Alfalfa Growth Research Articles

Endophytic strategies decoded by genome and transcriptome analysis of Fusarium nematophilum strain NQ8GII4

IntroductionFusarium nematophilum strain NQ8GII4 is an endophytic fungus with significant potential for improving growth and disease resistance of alfalfa. However, the molecular mechanisms underlying the symbiotic relationship between NQ8GII4 and alfalfa roots remain poorly understood.MethodsIn this study, we conducted (1) a comparative genomic analysis of selected saprophytic, pathogenic, and endophytic fungi, including molecular phylogeny analysis, whole-genome alignment, and divergence date estimation positioning, and (2) transcriptomic profiling of alfalfa roots infected with NQ8GII4.ResultsOur findings reveal that NQ8GII4 is genetically closely related to F. solani, suggesting it diverged from Fusarium phytopathogens. During the early stages of symbiosis establishment, genes encoding glycosyltransferases (GTs), fungal cell wall-degrading enzymes (FCWDEs), and steroid-14α-demethylase (CYP51) were significantly downregulated, potentially suppressing hyphal growth of the fungus. Once symbiosis was established, NQ8GII4 secreted effectors that activated plant immunity, which in turn could slow growth of the fungus. Moreover, genes involved in secondary metabolite biosynthesis, such as type I polyketide synthases (T1PKS) and non-ribosomal peptide synthetases (NRPSs), were significantly downregulated. Homologs of autophagy-related genes, including ATG1, ATG2, ATG11, and others, were also downregulated, suggesting that reduced phytotoxin production and autophagy inhibition is a consequence of NQ8GII4’s symbiosis.DiscussionThis study investigated the comprehensive molecular and genetic mechanisms governing the interaction between NQ8GII4 and alfalfa roots. Beyond the NQ8GII4-alfalfa system, these findings also provide a valuable molecular framework for understanding the mechanism of interactions between endophytic fungi and their host plants.

Hydrothermal biochar enhances the photosynthetic efficiency and yield of alfalfa by optimizing soil chemical properties and stimulating the activity of microbial communities

Hydrothermal biochar has demonstrated potential in enhancing crop growth by improving soil properties and microbial activity; however, its effectiveness varies with application rate, with excessive amounts potentially inhibiting plant growth. This study employed a pot experiment approach to compare varying application rates of hydrothermal biochar (ranging from 0 to 50 t/ha) and to analyze its effects on alfalfa biomass, photosynthetic efficiency, soil nutrient content, and microbial community composition. Biochar application increased alfalfa dry weight by 12.22-21.20% in leaves, 31.60-55.60% in stalks, and 5.62-38.05% in roots. It also enhanced the light utilization efficiency of photosystem II. However, excessive biochar (50 t/ha) reduced biomass and photosynthesis. The addition of biochar amendments enhances soil nutrient availability, particularly increasing the accessibility of carbon, nitrogen, and phosphorus, while also lowering soil pH and enriching microbial interactions within bacterial communities. However, the effects on fungal communities are not pronounced. In conclusion, a moderate application of biochar (10-20t/ha) is recommended to maximize the growth of alfalfa and improve soil health, offering a practical approach for the sustainable cultivation of alfalfa.

Effects of bio-based compound amendments on Na+, Ca2+, Mg2+, and K+ distribution in salt-tolerant alfalfa (Medicago sativa L.) grown in saline soils from two regions

ABSTRACT Ionic stress in saline soils has been demonstrated to result in a continuous decline in the quality and yield of alfalfas. Despite the widespread application of numerous soil amendments in saline soils management, the efficacy and general applicability of these novel amendments in mitigating ionic stress in alfalfas remains to be elucidated. To address this, bio-based compound amendments (BF), comprising earthworm manure, active enzyme treatments, and endophytic mycorrhizal fungi, were tested on two saline soils from the northern Jiangsu tidal flat (JS) and the Yellow River Delta (SD). The soils were treated with no amendments (CK) or BF in alfalfa potting experiments. We evaluated the effects of BF on alfalfa growth, as well as on the uptake, transport, and distribution of Na+, Ca2+, Mg2+, and K+, in the roots, stems, and leaves of alfalfa. BF application significantly increased aboveground and root dry weight of alfalfa on JS soils by 39.20% and 67.10%, respectively, and on SD soils by 78.38% and 45.58%, compared to CK. Additionally, BF application resulted in a significant decrease in Na+ concentration in stems and leaves and increased K+ concentration in all organs of alfalfa grown on both JS and SD saline soils. Applying BF also resulted in a significant increase in K+/Na+ ratios and Ca²⁺/Na⁺ transport selectivity ratios in each organ of alfalfa grown in JS and SD saline soils. However, in JS saline soils, BF application resulted in an increase in Mg²⁺ translocation from roots to stems and a decrease in translocation from stems to leaves. In contrast, the opposite was observed in SD. Overall, the application of BF can enhance the selective absorption and transportation of K+, Ca2+, and Mg2+ grown in JS and SD saline soils, inhibit the selective absorption and transportation of Na+ in alfalfa, maintain the balance of ion metabolism within the plant, and improve the nutritional status and salt tolerance of alfalfas, which is an effective method for improving saline soil and promoting the growth quality of alfalfas.

Total Water Consumption of Alfalfa as a Function of Active Soil Surface and Bioclimatic Coefficients

The paper highlights the significant impact of irrigation on crop yields, emphasizing that in the challenging climatic conditions of the dry-steppe Trans-Volga region, it is impossible to obtain a consistently high alfalfa yield without irrigation. Since irrigation presents both substantial benefits and potential drawbacks, it requires a highly skilled approach to managing irrigated lands and water resources. To estimate the total water consumption of alfalfa in the dry-steppe Trans-Volga region, it is proposed to use bioclimatic coefficients and soil surface activity indicators. (Research purpose) The study aims to determine the total water consumption of alfalfa by using bioclimatic coefficients and active soil surface values to improve the efficiency of irrigation regimes. (Materials and methods) The primary input factors influencing the total water consumption volume were analyzed. Changes in total water consumption during specific phases of alfalfa development were assessed based on evapotranspiration and the relative reserves of productive moisture, using experimental data on the crop's water consumption. (Results and discussion) The empirical coefficients representing the active surface state of alfalfa crops were determined based on the sum of average daily temperatures, calculated using Budyko-Zubenko method for estimating evaporation from the water surface. It was found that the coefficients of active surface state change significantly during plant development, increasing from 0.5 during the growth-branching period to 0.78 during branching-budding and reaching 0.99-1.0 during the budding-flowering period. (Conclusions) Curvilinear relationships between the ratio of total water consumption to evaporation and the relative reserves of productive moisture during different phases of alfalfa growth and development were experimentally established. Empirical coefficients were derived for calculating total water consumption during different periods of alfalfa vegetation grown on dark chestnut soils in the dry steppe Trans-Volga region.

Effects of karst environmental stresses on seed germination and seedling growth of alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) plays an important role in the development of animal husbandry in the karst region of southwestern China, and karst environmental stress has a significant impact on the germination of alfalfa seeds. This study subjected alfalfa seeds to calcium salt stress (0–100 mM), drought stress (0–0.53 MPa), and pH stress (pH 3–9). Germination indicators (germination rate, germination potential, germination index, and vigor index), seedling morphological indicators (shoot length and root length), and biomass indicators were measured to assess seed stress resistance. The results showed that mild drought stress (5% PEG solution) and weak alkaline stress (pH 8) promoted seed germination, and a 20 mM CaCl2 solution significantly increased the germination rate. The root system of seedlings was more sensitive to the three types of stress. Under moderate calcium stress (40 mM), only a slight decrease in tissue water content was observed. Under moderate drought stress (10% PEG), fresh weight and tissue water content decreased, but dry weight significantly increased. Under alkaline stress (pH 9), both biomass indicators and tissue water content increased. This study provides a theoretical reference for selecting plants suitable for cultivation in karst environments.

Growth, Quality, and Nitrogen Metabolism of Medicago sativa Under Continuous Light from Red-Blue-Green LEDs Responded Better to High Nitrogen Concentrations than Under Red-Blue LEDs.

Alfalfa is a widely grown forage with a high crude protein content. Clarifying the interactions between light quality and nitrogen level on yield and nitrogen metabolism can purposely improve alfalfa productivity in plant factories with artificial light (PFAL). In this study, the growth, quality, and nitrogen metabolism of alfalfa grown in PFAL were investigated using three nitrate-nitrogen concentrations (10, 15, and 20 mM, labeled as N10, N15, and N20) and continuous light (CL) with two light qualities (red-blue and red-blue-green light, labeled as RB-C and RBG-C). The results showed that the adaptation performance of alfalfa to nitrogen concentrations differed under red-blue and red-blue-green CL. Plant height, stem diameter, leaf area, yield, Chl a + b, Chl a, Chl b, crude protein contents, and NiR activity under the RB-CN15 treatment were significantly higher than RB-CN10 and RB-CN20 treatments. The RB-CN20 treatment showed morphological damage, such as plant dwarfing and leaf chlorosis, and physiological damage, including the accumulation of proline, H2O2, and MDA. However, the difference was that under red-blue-green CL, the leaf area, yield, and Chl a + b, carotenoid, nitrate, and glutamate contents under RBG-CN20 treatment were significantly higher than in the RBG-CN10 and RBG-CN15 treatments. Meanwhile, the contents of soluble sugar, starch, and cysteine were significantly lower. However, the crude protein content reached 21.15 mg·g-1. The fresh yield, dry yield, stomatal conductance, leaf area, plant height, stem diameter, crude protein, GS, and free amino acids of alfalfa were positively correlated with increased green light. In addition, with the increase in nitrogen concentration, photosynthetic capacity, NiR, and GOGAT activities increased, promoting growth and improving feeding value. The growth, yield, photosynthetic pigments, carbon, nitrogen substances, and enzyme activities of alfalfa were significantly affected by the interaction between nitrogen concentration and light quality, whereas leaf/stem ratio and DPPH had no effect. In conclusion, RB-CN15 and RBG-CN20 are suitable for the production of alfalfa in PFAL, and green light can increase the threshold for the nitrogen concentration adaptation of alfalfa.

Increasing the Growth and Production of Alfalfa by Supplementation with Various Types of Organic Fertilizers and Arbuscular Mycorrhizal Fungi

This study aimed to evaluate the effects of various organic fertilizers and arbuscular mycorrhizal fungi (AMF) on the growth and production of alfalfa plants in the tropics. This study used a completely randomized design (CRD) factorial pattern consisting of two factors with ten replications. Factor A included the types of organic fertilizer, including chicken manure, cow manure, goat manure, and vermicompost. Factor B was the dose of AMF, which included 0 g and 10 g. The results revealed an interaction effect (p<0,01) between the type of organic fertilizer and AMF factors on AMF colonization in alfalfa roots and leaf chlorophyll. The single factor of the type of organic fertilizer had a very significant effect (p<0,01) on increasing the pH, plant height, number of branches, number of leaves, root fresh weight, shoot fresh weight, and shoot dry weight of the media. Moreover, the single factor AMF had no significant effect (p>0,05) on the observed parameters. In conclusion, the application of various types of organic fertilizers with 20% media volume effectively increased the growth and production of alfalfa plants, and the type of organic fertilizer, cow manure, was the best treatment. The application of organic fertilizers alone or in combination with 10 g of AMF did not significantly affect the growth and production of alfalfa plants

Predicting alfalfa leaf area index by non-linear models and deep learning models.

Leaf area index (LAI) of alfalfa is a crucial indicator of its growth status and a predictor of yield. The LAI of alfalfa is influenced by environmental factors, and the limitations of non-linear models in integrating these factors affect the accuracy of LAI predictions. This study explores the potential of classical non-linear models and deep learning for predicting alfalfa LAI. Initially, Logistic, Gompertz, and Richards models were developed based on growth days to assess the applicability of nonlinear models for LAI prediction of alfalfa. In contrast, this study combines environmental factors such as temperature and soil moisture, and proposes a time series prediction model based on mutation point detection method and encoder-attention-decoder BiLSTM network (TMEAD-BiLSTM). The model's performance was analyzed and evaluated against LAI data from different years and cuts. The results indicate that the TMEAD-BiLSTM model achieved the highest prediction accuracy (R² > 0.99), while the non-linear models exhibited lower accuracy (R² > 0.78). The TMEAD-BiLSTM model overcomes the limitations of nonlinear models in integrating environmental factors, enabling rapid and accurate predictions of alfalfa LAI, which can provide valuable references for alfalfa growth monitoring and the establishment of field management practices.

Spring growth of lucerne (Medicago sativa L.) after different times of winter defoliation

The aim of this work was to determine if autumn/winter defoliation affected the time of lucerne stem extension in spring. Two experiments were established at the Field Research Centre, Lincoln University. Experiment 1 was established on 01/06/2022 using an existing two year old stand of “Force 4” lucerne. There were four canopy defoliation dates (1/06, 03/07, 17/07, 1/08). Experiment 2 repeated the treatments on the same paddock in the following year on eight defoliation dates (1/5, 15/5, 1/6, 15/6, 3/7, 17/7, 1/8 and 15/8). In both experiments, five stems were marked in each plot and stem height was measured weekly. The start of stem extension was unaffected by canopy defoliation date in either 2022 (P = 0.20) or 2023 (P = 0.17). Initiation of stem extension was triggered by a base photoperiod of 11.1 ± 0.2 h. This meant the thermal time accumulated from final defoliation to the start of stem extension differed among treatments. These results suggest the time of autumn/winter defoliation (the winter clean-up graze) will not affect the time of the first spring grazing, provided the developing basal buds are not removed. All stands reached 150 mm height by mid-September and produced 3.0 t/ha of dry matter by the end of the month. This suggests, grazing of the first paddock at Lincoln should commence in the first week of September to create a staggered start to rotational grazing that can maximise lucerne yield and quality for animal liveweight production.

Estimation of Malondialdehyde Content in Medicago truncatula under Salt Stress Based on Multi-Order Spectral Transformation Characteristics

Salt stress is a significant abiotic factor affecting the growth and development of alfalfa. Malondialdehyde (MDA) serves as a critical biomarker for assessing alfalfa’s salt tolerance. Traditional methods for measuring MDA are often time-consuming and labor-intensive. Recent advances in remote sensing technology have made non-destructive estimation of metabolites feasible, positioning the accurate estimation of MDA content in alfalfa as a key focus in intelligent breeding. To address the challenge of detecting subtle changes in MDA content, this study developed a partial least squares regression (PLSR) model specifically for Medicago truncatula. This study utilized leaf reflectance hyperspectral data across the visible near-infrared–shortwave infrared (VIR-NIR-SWIR) spectrum, applying multi-order spectral transformation methods, including continuous wavelet transform (CWT), fractional differential (FD), and multi-granularity spectral segmentation (MGSS). Feature selection techniques, such as sequential forward selection (SFS), Least-Squares Boosting (LSBoost), and feature selection using neighborhood component analysis for regression (FSRNCA), were employed to enhance the efficiency of the MDA estimation. The findings revealed that the optimal PLSR model for MDA estimation was achieved by integrating CWT features across orders 1–30 with the SFS method. This model demonstrated robust estimation capabilities under varying salt stress conditions, significantly outperforming the original spectral data (R2 = 0.654, RMSE = 22.567 vs. R2 = 0.242, RMSE = 33.411). A comparative analysis of feature selection methods confirmed that SFS was the most effective for estimating MDA content in alfalfa. These results provide valuable insights and methodologies for MDA estimation and evaluating salt tolerance in alfalfa.

MsFtsH8 Enhances the Tolerance of PEG-Simulated Drought Stress by Boosting Antioxidant Capacity in Medicago sativa L.

Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn2+-dependent enzyme that plays a significant character in the plant's response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa MsFtsH8 by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both MsFtsH8-overexpressing Arabidopsis and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H2O2 accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of MsFtsH8 enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that MsFtsH8 mitigates oxidative damage caused by drought by modulating the plant's antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering.

Priestia aryabhattai Improves Soil Environment and Promotes Alfalfa Growth by Enhancing Rhizosphere Microbial Carbon Sequestration Capacity Under Greenhouse Conditions.

Plant Growth-Promoting Rhizobacteria (PGPR) are gaining increasing attention, but their interactions with indigenous rhizosphere microbiomes remain unclear. To address this issue, we isolated a strain of Priestia aryabhattai with a growth-promoting effect. Under greenhouse conditions, its growth-promoting effect on alfalfa was evaluated, and amplicon sequencing was used to analyze changes in the rhizosphere microbial community to explore the growth promotion mechanism. Our study shows that inoculation with Priestia aryabhattai increases the α-diversity index of the alfalfa rhizosphere microbiome and enhances the abundance of beneficial bacterial genera. This is likely because inoculation with Priestia aryabhattai increased the abundance of carbon-sequestering genera, particularly Gemmatimonas, thereby improving the soil environment. The increased abundance of beneficial bacteria stimulates root development in alfalfa and enhances nutrient uptake, particularly phosphorus, which in turn boosts photosynthesis and promotes alfalfa growth. In summary, Priestia aryabhattai improves soil environment and promotes alfalfa growth by enhancing the carbon sequestration capacity of the rhizosphere microbial community. This work provides theoretical support and insight for the development of PGPR inoculants and for further research on their mechanisms.

Integration of physiology, genomics and microbiomics analyses reveal the biodegradation mechanism of petroleum hydrocarbons by Medicago sativa L. and growth-promoting bacterium Rhodococcus erythropolis KB1

Despite the effectiveness of microbial-phytoremediation for remediating total petroleum hydrocarbons (TPH)-contaminated soil, the underlying mechanisms remain elusive. This study investigated the whole-genome and biological activity of Rhodococcus erythropolis KB1, revealing its plant growth promotion (PGP), TPH degradation, and stress resistance capabilities. Phytoremediation (using alfalfa) and plant-microbial remediation (using alfalfa and KB1) were employed to degrade TPH. The highest TPH degradation rate, reaching 95%, was observed with plant-microbial remediation. This is attributed to KB1′s ability to promote alfalfa growth, induce the release of signaling molecules to activate plant antioxidant enzymes, actively recruit TPH-degrading bacteria (e.g., Sphingomonas, Pseudomonas, C1-B045), and increase soil nitrogen and phosphorus levels, thereby accelerating TPH degradation by both plants and microorganisms. This study demonstrates that R. erythropolis KB1 holds great potential for enhancing the remediation of TPH-contaminated soil through its multifaceted mechanisms, particularly in plant-microbial remediation strategies, providing valuable theoretical support for the application of this technology.

Solubilization of K and P nutrients from coal gangue by Bacillus velezensis.

Coal gangue piles not only occupy significant amounts of arable land but also cause serious environmental pollution. Therefore, finding sustainable methods for the clean utilization of CG is imperative. Although previous studies have shown that bacteria can promote the solubilization of available phosphorus and available potassium from CG, their impact on promoting plant growth remains understudied. To our knowledge, this study is the first to demonstrate the potential of Bacillus velezensis in enhancing the effectiveness of CG as a mineral fertilizer to support alfalfa growth. The evidence presented in this study provides an ecological strategy for the utilization of CG.

Investigating the Interactive Effect of Arbuscular Mycorrhizal Fungi and Different Chelating Agents (EDTA and DTPA) with Different Plant Species on Phytoremediation of Contaminated Soil

Heavy metal (HM) contamination in soil poses a severe environmental threat, jeopardizing ecosystem health and potentially entering the food chain through plant uptake. Phytoremediation, a bioremediation technique utilizing plants to remove or immobilize contaminants, offers a sustainable and eco-friendly solution for HM remediation. This study investigated the interactive effects of arbuscular mycorrhizal fungi (AMF) and chelating agents (EDTA and DTPA) on the growth of maize (Zea mays L.) and alfalfa (Medicago sativa L.) cultivated in metal-contaminated soil and their impact on HM uptake by these plants. The findings revealed that AMF and chelating agents have complex interactive effects on plant growth and metal accumulation. Maize (Zea mays L.) shoot dry matter increased with AMF and chelating agents at lower concentrations. Both plants generally showed a significant (p ≤ 0.05) increase in shoot dry matter with amendments, with AMF × EDTA (10 mmol/kg) being the most effective for alfalfa. DTPA and EDTA generally reduced the DTPA-extractable metals in soil, suggesting potential for metal removal. However, the effects of AMF on metal availability were variable. Metal concentrations in maize (Zea mays L.) shoots increased with increasing DTPA and EDTA concentrations, while the effects of AMF were more complex. The alfalfa shoot metal content showed varied responses, with EDTA (5 mmol/kg) effectively reducing the metal uptake. In general, treatments involving chelating agents (DTPA and EDTA) tend to result in higher bioaccumulation factor (BF) values compared to the non-treated controls for most HMs in both plant species. Mycorrhizal fungi (AMF) treatment alone or in combination with chelating agents also showed that varied effects on HM uptake in both the alfalfa and maize treatments with chelating agents, especially at higher concentrations, generally promoted the greater translocation of HMs in both plant species. Both alfalfa and maize responded differently to treatments, with some treatments showing higher translocation factor (TF) values for certain HMs in one species compared to the other. Mycorrhizal fungi (AMF) treatment alone or in combination with chelating agents also showed varied effects on HM uptake and translocation in both alfalfa and maize. Further research is required to optimize remediation strategies that balance plant health and metal mobilization.

Mining rhizobacteria from indigenous halophytes to enhance alfalfa (Medicago sativa L.) growth and soil reclamation in saline soils of Northwest China

Enhancing the growth of alfalfa (Medicago sativa L.) through inoculation with rhizobacteria represents a sustainable strategy for reclaiming saline soils. However, the lack of suitable strains and practical application guidelines poses significant challenges to the utilization of Plant Growth-Promoting Rhizobacteria (PGPR) in salt-affected soils of Northwest China. In this study, we selected four PGPR strains derived from indigenous halophytes based on their growth-promoting characteristics. These strains underwent further selection via a petri dish assay. Subsequently, the effects of the selected PGPR strains on alfalfa growth and soil fertility were rigorously examined through pot trials. The results demonstrated that Bacillus filamentosus HL3, B. filamentosus HL6, Bacillus subtilis subsp. stercoris HG12, and Paenibacillus peoriae HG24 significantly produced indole-3-acetic acid (IAA), solubilized phosphorus, and fixed nitrogen (except for B. filamentosus HL6, which did not significantly fix nitrogen). Compared to non-inoculated plants, B. filamentosus HL6 and B. subtilis subsp. stercoris HG12 significantly enhanced seed germination, root elongation, and seedling biomass in a 150 mmol/L NaCl saline solution. In saline-alkaline soils, PGPR inoculation under brackish water irrigation did not restore alfalfa growth to the levels observed under freshwater irrigation. Principal Component Analysis (PCA) condensed ten indicators into two indices, explaining 86.85% of the variance. Using these two indices as weights, an evaluation model for the PGPR-alfalfa symbiosis indicated that B. subtilis subsp. stercoris HG12 had the most substantial effect under freshwater irrigation, while co-inoculation with B. subtilis subsp. stercoris HG12 and B. filamentosus HL6 had the most significant impact on alfalfa growth and soil improvement under brackish water irrigation. Available phosphorus was identified as the primary factor influencing alfalfa growth, contributing 82.3% to the growth variation. These findings provide suitable microbial strains for the utilization of saline-alkali land and underscore the potential of applying indigenous PGPR-alfalfa symbiotic techniques to improve soil fertility and crop yield in the arid regions of Northwest China.

Hydrogen Peroxide Is Involved in Methane-Alleviated Cadmium Toxicity in Alfalfa (Medicago sativa L.) Seedlings by Enhancing Cadmium Chelation onto Root Cell Walls.

Although previous studies have demonstrated that methane (CH4) can mitigate the toxicity of cadmium (Cd) in alfalfa seedlings, the CH4-rich water used in these studies may create hypoxic conditions, potentially influencing the experimental outcomes. Therefore, this study aimed to investigate whether CH4 can reduce Cd toxicity in alfalfa seedlings without the interference of hypoxia and to analyze its underlying mechanisms. Here, it was observed that supplementing oxygen with saturated CH4-rich water can significantly alleviate the inhibition of 75 μM CdCl2 on the growth of alfalfa (Medicago sativa L.) seedlings. Less Cd accumulation was also observed in both root and shoot parts, which could be explained by the CH4-altered cell wall components in alfalfa seedling roots, including covalent and ionic soluble pectin, and the degree of demethylation in pectin, thus enabling a higher proportion of Cd binding to the cell walls and reducing the entry of Cd into the cells. The above actions of CH4 were accompanied by an increase in hydrogen peroxide (H2O2) content and NADPH oxidase activity, which could be blocked by the addition of the NADPH oxidase inhibitor diphenylene iodonium (DPI). Taken together, these results implied that exogenously applied CH4 could alleviate Cd toxicity in alfalfa seedlings by enhancing Cd chelation onto the root cell walls, which might be closely associated with NADPH oxidase-dependent H2O2 signals. These findings could provide insight into the mechanism through which CH4 alleviates Cd toxicity in alfalfa plants.

Halotolerant Enterobacter asburiae A103 isolated from the halophyte Salix linearistipularis: Genomic analysis and growth-promoting effects on Medicago sativa under alkali stress

Soil salinization negatively affects plant growth and threatens food security. Halotolerant plant growth-promoting bacteria (PGPB) can alleviate salt stress in plants via diverse mechanisms. In the present study, we isolated salt-tolerant bacteria with phosphate-solubilizing abilities from the rhizosphere of Salix linearistipularis, a halophyte distributed in saline-alkali soils. Strain A103 showed high phosphate solubilization activity and was identified as Enterobacter asburiae based on genome analysis. In addition, it can produce indole-3-acetic acid (IAA), siderophores, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Genome mining has also revealed the presence of several functional genes involved in the promotion of plant growth. Inoculation with A103 markedly improved alfalfa growth in the presence of 100 mM NaHCO3. Under alkali stress, the shoot and root dry weights after bacterial inoculation improved by 42.9 % and 21.9 %, respectively. Meanwhile, there was a 35.9–37.1 % increase in the shoot and root lengths after treatment with A103 compared to the NaHCO3-treated group. Soluble sugar content, peroxidase and catalase activities increased in A103-inoculated alfalfa under alkaline stress. A significant decrease in the malondialdehyde content was observed after treatment with strain A103. Metabolomic analysis indicated that strain A103 positively regulated alkali tolerance in alfalfa through the accumulation of metabolites, such as homocarnosine, panthenol, and sorbitol, which could reduce oxidative damage and act as osmolytes. These results suggest that halophytes are valuable resources for bioprospecting halotolerant beneficial bacteria and that the application of halotolerant growth-promoting bacteria is a natural and efficient strategy for developing sustainable agriculture.

Biological Decline of Alfalfa Is Accompanied by Negative Succession of Rhizosphere Soil Microbial Communities.

The growth and biological decline of alfalfa may be linked to the rhizosphere microbiome. However, plant-microbe interactions in the rhizosphere of alfalfa and associated microbial community variations with stand age remain elusive. This study explored the successional pattern of rhizosphere microbial communities across different aged alfalfa stands and its relationship with alfalfa decline. Rhizosphere soils were collected from 2- and 6-year-old alfalfa stands. Control soils were collected from interspaces between alfalfa plants in the same stands. Soil bacterial and fungal communities were characterized by 16S and ITS rRNA gene sequencing, respectively. Specific microbial taxa colonized the rhizosphere soils, but not the control soils. The rhizosphere-specific taxa mainly included potentially beneficial genera (e.g., Dechloromonas, Verrucomicrobium) in the young stand and harmful genera (e.g., Peziza, Campylocarpon) in the old stand. Alfalfa roots regulated soil microbial communities by selective promotion or inhibition of distinct taxa. The majority of time-enriched taxa were reported as harmful fungi, whose relative abundances were negatively correlated with plant traits. Time-depleted taxa were mostly known as beneficial bacteria, which had relative abundances positively correlated with plant traits. The relative abundances of functional bacterial genes associated with vancomycin biosynthesis, zeatin biosynthesis, and amino acid metabolism trended lower in rhizosphere soils from the old stand. An upward trend was observed for fungal pathogens and wood saprotrophs with increasing stand age. The results suggest that root activity drives the negative succession of rhizosphere microbial communities during alfalfa decline in old stands.

MsMIOX2, encoding a MsbZIP53-activated myo-inositol oxygenase, enhances saline-alkali stress tolerance by regulating cell wall pectin and hemicellulose biosynthesis in alfalfa.

Alfalfa is one of the most widely cultivated forage crops worldwide. However, soil salinization restricts alfalfa growth and development and affects global productivity. The plant cell wall is the first barrier against various stresses. Therefore, elucidating the alterations in cell wall architecture is crucial for stress adaptation. This study aimed to clarify the impact of myo-inositol oxygenase 2 (MsMIOX2) on cell wall pectin and hemicellulose biosynthesis under saline-alkali stress and identify the upstream transcription factors that govern MsMIOX2. MsMIOX2 activation induced cell wall pectin and hemicellulose accumulation under saline-alkali stress. The effects of MsMIOX2 in saline-alkali tolerance were investigated by characterizing its overexpression and RNA interference lines. MsMIOX2 overexpression positively regulated the antioxidant system and photosynthesis in alfalfa under saline-alkali stress. MsMIOX2 exhibited myo-inositol oxygenase activity, which increased polysaccharide contents, facilitated pectin and hemicellulose biosynthesis, and extended the cell wall thickness. However, MsMIOX2 RNA interference decreased cell wall thickness and alleviated alfalfa saline-alkali stress tolerance. In addition, MsbZIP53 was identified as an upstream transcriptional MsMIOX2 regulator by yeast one-hybrid, electrophoretic mobility shift assay, dual-luciferase, and beta-glucuronidase assays. MsbZIP53 overexpression increased MsMIOX2 expression, elevated MIOX activity, reinforced the antioxidant system and photosynthesis, and increased saline-alkali stress tolerance in alfalfa. In conclusion, this study presents a novel perspective for elucidating the molecular mechanisms of saline-alkali stress tolerance in alfalfa and emphasizes the potential use of MsMIOX2 in alfalfa breeding.