Genome-wide identification and characterization of NAC transcription factor family members in Trifolium pratense and expression analysis under lead stress.

BACKGROUND: The NAC TF family is widely involved in plant responses to various types of stress. Red clover (Trifolium pratense) is a high-quality legume, and the study of NAC genes in red clover has not been comprehensive. The aim of this study was to analyze the NAC gene family of red clover at the whole-genome level and explore its potential role in the Pb stress response. RESULTS: In this study, 72 TpNAC genes were identified from red clover; collinearity analysis showed that there were 5 pairs of large fragment replicators of TpNAC genes, and red clover was found to be closely related to Medicago truncatula. Interestingly, the TpNAC genes have more homologs in Arabidopsis thaliana than in soybean (Glycine max). There are many elements in the TpNAC genes promoters that respond to stress. Gene expression analysis showed that all the TpNAC genes responded to Pb stress. qRT-PCR showed that the expression levels of TpNAC29 and TpNAC42 were significantly decreased after Pb stress. Protein interaction network analysis showed that 21 TpNACs and 23 other genes participated in the interaction. In addition, the TpNAC proteins had three possible 3D structures, and the secondary structure of these proteins were mainly of other types. These results indicated that most TpNAC members were involved in the regulation of Pb stress in red clover. CONCLUSION: These results suggest that most TpNAC members are involved in the regulation of Pb stress in red clover. TpNAC members play an important role in the response of red clover to Pb stress.

First Report of Anthracnose Leaf Spot Caused by Colletotrichum americae-borealis on Potentilla anserina in China.

Potentilla anserina L. has an abundance of bioactive compounds and is widely recognized for its diverse applications in traditional medicine and as a food. In August 2023, typical symptoms of anthracnose were observed in 80% of P. anserina plants in Harbin, China. Symptoms, characterized by reddish-brown spots, tend to occur more frequently on leaves closer to the ground. They initially appeared as oval or irregular circles, measuring 1 to 3 mm in diameter, and later merged into larger patches surrounded by chlorotic areas on the leaves. Twenty leaves exhibiting characteristic symptoms were sampled. Each leaf was sectioned into 5×5 mm pieces at the interface between the diseased and healthy tissues. The sections were disinfected sequentially with 75% ethanol for 30 s, followed by 1% NaClO for 2 min, rinsed three times in sterilized distilled water. Post air-drying, samples were cultured on potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d, yielding nine morphologically similar single-spore isolates (JTC1 to JTC9). The colonies initially displayed gray aerial mycelia, becoming pale brown, accompanied by numerous black microsclerotia. The acervuli appeared black, protruded from the surface of the medium, and were adorned with dark brown setae. Setae (n=50) ranged from 58.4 to 188.2 µm in length, appearing dark brown to black, with smooth walls, rounded tips, swollen bases, and containing 1 to 4 septa. The conidia were hyaline, aseptate, cylindrical to spindle-shaped, with blunt and rounded ends, measuring 13.7 to 18.3 µm in length and 3.4 to 4.3 µm in width (n=50). Morphological analysis indicated a close affinity with Colletotrichum americae-borealis (Damm et al. 2014). For molecular identification, genomic DNA was extracted from three representative isolates (JTC1, JTC2, and JTC3).The ITS, HIS3，GAPDH, and ACT genes were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences were submitted to GenBank (ITS: PP338190 to PP338192; HIS3: PP355770 to PP355772; GAPDH: PP355773 to PP355775; ACT: PP355776 to PP355778). BLAST analysis showed 99 to 100% identity with C. americae-borealis type strain CBS 136232 (GenBank accessions: KM105224, KM105364, KM105579, and, KM105434, respectively). Multigene phylogenetic analysis positioned the three isolates close to C. americae-borealis. Pathogenicity tests were performed twice on 6-week-old P. anserina seedlings (cv. Qinghai Juema 1) in a greenhouse. A conidial suspension of the JTC1 isolate (1×105 conidia/ml) was sprayed applied to ten pots, each containing two seedlings, and the plants in the control pots were sprayed with sterile distilled water. Two weeks after inoculation under greenhouse conditions (26/22°C day/night temperature, 12-hour photoperiod, 90% relative humidity), the inoculated seedlings exhibited brown spots and necrotic lesions similar to those observed in the field, C. americae-borealis was successfully reisolated from these symptomatic tissues. To the best of our knowledge, this is the first report of C. americae-borealis causing leaf spot on P. anserina in China. Anthracnose caused by C. americae-borealis is associated with leaf spot disease in oats (Wang et al. 2022), alfalfa (Li et al. 2021), and licorice (Lyu et al.2020). However, C. americae-borealis poses a significant threat to P. anserina in China as well, highlighting the urgent need to develop effective disease management strategies.

Genome-wide investigation of NLP gene family members in alfalfa (Medicago sativa L.): evolution and expression profiles during development and stress.

BACKGROUND: NIN-like protein (NLP) transcription factors (TFs) compose a plant-specific gene family whose members play vital roles in plant physiological processes, especially in the regulation of plant growth and the response to nitrate-nitrogen. However, no systematic identification or analysis of the NLP gene family has been reported in alfalfa. The recently completed whole-genome sequence of alfalfa has allowed us to investigate genome-wide characteristics and expression profiles. RESULTS: 53 MsNLP genes were identified from alfalfa and renamed according to their respective chromosome distributions. Phylogenetic analysis demonstrated that these MsNLPs can be classified into three groups on the basis of their conserved domains. Gene structure and protein motif analyses showed that closely clustered MsNLP genes were relatively conserved within each subgroup. Synteny analysis revealed four fragment duplication events of MsNLPs in alfalfa. The ratios of nonsynonymous (Ka) and synonymous (Ks) substitution rates of gene pairs indicated that the MsNLP genes underwent purifying selection during evolution. Examination of the expression patterns of different tissues revealed specific expression patterns of the MsNLP genes in the leaves, indicating that these genes are involved in plant functional development. Prediction of cis-acting regulatory elements and expression profiles further demonstrated that the MsNLP genes might play important roles in the response to abiotic stress and in phytohormone signal transduction processes. CONCLUSION: This study represents the first genome-wide characterization of MsNLP in alfalfa. Most MsNLPs are expressed mainly in leaves and respond positively to abiotic stresses and hormonal treatments. These results provide a valuable resource for an improved understanding of the characteristics and biological roles of the MsNLP genes in alfalfa.

Identification and characterization of Colletotrichum truncatum and C. destructivum causing stem rot of white clover in China.

White clover (Trifolium repens L.) is an excellent quality forage legume species with superior planting efficiency, which reduces the cost of artificial weeding and nitrogen fertilizer inputs and has feeding and economic value. However, from June to September 2022, severe stem rot affected the yield and quality of white clover crops in Harbin, Heilongjiang Province, China. The aim of this study was to identify the causative agents of the disease. Overall, Colletotrichum truncatum (6 isolates) and C. destructivum (10 isolates) were obtained from rotten white clover stems and identified according to morpho-molecular characteristics and phylogenetic analyses. Pathogenicity tests of the isolates revealed that C. destructivum had a higher pathogenicity to white clover than C. truncatum. In addition, all isolates were highly pathogenic to broad bean, fodder soybean, soybean, pak choi, and chickpea, pathogenic to mint, and did not infect corn, wheat, or cilantro. C. destructivum and C. truncatum isolates were very sensitive to tebuconazole and pyraclostrobin, with EC50 values of 0.54 to 0.70 µg/ml and 0.42 to 0.62 µg/ml, respectively, efficacies ranging between 93.2 to 94.9% and 90.2 to 95.2% at 600 µg/ml and 450 µg/ml, respectively, and EC90 values of 1.88 to 13.36 µg/ml and 1.32 to 23.39 µg/ml, respectively. Therefore, intercropping of host and non-host plants and chemicals can be considered to control stem rot in white clover. These results provide a basis for controlling C. destructivum and C. truncatum in white clover in China.

Identification and Evolutionary Analysis of the Auxin Response Factor (ARF) Family Based on Transcriptome Data from Caucasian Clover and Analysis of Expression Responses to Hormones.

Caucasian clover (Trifolium ambiguum M. Bieb.) is an excellent perennial plant in the legume family Fabaceae, with a well-developed rhizome and strong clonal growth. Auxin is one of the most important phytohormones in plants and plays an important role in plant growth and development. Auxin response factor (ARF) can regulate the expression of auxin-responsive genes, thus participating in multiple pathways of auxin transduction signaling in a synergistic manner. No genomic database has been established for Caucasian clover. In this study, 71 TaARF genes were identified through a transcriptomic database of Caucasian clover rhizome development. Phylogenetic analysis grouped the TaARFs into six (1-6) clades. Thirty TaARFs contained a complete ARF structure, including three relatively conserved regions. Physical and chemical property analysis revealed that TaARFs are unstable and hydrophilic proteins. We also analyzed the expression pattern of TaARFs in different tissues (taproot, horizontal rhizome, swelling of taproot, rhizome bud and rhizome bud tip). Quantitative real-time RT-PCR revealed that all TaARFs were responsive to phytohormones (indole-3-acetic acid, gibberellic acid, abscisic acid and methyl jasmonate) in roots, stems and leaves. These results helped elucidate the role of ARFs in responses to different hormone treatments in Caucasian clover.

Genome-wide investigation of the PLD gene family in alfalfa (Medicago sativa L.): identification, analysis and expression.

BACKGROUND: External environmental factors, such as salt, alkali and drought, severely limit the acreage and yield of alfalfa. The mining of tolerance-related genes in alfalfa and improving the stress resistance of this plant are essential for increasing alfalfa yield. PLD is the main phospholipid hydrolase in plants and plays an important role in plant growth, development, signaling, and resistance to adverse stress. With the availability of whole genome sequences, the annotation and expression of PLDs in alfalfa can now be achieved. At present, few studies have investigated PLDs in alfalfa. Here, we conducted a study of PLDs in alfalfa and identified and analyzed the expression pattern of PLDs under different treatments. RESULTS: Fifty-nine MsPLDs were identified in alfalfa and classified into six subtypes: MsPLDα, ß, γ, δ and ε belong to the C2-PLD subfamily, and MsPLDζ belongs to the PXPH-PLD subfamily. Members of the same PLD subtype have similar physicochemical properties, sequence structure and domains, but their cis-acting elements are different. A qRT-PCR analysis revealed that MsPLDs are expressed in multiple tissues. MsPLDs can respond to alkali, drought, ABA, IAA, and GA3 treatments and particularly to salt stress. Different expression patterns were found for the same gene under different treatments and different genes under the same treatment. Expression of MsPLD05 improved salt tolerance in yeast. CONCLUSION: This study represents the first genome-wide characterization of MsPLDs in alfalfa. Most MsPLDs are expressed mainly in mature leaves and respond positively to abiotic stresses and hormonal treatments. This study further expands the resistance gene pool in legume forage grasses and provides a reference for further in-depth study of MsPLDs in alfalfa.

Validation of Appropriate Reference Genes for qRT-PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses.

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT-PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses.

Identification and Assessment of a Biocontrol Agent, Ochrobactrum intermedium I-5, for Management of Alfalfa Root Rot Caused by Fusarium tricinctum.

Alfalfa root rot caused by Fusarium tricinctum is one of the most important soilborne diseases, resulting in significant losses to alfalfa agriculture worldwide. Fungicides used in management of the disease affect the environment and human health. In this study, a strain of Ochrobactrum intermedium (I-5), isolated from alfalfa rhizosphere soil, exhibited strong antifungal activity against a number of causative pathogens of alfalfa root rot and showed the strongest antagonistic activity against F. tricinctum (a longest radius/shortest radius ratio of 3.09). When applied at 10%, a filtrate of the strain liquid culture significantly reduced the spore production and germination and mycelial growth of F. tricinctum, and the inhibition rates were 76.67, 78.93, and 55.77%, respectively. Furthermore, a filtrate and suspension of the strain, when applied at 10%, reduced alfalfa root rot by >73% in repeated experiments. The strain clearly promoted the activities of invertase, urease, cellulose, and neutral phosphatase in alfalfa rhizosphere soil and significantly reduced the damage to rhizosphere soil quality attributable to alfalfa root rot. Moreover, the strain clearly promoted the growth of alfalfa without causing any evident damage to plants. The active substance produced by the strain was insensitive to heat and ultraviolet irradiation and displayed optimal efficacy at pH 8. To the best of our knowledge, this is the first study describing the use of O. intermedium for the biological control of alfalfa root rot. O. intermedium (I-5) has potential for application in the control of alfalfa root rot and improvement of the quality of cultivated alfalfa.

Revealing the full-length transcriptome of caucasian clover rhizome development.

BACKGROUND: Caucasian clover (Trifolium ambiguum M. Bieb.) is a strongly rhizomatous, low-crowned perennial leguminous and ground-covering grass. The species may be used as an ornamental plant and is resistant to cold, arid temperatures and grazing due to a well-developed underground rhizome system and a strong clonal reproduction capacity. However, the posttranscriptional mechanism of the development of the rhizome system in caucasian clover has not been comprehensively studied. Additionally, a reference genome for this species has not yet been published, which limits further exploration of many important biological processes in this plant. RESULT: We adopted PacBio sequencing and Illumina sequencing to identify differentially expressed genes (DEGs) in five tissues, including taproot (T1), horizontal rhizome (T2), swelling of taproot (T3), rhizome bud (T4) and rhizome bud tip (T5) tissues, in the caucasian clover rhizome. In total, we obtained 19.82 GB clean data and 80,654 nonredundant transcripts were analysed. Additionally, we identified 78,209 open reading frames (ORFs), 65,227 coding sequences (CDSs), 58,276 simple sequence repeats (SSRs), 6821 alternative splicing (AS) events, 2429 long noncoding RNAs (lncRNAs) and 4501 putative transcription factors (TFs) from 64 different families. Compared with other tissues, T5 exhibited more DEGs, and co-upregulated genes in T5 are mainly annotated as involved in phenylpropanoid biosynthesis. We also identified betaine aldehyde dehydrogenase (BADH) as a highly expressed gene-specific to T5. A weighted gene co-expression network analysis (WGCNA) of transcription factors and physiological indicators were combined to reveal 11 hub genes (MEgreen-GA3), three of which belong to the HB-KNOX family, that are up-regulated in T3. We analysed 276 DEGs involved in hormone signalling and transduction, and the largest number of genes are associated with the auxin (IAA) signalling pathway, with significant up-regulation in T2 and T5. CONCLUSIONS: This study contributes to our understanding of gene expression across five different tissues and provides preliminary insight into rhizome growth and development in caucasian clover.

Full-length transcriptome sequencing reveals the low-temperature-tolerance mechanism of Medicago falcata roots.

BACKGROUND: Low temperature is one of the main environmental factors that limits crop growth, development, and production. Medicago falcata is an important leguminous herb that is widely distributed worldwide. M. falcata is related to alfalfa but is more tolerant to low temperature than alfalfa. Understanding the low temperature tolerance mechanism of M. falcata is important for the genetic improvement of alfalfa. RESULTS: In this study, we explored the transcriptomic changes in the roots of low-temperature-treated M. falcata plants by combining SMRT sequencing and NGS technologies. A total of 115,153 nonredundant sequences were obtained, and 8849 AS events, 73,149 SSRs, and 4189 lncRNAs were predicted. A total of 111,587 genes from SMRT sequencing were annotated, and 11,369 DEGs involved in plant hormone signal transduction, protein processing in endoplasmic reticulum, carbon metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, and endocytosis pathways were identified. We characterized 1538 TF genes into 45 TF gene families, and the most abundant TF family was the WRKY family, followed by the ERF, MYB, bHLH and NAC families. A total of 134 genes, including 101 whose expression was upregulated and 33 whose expression was downregulated, were differentially coexpressed at all five temperature points. PB40804, PB75011, PB110405 and PB108808 were found to play crucial roles in the tolerance of M. falcata to low temperature. WGCNA revealed that the MEbrown module was significantly correlated with low-temperature stress in M. falcata. Electrolyte leakage was correlated with most genetic modules and verified that electrolyte leakage can be used as a direct stress marker in physiological assays to indicate cell membrane damage from low-temperature stress. The consistency between the qRT-PCR results and RNA-seq analyses confirmed the validity of the RNA-seq data and the analysis of the regulatory mechanism of low-temperature stress on the basis of the transcriptome. CONCLUSIONS: The full-length transcripts generated in this study provide a full characterization of the transcriptome of M. falcata and may be useful for mining new low-temperature stress-related genes specific to M. falcata. These new findings could facilitate the understanding of the low-temperature-tolerance mechanism of M. falcata.

Genome-wide investigation of the PIF gene family in alfalfa (Medicago sativa L.) expression profiles during development and stress.

BACKGROUND: Phytochrome-interacting factors (PIFs) plays an important role in plants as hubs for intracellular signaling regulation. The PIF gene family has been identified and characterized in many plants, but alfalfa (Medicago sativa L.), an important perennial high-quality legume forage, has not been reported on the PIF gene family. RESULTS: In this study, we presented the identification and characterization of five MsPIF genes in alfalfa (Medicago sativa L.). Phylogenetic analysis indicated that PIFs from alfalfa and other four plant species could be divided into three groups supported by similar motif analysis. The collinearity analysis of the MsPIF gene family showed the presence of two gene pairs, and the collinearity analysis with AtPIFs showed three gene pairs, indicating that the evolutionary process of this family is relatively conservative. Analysis of cis-acting elements in promoter regions of MsPIF genes indicated that various elements were related to light, abiotic stress, and plant hormone responsiveness. Gene expression analyses demonstrated that MsPIFs were primarily expressed in the leaves and were induced by various abiotic stresses. CONCLUSION: This study conducted genome-wide identification, evolution, synteny analysis, and expression analysis of the PIFs in alfalfa. Our study lays a foundation for the study of the biological functions of the PIF gene family and provides a useful reference for improving abiotic stress resistance in alfalfa.

The Multiple Promoting Effects of Suaeda glauca Root Exudates on the Growth of Alfalfa under NaCl Stress.

Under abiotic stress, plant root exudates can improve plant growth performance. However, studies on the effect of root exudates on the stress resistance of another plant are insufficient. In this study, root exudates (REs) were extracted from Suaeda glauca to explore their effect on alfalfa seedlings under salt stress. The results showed that the plant height and fresh weight of alfalfa significantly increased by 47.72% and 53.39% after 7 days of RE treatment at a 0.4% NaCl concentration. Under 1.2% salt stress, REs reduced the Malondialdehyde content in alfalfa by 30.14% and increased the activity of its antioxidant enzymes (peroxidase and catalase) and the content of its osmotic regulators (soluble sugar and proline) by 60.68%, 52%, 45.67%, and 38.67%, respectively. Soil enzyme activity and the abundance of soil-beneficial bacteria were increased by REs. Spearman analysis showed that urease and neutral phosphatase were related to the richness of beneficial bacteria. Redundancy analysis confirmed that urease affected the composition of the soil bacterial community. The partial least squares structural equation model (PLS-SEM) revealed that REs had a direct positive effect on alfalfa growth under salt stress by regulating the plant's injury and antioxidant systems, and the soil bacterial community had an indirect positive effect on alfalfa growth through soil enzyme activity.

Mechanism of action, benefits, and research gap in fermented soybean meal utilization as a high-quality protein source for livestock and poultry.

Animal nutritionists have incessantly worked towards providing livestock with high-quality plant protein feed resources. Soybean meal (SBM) has been an essential and predominantly adopted vegetable protein source in livestock feeding for a long time; however, several SBM antinutrients could potentially impair the animal's performance and growth, limiting its use. Several processing methods have been employed to remove SBM antinutrients, including fermentation with fungal or bacterial microorganisms. According to the literature, fermentation, a traditional food processing method, could improve SBM's nutritional and functional properties, making it more suitable and beneficial to livestock. The current interest in health-promoting functional feed, which can enhance the growth of animals, improve their immune system, and promote physiological benefits more than conventional feed, coupled with the ban on the use of antimicrobial growth promoters, has caused a renewed interest in the use of fermented SBM (FSBM) in livestock diets. This review details the mechanism of SBM fermentation and its impacts on animal health and discusses the recent trend in the application and emerging advantages to livestock while shedding light on the research gap that needs to be critically addressed in future studies. FSBM appears to be a multifunctional high-quality plant protein source for animals. Besides removing soybean antinutrients, beneficial bioactive peptides and digestive enzymes are produced during fermentation, providing probiotics, antioxidants, and immunomodulatory effects. Critical aspects regarding FSBM feeding to animals remain uncharted, such as the duration of fermentation, the influence of feeding on digestive tissue development, choice of microbial strain, and possible environmental impact.

Effects of the fermentation quality and microbial community of waxy maize mixed with fodder soybean silage.

Waxy maize (Zea mays L. sinensis Kulesh) is highly regarded for its high nutritional content and unique taste. Although the stalks and leaves contain high carbohydrate levels after ear harvesting, inadequate crude protein (CP) limits the utilization and promotion of waxy maize silage in animal husbandry. In this study, waxy maize and fodder soybeans were mixed for sowing in different proportions [1:0 (CK), 1:1 (A1), 1:2 (A2), 1:3 (A3), and 1:4 (A4)] to investigate the effects of different mixing ratios on the growth of the waxy maize, the chemical indices, fermentation quality, and the microbial community of the mixed silage after ear harvesting. The mixed planting of waxy maize and fodder soybeans in different proportions had no effect on the yield and quality of the waxy maize ears and increased the aboveground biomass after ear harvesting. After ear harvesting, the neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents significantly decreased, and the CP content and relative feeding value (RFV) gradually increased in the mixed silage. The pH of the treatments was lower than 4.2 except for A4, and the lowest ammonia nitrogen (AN) concentration was observed in A3. With increasing proportions of fodder soybeans, the abundance of beneficial bacteria increased and that of harmful bacteria decreased; Firmicutes and Lactobacillus were the dominant phylum and genus, respectively, and both increased gradually. Redundancy analysis (RDA) revealed that the fermentation indices affecting the microbial community composition in the silage were inconsistent among the different mixed sowing combinations. The Mantel test showed that the composition of the microbial communities in the treatments was significantly correlated with the ADF, water-soluble carbohydrate (WSC), and propionic acid (PA) contents. Comprehensive analysis revealed that the optimal mixed sowing ratio of waxy maize to fodder soybeans was 1:3, and waxy maize and fodder soybeans silage can increase the utilization of aboveground biomass and improve the fermentation quality and feeding quality of silage by changing the microbial community. These findings lay a certain theoretical foundation for improving the utilization of waxy maize.

Arbuscular mycorrhizal fungi communities and promoting the growth of alfalfa in saline ecosystems of northern China.

Arbuscular mycorrhizal fungi (AMF) are universally distributed in soils, including saline soils, and can form mycorrhizal symbiosis with the vast majority of higher plants. This symbiosis can reduce soil salinity and influence plant growth and development by improving nutrient uptake, increasing plant antioxidant enzyme activity, and regulating hormone levels. In this study, rhizosphere soil from eight plants in the Songnen saline-alkaline grassland was used to isolate, characterize, and screen the indigenous advantageous AMF. The promoting effect of AMF on alfalfa (Medicago sativa L.) under salt treatment was also investigated. The findings showed that 40 species of AMF in six genera were identified by high-throughput sequencing. Glomus mosseae (G.m) and Glomus etunicatum (G.e) are the dominant species in saline ecosystems of northern China. Alfalfa inoculated with Glomus mosseae and Glomus etunicatum under different salt concentrations could be infested and form a symbiotic system. The mycorrhizal colonization rate and mycorrhizal dependence of G.m inoculation were significantly higher than those of G.e inoculation. With increasing salt concentration, inoculation increased alfalfa plant height, fresh weight, chlorophyll content, proline (Pro), soluble sugar (SS), soluble protein (SP), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activity while decreasing the malondialdehyde (MDA) content and superoxide anion production rate. The results highlight that inoculation with G.m and G.e effectively alleviated salinity stress, with G.m inoculation having a significant influence on salt resistance in alfalfa. AMF might play a key role in alfalfa growth and survival under harsh salt conditions.

Effects of different concentrations of biochar amendments and Pb toxicity on rhizosphere soil characteristics and bacterial community of red clover (Trifolium pretense L.).

Amending soil with biochar can reduce the toxic effects of heavy metals (HM) on plants and the soil. However, the effects of different concentrations of biochar on the properties and microbial activities in lead (Pb)-contaminated soils are unclear. In this study, two Pb concentrations were set (low, 1000 mg/kg; high, 5000 mg/kg), and five corn straw biochar (CSB) concentrations (0, 2.5, 5, 10 and 15%) were used to determine the response of the growth and rhizosphere of red clover (Trifolium pretense L.) (in terms of soil properties and bacteria) to CSB and Pb application. The results showed that 5% CSB better alleviated the toxicity of Pb on the shoot length of red clover, the biomass increased by 74.55 and 197.76% respectively and reduced the enrichment factor (BCF) and transport factor (TF) of red clover. Pb toxicity reduced soil nutrients, catalase (CAT), acid phosphatase (ACP) and urease activity, while the addition of CSB increased soil pH, soil organic matter (SOM) content and soil enzyme activity. 16S rDNA amplicon sequencing analysis showed that Pb toxicity reduced the diversity of rhizosphere bacteria in red clover and reduced the relative abundance of plant growth-promoting rhizobacteria such as Gemmatimonas, Devosia and Bryobacter. Spearman correlation analysis showed that the addition of alkaline CSB restored the relative abundance of rhizobacteria positively correlated with pH, such as Chitinophaga, Sphingomonas, Devosia and Pseudomonas, and thus restored the rhizosphere soil environment. This study demonstrates that 5% CSB can better alleviate the toxicity of Pb to red clover and soil. We also provide a theoretical basis for the subsequent use of beneficial bacteria to regulate the repair efficiency of red clover.

Integrated physiological, transcriptomic and metabolomic analysis of the response of Trifolium pratense L. to Pb toxicity.

Lead (Pb) interferes with plant gene expression, alters metabolite contents and affects plant growth. However, the molecular mechanism underlying the plant response to Pb is not completely understood. In the present study, Trifolium pratense L. was exposed to Pb concentrations of 0 (Pb0), 500 (Pb500), 1000 (Pb1000), 2000 (Pb2000) and 3000 (Pb3000) mg/kg in soils. Pb stress affected the ability of T. pratense to accumulate and transport Pb, increased the activity of peroxidase (POD) and the contents of malondialdehyde (MDA) and proline, decreased the amount of photosynthetic pigments and soluble proteins, and led to changes in growth and biomass. Transcriptomic and metabolomic analyses showed that Pb mainly affected eight pathways, and LHC, flavonoids, organic acids, amino acids and carbohydrates were upregulated or downregulated. Moreover, Pb500 induced the upregulation of serA, promoted the synthesis of citric acid, maintained photosynthetic pigment levels, and ultimately promoted an increase in stem length. Pb3000 induced the upregulation of ARF, GH3 and SAUR genes, but the saccharide contents and stem length decreased in response to Pb stress. We used a variety of methods to provide a molecular perspective on the mechanism underlying the response of T. pratense to Pb stress.

The Caucasian Clover Gene TaMYC2 Responds to Abiotic Stress and Improves Tolerance by Increasing the Activity of Antioxidant Enzymes.

Abiotic stress affects metabolic processes in plants and restricts plant growth and development. In this experiment, Caucasian clover (Trifolium ambiguum M. Bieb.) was used as a material, and the CDS of TaMYC2, which is involved in regulating the response to abiotic stress, was cloned. The CDS of TaMYC2 was 726 bp in length and encoded 241 amino acids. The protein encoded by TaMYC2 was determined to be unstable, be highly hydrophilic, and contain 23 phosphorylation sites. Subcellular localization results showed that TaMYC2 was localized in the nucleus. TaMYC2 responded to salt, alkali, cold, and drought stress and could be induced by IAA, GA3, and MeJA. By analyzing the gene expression and antioxidant enzyme activity in plants before and after stress, we found that drought and cold stress could induce the expression of TaMYC2 and increase the antioxidant enzyme activity. TaMYC2 could also induce the expression of ROS scavenging-related and stress-responsive genes and increase the activity of antioxidant enzymes, thus improving the ability of plants to resist stress. The results of this experiment provide references for subsequent in-depth exploration of both the function of TaMYC2 in and the molecular mechanism underlying the resistance of Caucasian clover.

Identification and responding to exogenous hormone of HB-KNOX family based on transcriptome data of Caucasian clover.

Caucasian clover (Trifolium ambiguum M. Bieb.) is a strongly rhizomatous, low-crowned perennial leguminous and ground-covering grass. The species is resistant to cold, arid temperatures and grazing due to a well-developed underground rhizome system and a strong clonal reproduction capacity. KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors with important roles in plant development. Preliminary transcriptome analysis enabled us to understand the gene expression in five different tissues, which helped us to screen the predetermined genes of the HB-KNOX family genes for the rhizome growth and development of Caucasian clover. The study identified 41 TaKNOX genes from the Caucasian clover transcriptome database. Gene length, MW and pl of TaKNOX family transcription factors varied, but the gene structure and motifs were relatively conserved in bioinformatics analysis. Phylogenetic analyses of Arabidopsis thaliana, soybean, Medicago truncatula and Caucasian clover were performed to study the evolutionary and functional relationships in various species. Prediction and verification of the subcellular localizations revealed the diverse subcellular localization of these 41 TaKNOX proteins. The expression profile of exogenous hormones showed that the TaKNOX gene showed multiple expression regulation patterns, and was involved in 6-BA, IAA and KT signaling pathways. Our results reveal the characteristics of the TaKNOX gene family, thus laying a foundation for further functional analysis of the KNOX family in Caucasian clover.

Alfalfa modified the effects of degraded black soil cultivated land on the soil microbial community.

Legume alfalfa (Medicago sativa L.) is extensively planted to reduce chemical fertilizer input to the soil and remedy damaged fields. The soil mechanism of these effects is potentially related to the variations in alfalfa-mediated interactions of the soil microbial community. To understand the impact of planting alfalfa on the soil microbial community in degraded black soil cultivated land, a 4-year experiment was conducted in degraded black soil cultivated land. We assessed soil parameters and characterized the functional and compositional diversity of the microbial community by amplicon sequencing that targeted the 16S rDNA gene of bacteria and ITS of fungi in four systems under corn cultivation at the Harbin corn demonstration base (Heilongjiang, China): multiyear corn planting (more than 30 years, MC1); 2 years of alfalfa-corn rotation (OC); 3 years of alfalfa planting (TA); and 4 years of alfalfa planting (FA). It was found out that alfalfa led to changes in the alpha diversity of soil bacteria rather than in fungi in the degraded arable land. The abundance of the bacterial groups Gemmatimonadetes, Actinobacteria, Planctomycetes, and Chloroflexi was increased in OC, while Proteobacteria and Acidobacteria and the fungal group Glomeromycota were increased in TA and FA. OC, TA, and FA significantly increased the pH level but reduced soil electrical conductivity, but they had no impact on soil available nitrogen and soil available potassium at the 0-15 cm soil depth. However, with the years of alfalfa planting, soil available nitrogen and soil available potassium were reduced at the 15-30 cm soil depth. OC, TA, and FA significantly reduced the soil available phosphorus and soil total phosphorus at the 15-30 cm soil depth. There was no significant impact made on soil total nitrogen. FA significantly reduced the soil organic matter at the 15-30 cm soil depth. Planting alfalfa in degraded black soil cultivated land can reduce the salt content of the soil, and the nutrient content of soil planted with alfalfa without fertilization was equivalent to that of degraded corn cultivated land with annual fertilization. Besides, alfalfa recruited and increased contained taxa with the capacity to improve soil nutrient utilization and inhibit the harmful influences of pathogens for subsequent crops. Meanwhile, the planting of alfalfa can modify soil conditions by promoting the proliferation of specific beneficial microbiota groups.