents, and expression of representative genes associated with antioxidant pathways in Achnatherum inebrians (Hance) Keng plants subjected to low (15%) and high (60%) soil moisture conditions. Gene expression levels were measured using RNA-seq. As expected, the endophyte reduced the oxidative damage by 17.55% and increased the antioxidant contents by 53.14% (on average) in plants subjected to low soil moisture. In line with the accumulation of antioxidants in plant tissues, the endophyte increased the expression of most plant genes associated with the biosynthesis of antioxidants (e.g., MIOX, crtB, gpx) while it reduced the expression of plant genes related to the metabolization of antioxidants (e.g., GST, PRODH, ALDH). Our findings suggest that endophyte ability of increasing antioxidant contents in plants may reduce the oxidative damage caused by stresses and that the fungal regulation of plant antioxidants would partly explain the accumulation of these compounds in plant tissues.

Warming and altered precipitation independently and interactively suppress alpine soil microbial growth in a decadal-long experiment.

Warming and precipitation anomalies affect terrestrial carbon balance partly through altering microbial eco-physiological processes (e.g., growth and death) in soil. However, little is known about how such processes responds to simultaneous regime shifts in temperature and precipitation. We used the 18O-water quantitative stable isotope probing approach to estimate bacterial growth in alpine meadow soils of the Tibetan Plateau after a decade of warming and altered precipitation manipulation. Our results showed that the growth of major taxa was suppressed by the single and combined effects of temperature and precipitation, eliciting 40-90% of growth reduction of whole community. The antagonistic interactions of warming and altered precipitation on population growth were common (~70% taxa), represented by the weak antagonistic interactions of warming and drought, and the neutralizing effects of warming and wet. The members in Solirubrobacter and Pseudonocardia genera had high growth rates under changed climate regimes. These results are important to understand and predict the soil microbial dynamics in alpine meadow ecosystems suffering from multiple climate change factors.

Overexpression of P5CDH from Cleistogenes songorica improves alfalfa growth performance under field drought conditions.

Water stress affects the metabolic regulation and delays the growth and development of alfalfa, causing a reduction in biomass. New alfalfa germplasm was created with improved drought tolerance in greenhouse conditions by introducing the key gene P5CDH1 from C. songorica, a xerophytic grass. However, the field adaptability and response mechanism of new drought-tolerant alfalfa germplasms under water stress are still unclear. In the present study, the yield and quality traits of transgenic CsP5CDH1 alfalfa lines under water stress and normal irrigation conditions were measured and analyzed for two years. The genetic variance components of the tested traits were calculated from the data fitted by the mixed linear model. The plant height of all lines showed significant genotypic variation (σ2g) (P < 0.05), and the stem diameter, stem number, and dry weight of all lines had a significant genotype × environment interaction (σ2ge) (P < 0.05). The heritability (H) of plant height, stem diameter, stem number, dry weight and leaf-to-stem ratio of alfalfa lines were 0.87, 0.52, 0.59, 0.52 and 0.50, respectively. There were significant genotype × environment interactions (σ2ge) (P < 0.05) for the quality traits of all lines. The heritabilities (H) of acid detergent fiber and neutral detergent fiber were 0.65 and 0.64, respectively. The results of transcriptional expression analysis with RNA-seq showed that the genes MsProDH1, MsProDH4, MsProDH5, MsP5CDH1, MsP5CS5, MsP5CS9, and MsP5CR1, which are involved in the proline metabolism pathway, played an important role in the drought tolerance of innovative alfalfa germplasm. Under water stress, with the regulation of key genes in the proline metabolism pathway, the proline content of all alfalfa lines increased to varying degrees. Among them, the proline content in the shoots and roots of transgenic line L6 was 7.29 times and 12.22 times that under normal irrigation conditions, respectively. The present study helped to clarify that the new germplasm of alfalfa transformed with the CsP5CDH gene synthesized a large amount of proline under water stress, and effectively slowed leaf water loss, thus improving the drought resistance of alfalfa.

Interactive effects of grassland utilization and climatic factors govern the plant diversity-soil C relationship in steppe of North China.

The relationship between plant diversity and the ecosystem carbon pool is important for understanding the role of biodiversity in regulating ecosystem functions. However, it is not clear how the relationship between plant diversity and soil carbon content changes under different grassland use patterns. In a 3-year study from 2013 to 2015, we investigated plant diversity and soil total carbon (TC) content of grasslands in northern China under different grassland utilization methods (grazing, mowing, and enclosure) and climatic conditions. Shannon-Wiener and Species richness index of grassland were significantly decreased by grazing and mowing. Plant diversity was positively correlated with annual precipitation (AP) and negatively correlated with annual mean temperature (AMT). AP was the primary regulator of plant diversity. Grazing and mowing decreased TC levels in grasslands compared with enclosures, especially in topsoil (0-20 cm). The average TC content was decreased by 58 % and 36 % in the 0-10 cm soil layer, while it was decreased by 68 % and 39 % in 10-20 cm soil layer. TC was positively correlated with AP and negatively correlated with AMT. Principal component analysis (PCA) showed that plant diversity was positively correlated with soil TC, and the correlation decreased with an increase in the soil depth. Overall, this study provides a theoretical basis for predicting soil carbon storage in grasslands under human disturbances and climate change impacts.

Alpine grassland degradation intensifies the burrowing behavior of small mammals: evidence for a negative feedback loop.

Globally, grassland degradation is an acute ecological problem. In alpine grassland on the Tibetan Plateau, increased densities of various small mammals in degraded grassland are assumed to intensify the degradation process and these mammals are subject to lethal control. However, whether the negative impact of small mammals is solely a result of population size or also a result of activity and behavior has not been tested. In this study, we use plateau pika as a model to compare population size, core area of colony, and the number of burrow entrances and latrines between lightly and severely degraded grassland. We test whether the alleged contribution of pika to grassland degradation is a result of increased population size or increased burrowing activities of individuals in response to lower food abundance. We found that grassland degradation resulted in lower plant species richness, plant height, and biomass. Furthermore, the overall population size of pika was not significantly affected by location in lightly and severely degraded grassland. However, pika core areas in severely grassland degradation were significantly larger and had significantly higher densities of burrows and latrines. Our study provides convincing evidence that habitat-induced changes in the behavior of small, burrowing mammals, such as pika, can exacerbate grassland degradation. This finding has significant implications for managing small mammals and restoring degraded grassland ecosystems.

Contrasting Effects of Grazing in Shaping the Seasonal Trajectory of Foliar Fungal Endophyte Communities on Two Semiarid Grassland Species.

Fungal endophytes are harboured in the leaves of every individual plant host and contribute to plant health, leaf senescence, and early decomposition. In grasslands, fungal endophytes and their hosts often coexist with large herbivores. However, the influence of grazing by large herbivores on foliar fungal endophyte communities remains largely unexplored. We conducted a long-term (18 yr) grazing experiment to explore the effects of grazing on the community composition and diversity of the foliar fungal endophytes of two perennial grassland species (i.e., Artemisia capillaris and Stipa bungeana) across one growing season. Grazing significantly increased the mean fungal alpha diversity of A. capillaris in the early season. In contrast, grazing significantly reduced the mean fungal alpha diversity of endophytic fungi of S. bungeana in the late season. Grazing, growing season, and their interactions concurrently structured the community composition of the foliar fungal endophytes of both plant species. However, growing season consistently outperformed grazing and environmental factors in shaping the community composition and diversity of both plant species. Overall, our findings demonstrate that the foliar endophytic fungal community diversity and composition differed in response to grazing between A. capillaris and S. bungeana during one growing season. The focus on this difference will enhance our understanding of grazing's impact on ecological systems and improve land management practices in grazing regions. This variation in the effects of leaf nutrients and plant community characteristics on foliar endophytic fungal community diversity and composition may have a pronounced impact on plant health and plant-fungal interactions.

Microbial carbon use efficiency in different ecosystems: A meta-analysis based on a biogeochemical equilibrium model.

Soil microbial carbon use efficiency (CUE) is a crucial parameter that can be used to evaluate the partitioning of soil carbon (C) between microbial growth and respiration. However, general patterns of microbial CUE among terrestrial ecosystems (e.g., farmland, grassland, and forest) remain controversial. To address this knowledge gap, data from 41 study sites (n = 197 soil samples) including 58 farmlands, 95 forests, and 44 grasslands were collected and analyzed to estimate microbial CUEs using a biogeochemical equilibrium model. We also evaluated the metabolic limitations of microbial growth using an enzyme vector model and the drivers of CUE across different ecosystems. The CUEs obtained from soils of farmland, forest, and grassland ecosystems were significantly different with means of 0.39, 0.33, and 0.42, respectively, illustrating that grassland soils exhibited higher microbial C sequestration potentials (p < .05). Microbial metabolic limitations were also distinct in these ecosystems, and carbon limitation was dominant exhibiting strong negative effects on CUE. Exoenzyme stoichiometry played a greater role in impacting CUE values than soil elemental stoichiometry within each ecosystem. Specifically, soil exoenzymatic ratios of C:phosphorus (P) acquisition activities (EEAC:P ) and the exoenzymatic ratio of C:nitrogen (N) acquisition activities (EEAC:N ) imparted strong negative effects on soil microbial CUE in grassland and forest ecosystems, respectively. But in farmland soils, EEAC:P exhibited greater positive effects, showing that resource constraints could regulate microbial resource allocation with discriminating patterns across terrestrial ecosystems. Furthermore, mean annual temperature (MAT) rather than mean annual precipitation (MAP) was a critical climate factor affecting CUE, and soil pH as a major factor remained positive to drive the changes in microbial CUE within ecosystems. This research illustrates a conceptual framework of microbial CUEs in terrestrial ecosystems and provides the theoretical evidence to improve soil microbial C sequestration capacity in response to global change.

Drought Eliminates the Difference in Root Trait Plasticity and Mycorrhizal Responsiveness of Two Semiarid Grassland Species with Contrasting Root System.

Root traits and arbuscular mycorrhizal (AM) fungi are important in determining the access of plants to soil resources. However, whether plants with different root systems (i.e., taproot vs. fibrous-root) exhibit different root trait plasticity and mycorrhizal responsiveness under drought remains largely unexplored. Tap-rooted Lespedeza davurica and fibrous-rooted Stipa bungeana were grown in monocultures in sterilized and live soils, followed by a drought treatment. Biomass, root traits, root colonization by AM fungi, and nutrient availability were evaluated. Drought decreased biomass and root diameter but increased the root:shoot ratio (RSR), specific root length (SRL), soil NO3--N, and available P for the two species. Under control and drought conditions, soil sterilization significantly increased the RSR, SRL, and soil NO3--N for L. davurica, but this only occurs under drought condition for S. bungeana. Soil sterilization significantly reduced AM fungal root colonization of both species, but drought significantly increased it in live soil. In water-abundant conditions, tap-rooted L. davurica may depend more on AM fungi than fibrous-rooted S. bungeana; however, under drought conditions, AM fungi are of equal importance in favoring both plant species to forage soil resources. These findings provide new insights for understanding the resource utilization strategies under climate change.

Arbuscular mycorrhizal fungus changes alfalfa (Medicago sativa) metabolites in response to leaf spot (Phoma medicaginis) infection, with subsequent effects on pea aphid (Acyrthosiphon pisum) behavior.

Plant disease occurs simultaneously with insect attack. Arbuscular mycorrhizal fungi (AMF) modify plant biotic stress response. Arbuscular mycorrhizal fungi and pathogens may modify plant volatile organic compound (VOC) production and insect behavior. Nevertheless, such effects are rarely studied, particularly for mesocosms where component organisms interact with each other. Plant-mediated effects of leaf pathogen (Phoma medicaginis) infection on aphid (Acyrthosiphon pisum) infestation, and role of AMF (Rhizophagus intraradices) in modifying these interactions were elucidated in a glasshouse experiment. We evaluated alfalfa disease occurrence, photosynthesis, phytohormones, trypsin inhibitor (TI) and total phenol response to pathogen and aphid attack, with or without AMF, and aphid behavior towards VOCs from AMF inoculated and non-mycorrhizal alfalfa, with or without pathogen infection. AM fungus enhanced alfalfa resistance to pathogen and aphid infestation. Plant biomass, root : shoot ratio, net photosynthetic rate, transpiration rate, stomatal conductance, salicylic acid, and TI were significantly increased in AM-inoculated alfalfa. Arbuscular mycorrhizal fungi and pathogen significantly changed alfalfa VOCs. Aphids preferred VOCs of AM-inoculated and nonpathogen-infected to nonmycorrhizal and pathogen-infected alfalfa. We propose that AMF alter plant response to multiple biotic stresses in ways both beneficial and harmful to the plant host, providing a basis for strategies to manage pathogens and herbivore pests.

Leaf morphology, functional trait and altitude response in perennial vetch (Vicia unijuga A. Braun), alfalfa (Medicago sativa L.) and sainfoin (Onobrychis viciifolia Scop.).

MAIN CONCLUSION: Species have plasticity across altitude gradients in leaf morphology and function, and their response to high altitude conditions was mainly reflected in leaf cell metabolism and gas exchange. Leaf morphological and functional adaptation to altitude has received research attention in recent years, but there are no studies for forage legumes. Here we report differences in 39 leaf morphology and functional traits of three leguminous forages (alfalfa, sainfoin and perennial vetch) at three sites in Gansu Province, China, ranging from 1768 to 3074 m altitude to provide information for potential use in breeding programmes. With increasing altitude, plant water status increased, reflecting increase in soil water content and decreased average temperature, which lead to leaf intercellular CO2 concentration. Stomatal conductance and evapotranspiration increased significantly but water-use efficiency decreased. At high altitude, ΦPSII decreased but non-photochemical quenching and chlorophyll a:b ratio increased while spongy mesophyll tissue and leaf thickness increased. These changes may be due to UV or low-temperature damage of leaf protein and metabolic cost of plant protection or defence responses. Contrary to many other studies, leaf mass per area decreased significantly at higher altitude. This was consistent with predictions under the worldwide leaf economic spectrum on the basis that soil nutrients increased with increasing altitude. The key species differences were more irregularly shaped epidermal cells and larger stomatal size in perennial vetch compared to alfalfa or sainfoin that enhanced gas exchange and photosynthesis by generating mechanical force, increasing guard cell turgor, and promoting stomatal operation. The lower adaxial stomatal density also enhanced water-use efficiency. These adaptations might confer perennial vetch an advantage in environments with extreme diurnal temperature fluctuation or in frigid conditions.

Effect of Epichloë gansuensis Endophyte on Seed-Borne Microbes and Seed Metabolites in Achnatherum inebrians.

The seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.

Metagenomics reveals the response of antibiotic resistance genes to elevated temperature in the Yellow River.

Climate warming may aggravate the threat of antibiotic resistance genes (ARGs) to environmental and human health. However, whether temperature can predict ARGs and influence their assembly processes remains unknown. Here, we used metagenomic sequencing to explore how gradually elevated water temperature (23 °C, 26 °C, 29 °C, 32 °C, 35 °C) influences ARG and mobile genetic element (MGE) profiles in the Yellow River. In total, 30 ARG types including 679 subtypes were detected in our water samples. Gradually increased temperature remarkably reduced ARG diversity but increased ARG abundance. Approximately 37 % of ARGs and 42 % of MGEs were predicted by temperature, while most others were not sensitive to temperature. For each 1 °C increase in temperature, the ARG abundance rose by 2133 TPM (Transcripts Per kilobase of exon model per Million mapped reads) abundance, and multidrug, tetracycline and peptide resistance genes had the fastest increases. Proteobacteria and Actinobacteria were the primary ARG hosts, with 558 and 226 ARG subtypes, respectively. Although ARG profiles were mainly governed by stochastic process, elevated temperature increased the deterministic process of ARGs in the Yellow River. The abundance of five high-risk ARGs (tetM, mecA, bacA, vatE and tetW) significantly increased with elevated water temperature, and these ARGs co-occurred with several opportunistic pathogens (Delftia, Legionella and Pseudomonas), implying that antibiotic resistance risk may increase under climate warming. Our study explored the possibility of predicting resistomes and their health risks through temperature, providing a novel approach to predict and control ARGs in water environments under climate warming.

Abscisic Acid May Play a Critical Role in the Moderating Effect of Epichloë Endophyte on Achnatherum inebrians under Drought Stress.

Water scarcity is a major constraint that adversely affects plant development and growth. Abscisic acid (ABA) is a plant stress hormone that is rapidly synthesized and can induce stomatal closure to conserve water, thereby alleviating the drought stress of plants. The Epichloë endophyte enhances the drought tolerance of Achnatherum inebrians (drunken horse grass, DHG). To better understand how the Epichloë endophyte enhances drought tolerance, DHG plants without (EF) and with (EI), an Epichloë endophyte, were grown under 20% and 60% soil water conditions (SWC), and the leaves of the three treatments of EF and EI plants were sprayed with ABA solution (1 mg/L); fluridone (FLU), the ABA biosynthesis inhibitor solution (1 mg/L); and distilled water, respectively. Four-weeks later, the results indicated that the exogenous ABA application promoted plant growth, stomatal conductance, and photosynthetic rate, while the opposite effect occurred with plants sprayed with FLU. The differences between EI and EF plants in tiller number, height, chlorophyll content, stomata conductance, and photosynthetic rate were highest when sprayed with ABA. Thus, it is concluded that ABA might be involved in the moderating effect of Epichloë endophytes on DHG plants exposed to drought by maintaining growth and improving photosynthetic efficiency.

Transcriptomic Analyses Reveals Molecular Regulation of Photosynthesis by Epichloë endophyte in Achnatherum inebrians under Blumeria graminis Infection.

Photosynthesis is essential for the growth of all green plants, and the presence of an Epichloë endophyte enhances the photosynthesis of Achnatherum inebrians (drunken horse grass, DHG), including when it is under attack by fungal pathogens. However, few studies have examined the mechanism of the increased photosynthetic activity at the molecular level of A. inebrians when it is under pathogen stress. The present study investigated the effects of the presence of the Epichloë endophyte on the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance, and transpiration rate of DHG plants under a Blumeria graminis infection condition, and we compared the transcriptomes using RNA sequencing. The results showed that the photosynthetic rate of Epichloë endophyte-infected (E+) plants was higher under the B. graminis infection condition, and also without this pathogen, when it was compared with Epichloë endophyte-free (E-) plants. The E+ plants uninfected with B. graminis had 15 up-regulated unigenes that are involved in photosynthesis which were compared to the E- plants that were uninfected with this pathogen. This suggests that the presence of an Epichloë endophyte up-regulates the genes that are involved in the process of photosynthesis.

The microbiota diversity of Festuca sinensis seeds in Qinghai-Tibet Plateau and their relationship with environments.

A total of 14 Festuca sinensis seed lots were collected from different geographical locations on the Qinghai-Tibet Plateau to study the seed microbiota and determine the abiotic (temperature, precipitation, and elevation) and biotic (Epichloë sinensis infection rate) factors likely to shape the seed microbiome. The 14 seed lots had different bacterial and fungal structures and significantly different diversities (p < 0.05). The α-diversity indices of the bacteria were significantly correlated with precipitation (p < 0.05), whereas those of the fungi were significantly correlated with temperature (p < 0.05). Microbiota analysis showed that Proteobacteria, Cyanobacteria, and Bacteroidetes were the most abundant bacteria at the phylum level in the seeds, and Ascomycota and Basidiomycota were the most abundant fungi. ß-diversity analysis suggested large differences in the microbial communities of each sample. Redundancy analysis showed that temperature and precipitation were the main environmental factors that drive variations in the microbial community, at the medium-high elevation (3,000-4,500 m), the impact of temperature and precipitation on microbial community is different, and the other elevations that effect on microbial community were basically identical. Spearman's correlation analysis showed that the relative abundances of the most abundant bacterial phyla were significantly correlated with temperature (p < 0.05), whereas those of the most abundant fungal phyla were significantly correlated with precipitation (p < 0.05). E. sinensis infection rates were significantly correlated with elevation and temperature (p < 0.05). These results suggest that temperature and precipitation are the key factors driving the microbial community, that temperature and elevation also had a great influence on the E. sinensis infection rate, and that environmental factors (temperature and elevation) may further affect the microbial community by regulating the E. sinensis infection rate.

Cuticular Wax Modification by Epichloë Endophyte in Achnatherum inebrians under Different Soil Moisture Availability.

The cuticular wax serves as the outermost hydrophobic barrier of plants against nonstomatal water loss and various environmental stresses. An objective of this study was to investigate the contribution of the mutualistic fungal endophyte Epichloë gansuensis to leaf cuticular wax of Achnatherum inebrians under different soil moisture availability. Through a pot experiment and gas chromatography-mass spectrometry (GC-MS) analysis, our results indicated that the hydrocarbons were the dominant components of leaf cuticular wax, and the proportion of alcohols, aldehydes, amines, and ethers varied with the presence or absence of E. gansuensis and different soil moisture availability. Amines and ethers are unique in endophyte-free (EF) A. inebrians plants and endophyte-infected (EI) A. inebrians plants, respectively. By transcriptome analysis, we found a total of 13 differentially expressed genes (DEGs) related to cuticular biosynthesis, including FabG, desB, SSI2, fadD, BiP, KCS, KAR, FAR, and ABCB1. A model is proposed which provides insights for understanding cuticular wax biosynthesis in the association of A. inebrians plants with E. gansuensis. These results may help guide the functional analyses of candidate genes important for improving the protective layer of cuticular wax of endophyte-symbiotic plants.

Integrative Analyses of Transcriptomes and Metabolomes Reveal Associated Genes and Metabolites with Flowering Regulation in Common Vetch (Vicia sativa L.).

As an important source of protein for livestock and human consumption, Vicia sativa is cultivated worldwide, but its seed production is hampered at high altitudes because of the short frost-free period. Flowering represents the transition from a vegetative to a reproductive period, and early flowering benefits plant seed production at high altitudes. However, the molecular mechanisms of flowering regulation in V. sativa remain elusive. In the present study, two V. sativa accessions with different flowering characteristics were used: Lan3 (early-flowering) was cultivated by our laboratory, and 503 (late-flowering) was selected from 222 V. sativa accessions after three years of field experiments. The shoot samples (shoot tip length = 10 cm) of these two accessions were collected 63, 70, and 77 days after sowing, and the molecular regulatory mechanism of the flowering process was identified by integrative analyses of the transcriptomes and metabolomes. Kyoto Encyclopedia of Genes and Genomes enrichment showed that the synthesis and signal transduction of plant hormone pathways were the most enriched pathways in 4274 differentially expressed genes (DEGs) and in 259 differential metabolites between Lan3 and 503. Moreover, the contents of three metabolites related to salicylic acid biosynthesis and the transcription levels of two DEGs related to salicylic acid signal transduction in Lan3 were higher than those in 503. Further verification in various accessions indicated that salicylic acid metabolism may be involved in the flowering regulation process of V. sativa. These findings provide valuable information for understanding the flowering mechanism and for promoting breeding research in V. sativa.

Vertically Transmitted Epichloë Systemic Endophyte Enhances Drought Tolerance of Achnatherum inebrians Host Plants through Promoting Photosynthesis and Biomass Accumulation.

Achnatherum inebrians (drunken horse grass, DHG) plants, a dominant grass species in the arid and semi-arid regions of northwest China, symbiotic with an Epichloë fungal endophyte, is well adapted to drought. However, little is known about how the presence of the foliar Epichloë endophyte enhances the tolerance of DHG to drought at the molecular level. This study explored the positive effects of the presence of the Epichloë endophyte on plant growth, biomass, and photosynthetic efficiency and processes of DHG under non-drought and two drought (moderate and severe) treatments, using RNA sequencing to compare transcriptomes. The transcriptome results showed that 32 selected unigenes involved in the photosynthesis processes within Epichloë symbiotic plants were differently expressed (DEGs) versus non-symbiotic plants. The majority of these selected DEGs were upregulated in Epichloë symbiotic plants versus non-symbiotic plants, such as upregulated unigenes (c51525.graph_c1, c47798.graph_c0 & c64087.graph_c0) under drought conditions. In line with the transcriptomes data, the presence of the Epichloë endophyte promoted the photosynthetic rate and biomass accumulation of DHG plants, and the relationship between the photosynthetic rate and biomass is linear and significant. The presence of the endophyte only increased the biomass per tiller of DHG plants under drought. This study provides further insights into the molecular mechanisms that underlie the enhanced plant growth and drought tolerance of Epichloë-symbiotic DHG plants.