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Background: Researchers have investigated different techniques for synthesis of carbon dots. These techniques include Arc discharge, laser ablation, oxidation, water/solvothermal, and chemical vapor deposition. However, these techniques suffer from some limitations like the utilization of gaseous charged particles, high current, high temperature, potent oxidizing agents, non-environmentally friendly carbon sources, and the generation of uneven particle size. Therefore, there was a significant demand for the adoption of a new technology that combines the environmentally friendly aspects of both bio-based carbon sourcing and synthesis technique., Results: Medicago sativa L (alfalfa)-derived N, S-CDs have been successfully synthesized via microwave irradiation. The N,S-CDs exhibit strong fluorescence (λ ex/em of 320/420 nm) with fluorescence quantum yield of 2.2 % and high-water solubility. The produced N,S-CDs were characterized using TEM, EDX, Zeta potential analysis, IR, UV-Visible, and fluorescence spectroscopy. The average diameter of the produced N, S-CDs was 4.01 ± 1.2 nm, and the Zeta potential was -24.5 ± 6.63 mv. The stability of the produced nano sensors was also confirmed over wide pH range, long time, and in presence of different ions. The synthesized N, S-CDs were employed to quantify the antibacterial drug, nifuroxazide (NFZ), by fluorescence quenching via inner filter effect mechanism. The method was linear with NFZ concentration ranging from 1.0 to 30.0 μM. LOD and LOQ were 0.16 and 0.49 μM, respectively. The method was applied to quantify NFZ in simulated gastric juice (SGJ) with % recovery 99.59 ± 1.4 in addition to pharmaceutical dosage forms with % recovery 98.75 ± 0.61 for Antinal Capsules® and 100.63 ± 1.54 for Antinal suspension®. The Method validation was performed in compliance with the criteria outlined by ICH., Significance and Novelty: The suggested approach primarily centers on the first-time use of alfalfa, an ecologically sustainable source of dopped-CDs, and a cost-effective synthesis technique via microwave irradiation, which is characterized by low energy consumption, minimized reaction time, and the ability to control the size of the produced CDs. This is in line with the growing global recognition of the implementation of green analytical chemistry principles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Drought stress restricts the growth, distribution and productivity of alfalfa (Medicago sativa L.). In order to study the response differences of alfalfa cultivars to drought stress, we previously carried out physiological and molecular comparative analysis on two alfalfa varieties with contrasting drought resistance (relatively drought-tolerant Longdong and drought-sensitive Algonquin). However, the differences in proteomic factors of the two varieties in response to drought stress still need to be further studied. Therefore, TMT-based quantitative proteomic analysis was performed using leaf tissues of the two alfalfa cultivars to identify and uncover differentially abundant proteins (DAPs)., Results: In total, 677 DAPs were identified in Algonquin and 277 in Longdong under drought stress. Subsequently, we conducted various bioinformatics analysis on these DAPs, including subcellular location, functional classification and biological pathway enrichment. The first two main COG functional categories of DAPs in both alfalfa varieties after drought stress were 'Translation, ribosomal structure and biogenesis' and 'Posttranslational modification, protein turnover, chaperones'. According to KEGG database, the DAPs of the two alfalfa cultivars after drought treatment were differentially enriched in different biological pathways. The DAPs from Algonquin were enriched in 'photosynthesis' and 'ribosome'. The pathways of 'linoleic acid metabolism', 'protein processing in endoplasmic reticulum' and 'RNA transport' in Longdong were significantly enriched. Finally, we found significant differences in DAP enrichment and expression patterns between Longdong and Algonquin in glycolysis/glycogenesis, TCA cycle, photosynthesis, protein biosynthesis, flavonoid and isoflavonoid biosynthesis, and plant-pathogen interaction pathway after drought treatment., Conclusions: The differences of DAPs involved in various metabolic pathways may explain the differences in the resistance of the two varieties to drought stress. These DAPs can be used as candidate proteins for molecular breeding of alfalfa to cultivate new germplasm with more drought tolerance to adapt to unfavorable environments., (© 2024. The Author(s).)

Multi-spectral imaging by unoccupied aerial vehicles provides a non-destructive, high throughput approach to measuring biomass accumulation over successive alfalfa (Medicago sativa L. subsp. sativa) harvests. Information from estimated growth curves can be used to infer harvest biomass and to gain insights into the relationship between growth dynamics and forage biomass stability across cuttings and years. In this study, multi-spectral imaging and several common vegetation indices were used to estimate genetic parameters and model growth of alfalfa cultivars to determine the longitudinal relationship between vegetation indices and forage biomass. Results showed moderate heritability for vegetation indices, with median plot level heritability ranging from 0.11-0.64, across multiple cuttings in three trials planted in Ithaca, NY, and Las Cruces, NM. Genetic correlations between the normalized difference vegetation index and forage biomass were moderate to high across trials, cuttings, and the timing of multi-spectral image capture. To evaluate the relationship between growth parameters and forage biomass stability across cuttings and environmental conditions, random regression modeling approaches were used to estimate the growth parameters of cultivars for each cutting and the variance in growth was compared to the variance in genetic estimates of forage biomass yield across cuttings. These analyses revealed high correspondence between stability in growth parameters and stability of forage yield. The results of this study indicate that vegetation indices are effective at modeling genetic components of biomass accumulation, presenting opportunities for more efficient screening of cultivars and new longitudinal modeling approaches that can provide insights into temporal factors influencing cultivar stability., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Saline-alkali stress is one of the main abiotic stresses that limits plant growth. Salt stress has been widely studied, but alkaline salt degradation caused by NaHCO 3 has rarely been investigated. In the present study, the alfalfa cultivar 'Zhongmu No. 1' was treated with 50 mM NaHCO 3 (0, 4, 8, 12 and 24 h) to study the resulting enzyme activity and changes in mRNA, miRNA and metabolites in the roots. The results showed that the enzyme activity changed significantly after alkali stress treatment. The genomic analysis revealed 14,970 differentially expressed mRNAs (DEMs), 53 differentially expressed miRNAs (DEMis), and 463 differentially accumulated metabolites (DAMs). Combined analysis of DEMs and DEMis revealed that 21 DEMis negatively regulated 42 DEMs. In addition, when combined with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEMs and DAMs, we found that phenylpropanoid biosynthesis, flavonoid biosynthesis, starch and sucrose metabolism and plant hormone signal transduction played important roles in the alkali stress response. The results of this study further elucidated the regulatory mechanism underlying the plant response to alkali stress and provided valuable information for the breeding of new saline-alkaline tolerance plant varieties., Competing Interests: Declaration of competing interest All the authors declare that there are no conflicts of interest., (Copyright © 2024. Published by Elsevier Masson SAS.)

Atrazine, a commonly employed herbicide for corn production, can leave residues in soil, resulting in photosynthetic toxicity and impeding growth in subsequent alfalfa ( Medicago sativa L.) crops within alfalfa-corn rotation systems. The molecular regulatory mechanisms by which atrazine affects alfalfa growth and development, particularly its impact on the microbial communities of the alfalfa rhizosphere, are not well understood. This study carried out field experiments to explore the influence of atrazine stress on the biomass, chlorophyll content, antioxidant system, and rhizosphere microbial communities of the atrazine-sensitive alfalfa variety WL-363 and the atrazine-resistant variety JN5010. The results revealed that atrazine significantly reduced WL-363 growth, decreasing plant height by 8.58 cm and root length by 5.42 cm ( p  < 0.05). Conversely, JN5010 showed minimal reductions, with decreases of 1.96 cm in height and 1.26 cm in root length. Chlorophyll content in WL-363 decreased by 35% under atrazine stress, while in JN5010, it was reduced by only 10%. Reactive oxygen species (ROS) accumulation increased by 60% in WL-363, compared to a 20% increase in JN5010 ( p  < 0.05 for both). Antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT), were significantly elevated in JN5010 ( p  < 0.05), suggesting a more robust defense mechanism. Although the predominant bacterial and fungal abundances in rhizosphere soils remained generally unchanged under atrazine stress, specific microbial groups exhibited variable responses. Notably, Promicromonospora abundance declined in WL-363 but increased in JN5010. FAPROTAX functional predictions indicated shifts in the abundance of microorganisms associated with pesticide degradation, resistance, and microbial structure reconstruction under atrazine stress, displaying different patterns between the two varieties. This study provides insights into how atrazine residues affect alfalfa rhizosphere microorganisms and identifies differential microbial responses to atrazine stress, offering valuable reference data for screening and identifying atrazine-degrading bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Lu, Dong, Wang, Liu, Li, Cui, Wang, Ma, Shi, Wang, Zhu and Sun.)

Leaves are a key forage part for livestock, and the aging of leaves affects forage biomass and quality. Preventing or delaying premature leaf senescence leads to an increase in pasture biomass accumulation and an improvement in alfalfa quality. NAC transcription factors have been reported to affect plant growth and abiotic stress responses. In this study, 48 NAC genes potentially associated with leaf senescence were identified in alfalfa under dark or salt stress conditions. A phylogenetic analysis divided MsNACs into six subgroups based on similar gene structure and conserved motif. These MsNACs were unevenly distributed in 26 alfalfa chromosomes. The results of the collinearity analysis show that all of the MsNACs were involved in gene duplication. Some cis -acting elements related to hormones and stress were screened in the 2-kb promoter regions of MsNACs . Nine of the MsNAC genes were subjected to qRT-PCR to quantify their expression and Agrobacterium -mediated transient expression to verify their functions. The results indicate that Ms.gene031485, Ms.gene032313, Ms.gene08494, and Ms.gene77666 might be key NAC genes involved in alfalfa leaf senescence. Our findings extend the understanding of the regulatory function of MsNACs in leaf senescence.

High temperature stress is one of the most severe forms of abiotic stress in alfalfa. With the intensification of climate change, the frequency of high temperature stress will further increase in the future, which will bring challenges to the growth and development of alfalfa. Therefore, untargeted metabolomic and RNA-Seq profiling were implemented to unravel the possible alteration in alfalfa seedlings subjected to different temperature stress (25 ℃, 30 ℃, 35 ℃, 40 ℃) in this study. Results revealed that High temperature stress significantly altered some pivotal transcripts and metabolites. The number of differentially expressed genes (DEGs) markedly up and down-regulated was 1876 and 1524 in T30&#95;vs&#95;CK, 2, 815 and 2667 in T35&#95;vs&#95;CK, and 2115 and 2, 226 in T40&#95;vs&#95;CK, respectively. The number for significantly up-regulated and down-regulated differential metabolites was 173 and 73 in T30&#95;vs&#95;CK, 188 and 57 in T35&#95;vs&#95;CK, and 220 and 66 in T40&#95;vs&#95;CK, respectively. It is worth noting that metabolomics and transcriptomics co-analysis characterized enriched in plant hormone signal transduction (ko04705), glyoxylate and dicarboxylate metabolism (ko00630), from which some differentially expressed genes and differential metabolites participated. In particular, the content of hormone changed significantly under T40 stress, suggesting that maintaining normal hormone synthesis and metabolism may be an important way to improve the HTS tolerance of alfalfa. The qRT-PCR further showed that the expression pattern was similar to the expression abundance in the transcriptome. This study provides a practical and in-depth perspective from transcriptomics and metabolomics in investigating the effects conferred by temperature on plant growth and development, which provided the theoretical basis for breeding heat-resistant alfalfa., (© 2024. The Author(s).)

Flower development is a crucial and complex process in the reproductive stage of plants, which involves the interaction of multiple endogenous signals and environmental factors. However, regulatory mechanism of flower development was unknown in alfalfa ( Medicago sativa ). In this study, the three stages of flower development of ' M. sativa cv. Gannong No. 5' (G5) and its early flowering and multi flowering mutant (MG5) were comparatively analyzed by transcriptomics. The results showed that compared with late bud stage (S1), 14287 and 8351 differentially expressed genes (DEGs) were identified at early flower stage (S2) in G5 and MG5, and 19941 and 19469 DEGs were identified at late flower stage (S3). Compared with S2, 9574 and 10870 DEGs were identified at S3 in G5 and MG5, respectively. Venn analysis revealed that 547 DEGs were identified among the three comparison groups. KEGG pathway enrichment analysis showed that these genes were involved in the development of alfalfa flowers through redox pathways and plant hormone signaling pathways. Key candidate genes including SnRK2 , BSK , GID1 , DELLA and CRE1 , for regulating the development from buds to mature flowers in alfalfa were screened. In addition, differential expression of transcription factors such as MYB, AP2, bHLH, C2C2, MADS-box, NAC, bZIP, B3 and AUX/IAA also played an important role in this process. The results laid a theoretical foundation for studying the molecular mechanisms of the development process from buds to mature flowers in alfalfa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Niu and Ma.)

Odontothrips loti (Haliday) (Thysanoptera: Thripidae) is one of the most serious pests on alfalfa, causing direct damage by feeding and indirect damage by transmitting plant viruses. This damage causes significant loss in alfalfa production. Semiochemicals offer opportunities to develop new approaches to thrips management. In this study, behavioral responses of female and male adults of O. loti to headspace volatiles from live female and male conspecifics were tested in a Y-tube olfactometer. The results showed that both male and female adults of O. loti were attracted to the odors released by conspecific males but not those released by females. Headspace volatiles released by female and male adults were collected using headspace solid-phase microextraction (HS-SPME). The active compound in the volatiles was identified by gas chromatography-mass spectrometry (GC-MS). The analysis showed that there was one major compound, (R)-lavandulyl (R)-2-methylbutanoate. The attractive activity of the synthetic aggregation pheromone compound was tested under laboratory and field conditions. In an olfactometer, both male and female adults showed significant preference for synthetic (R)-lavandulyl (R)-2-methylbutanoate at certain doses. Lures with synthetic (R)-lavandulyl (R)-2-methylbutanoate significantly increased the trap catches of sticky white traps at doses of 40-80 µg in the field. This study confirmed the production of aggregation pheromone by O. loti male adults and identified its active compound as (R)-lavandulyl (R)-2-methylbutanoate, providing a basis for population monitoring and mass trapping of this pest., (© 2024. The Author(s).)

Numerous studies have investigated seed aging, with a particular emphasis on the involvement of reactive oxygen species. Reactive oxygen species diffuse into the nucleus and damage telomeres, resulting in loss of genetic integrity. Telomerase reverse transcriptase (TERT) plays an essential role in maintaining plant genomic stability. Genome-wide analyses of TERT genes in alfalfa (Medicago sativa) have not yet been conducted, leaving a gap in our understanding of the mechanisms underlying seed aging associated with TERT genes. In this study, four MsTERT genes were identified in the alfalfa genome. The expression profiles of the four MsTERT genes during seed germination indicated that MS. gene79077 was significantly upregulated by seed aging. Transgenic seeds overexpressing MS. gene79077 in Arabidopsis exhibited enhanced tolerance to seed aging by reducing the levels of H 2 O 2 and increasing telomere length and telomerase activity. Furthermore, transcript profiling of aging-treated Arabidopsis wild-type and overexpressing seeds showed an aging response in genes related to glutathione-dependent detoxification and antioxidant defense pathways. These results revealed that MS. gene79077 conferred Arabidopsis seed-aging tolerance via modulation of antioxidant defense and telomere homeostasis. This study provides a new way to understand stress-responsive MsTERT genes for the potential genetic improvement of seed vigor., Competing Interests: Declaration of competing interest The authors assert that this research was conducted without any affiliations or financial involvements that might be interpreted as potential conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Background: Daye No.3 is a novel cultivar of alfalfa (Medicago sativa L.) that is well suited for cultivation in high-altitude regions such as the Qinghai‒Tibet Plateau owing to its high yield and notable cold resistance. However, the limited availability of transcriptomic information has hindered our investigation into the potential mechanisms of cold tolerance in this cultivar. Consequently, we conducted de novo transcriptome assembly to overcome this limitation. Subsequently, we compared the patterns of gene expression in Daye No. 3 during cold acclimatization and exposure to cold stress at various time points., Results: A total of 15 alfalfa samples were included in the transcriptome assembly, resulting in 141.97 Gb of clean bases. A total of 441 DEGs were induced by cold acclimation, while 4525, 5016, and 8056 DEGs were identified at 12 h, 24 h, and 36 h after prolonged cold stress at 4 °C, respectively. The consistency between the RT‒qPCR and transcriptome data confirmed the accuracy and reliability of the transcriptomic data. KEGG enrichment analysis revealed that many genes related to photosynthesis were enriched under cold stress. STEM analysis demonstrated that genes involved in nitrogen metabolism and the TCA cycle were consistently upregulated under cold stress, while genes associated with photosynthesis, particularly antenna protein genes, were downregulated. PPI network analysis revealed that ubiquitination-related ribosomal proteins act as hub genes in response to cold stress. Additionally, the plant hormone signaling pathway was activated under cold stress, suggesting its vital role in the cold stress response of alfalfa., Conclusions: Ubiquitination-related ribosomal proteins induced by cold acclimation play a crucial role in early cold signal transduction. As hub genes, these ubiquitination-related ribosomal proteins regulate a multitude of downstream genes in response to cold stress. The upregulation of genes related to nitrogen metabolism and the TCA cycle and the activation of the plant hormone signaling pathway contribute to the enhanced cold tolerance of alfalfa., (© 2024. The Author(s).)

Feeding ruminants with high-quality forage can enhance digestibility and reduce methane production. Development of high-quality silage from leguminous plants with lactic acid bacteria can improve digestibility and it mitigate the greenhouse gas emissions. In this study, we developed a high-quality alfalfa silage with improved fermentation index and microbial dynamics using Levilactobacillus brevis-KCC-44 at low or high moisture (LM/HM) conditions and preserved it for 75 or 150 days. Alfalfa fermentation with L. brevis enhances acidification and fermentation characteristics primarily due to the dominance of lactic acid bacteria (LAB) L. brevis (>95%) compared to alfalfa fermented with epiphytic LAB. The inoculant L. brevis improved the anaerobic fermentation indexes resulting in a higher level of lactic acid in both high (10.0 ± 0.12 & 8.90 ± 0.31%DM) and low moisture (0.55 ± 0.08 & 0.39 ± 0.0 %DM) in 75 and 150 days respectively, compared to control silage. In addition, the marginal amount of acetic acid (range from 0.23 ± 0.07 to 2.04 ± 0.27 %DM) and a reduced level of butyric acid (range between 0.03 ± 0.0 to 0.13 ± 02 %DM) was noted in silage treated with LAB than the control. The LAB count and abundance of Levilactobacillus were higher in alfalfa silage fermented with L. brevis. Microbial richness and diversity were reduced in alfalfa silage treated with L. brevis which prompted lactic acid production at a higher level even for a prolonged period of time. Therefore, this L.brevis is an effective inoculant for producing high-quality alfalfa silage since it improves fermentation indexes and provides reproducible ensiling properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes' expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring F v /F m and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Shangang Jia, Peisheng Mao reports financial support was provided by the Key R & D project of Sichuan Science and Technology Program (2023YFSY0012), earmarked fund for CARS (CARS-34), and National Key R&D Program of China (2022YFD1300804). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

This study investigates how extracellular polymeric substances (EPS) synthesized by dark septate endophytic (DSE) improve alfalfa's drought resistance. Drought stress was simulated in hydroponic culture, and roots were treated with different EPS concentrations to determine their effects on drought tolerance and applicable concentrations. Hydroponic solutions with 0.25 and 0.50% EPS concentrations alleviated leaf wilting and increased total plant fresh weight by 35.8 and 57.7%, respectively. SEM shows that EPS attached to the roots and may have served to protect the root system. EPS treatment significantly depressed the MDA contents of the roots, stems, and leaves. Roots responded to drought stress by increasing soluble sugar contents and antioxidant enzyme activities, while mitigating stem and leaf stress by synthesizing lipid compounds, amino acids, and organic acid metabolites. Five metabolites in the stem have been reported to be associated with plant stress tolerance and growth, namely 3- O -methyl 5- O -(2-methyl propyl) (4S)-2,6-dimethyl-4-(2-nitrophenyl)-3,4-dihydropyridine-3,5-dicarboxylate, malic acid, PA (20:1(11Z)/15:0), N -methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and 2-( S -glutathionyl) acetyl glutathione. In summary, EPS treatment induced oxidative stress and altered plant metabolism, and this in turn increased plant antioxidant capacity. The results provide a theoretical basis for the application of EPS in commercial products that increase plant resistance and ecological restoration.

Alfalfa often suffers from low temperature during spring rejuvenation, so it is important to improve the cold tolerance of alfalfa leaves for its smooth rejuvenation, and the alternative pathway (AP) could effectively improve the plant's tolerance. In this study, the contribution of AP on spring rejuvenation of alfalfa was investigated in Xinmu No.4 and Gannong No.5 with different fall dormancy levels. Though the protein and AP capacity were decreased during the rejuvenation, the ratio of AP/TP were increased in two alfalfa varieties, compared to those in alfalfa before overwintering. This indicated that AP had positive response to alfalfa rejuvenation. The limitation of AP significantly affected the leaf length, leaf width and growth rate of greening alfalfa, showing that AP played an important role in alfalfa rejuvenation. Inhibition of AP resulted in a significant decrease in Pn, Ci, Gs and stomatal structure deformity, suggestion that AP affected photosynthesis by influencing stomatal development during rejuvenation. AP reduces oxidative damage to PSII core protein repair in alfalfa leaves and optimizes photosynthesis by up-regulating NADP-MDH activity, decreasing the accumulation of excess reducing power in the chloroplasts, and by increasing SOD and POD activities and decreasing the accumulation of hydrogen peroxide. The higher proportion of AP keeps it more tolerant to low temperature for rejuvenation in Xinmu No.4 with a lower fall dormancy level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Alfalfa is widely recognized as an important forage crop. To understand the morphological characteristics and genetic basis of seed morphology in alfalfa, we screened 318 Medicago spp., including 244 Medicago sativa subsp. sativa (alfalfa) and 23 other Medicago spp., for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness & red-green-blue (RGB) intensities. The results revealed phenotypic diversity and correlations among the tested accessions. Based on the phenotypic data of M. sativa subsp. sativa, a genome-wide association study (GWAS) was conducted using single nucleotide polymorphisms (SNPs) called against the Medicago truncatula genome. Genes in proximity to associated markers were detected, including CPR1, MON1, a PPR protein, and Wun1(threshold of 1E-04). Machine learning models were utilized to validate GWAS, and identify additional marker-trait associations for potentially complex traits. Marker S7&#95;33375673, upstream of Wun1, was the most important predictor variable for red color intensity and highly important for brightness. Fifty-two markers were identified in coding regions. Along with strong correlations observed between seed morphology traits, these genes will facilitate the process of understanding the genetic basis of seed morphology in Medicago spp., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

This article follows-up on our recently published work, which evaluated the impact of the addition of an alfalfa leaf-derived adsorbent in the aflatoxin B 1 (AFB 1 )-contaminated diet in regard to the production parameters, blood cell count, serum biochemistry, liver enzymes, and liver histology of turkey poults. This paper presents complementary results on microbial community, ileal morphology, barrier function, and immunity. For this purpose, 350 1-day-old female turkey poults were randomly distributed into five groups: (1) Control, AFB 1 -free diet; (2) AF, AFB 1 -contaminated diet at 250 ng/g; (3) alfalfa, AFB 1 -free diet + 0.5% ( w / w ) adsorbent; (4) alfalfa + AF, AFB 1 -contaminated diet at 250 ng/g + 0.5% ( w / w ) adsorbent; and (5) YCW + AF, AFB 1 -contaminated diet at 250 ng/g + 0.5% ( w / w ) commercial yeast cell wall-based adsorbent (reference group). In general, in the AF group, the growth of opportunistic pathogens was promoted, which lead to gut dysbacteriosis, mainly influenced by Streptococcus lutetiensis . Conversely, a significant increase in beneficial bacteria ( Faecalibacterium and Coprococcus catus ) was promoted by the addition of the plant-based adsorbent. Moreover, the AF group had the lowest villus height and a compromised barrier function, as evidenced by a significant ( p < 0.05) increase in fluorescein isothiocyanate dextran (FITC-d), but these negative effects were almost reversed by the addition of the alfalfa adsorbent. Furthermore, the AF + YCW and alfalfa + AF groups exhibited a significant increase in the cutaneous basophil hypersensitivity response compared to the rest of the experimental groups. Taken together, these results pointed out that the alfalfa counteracts the adverse effects of AFB 1 in poults, facilitating the colonization of beneficial bacteria and improving the barrier function of the turkey poults.

Soil salinization poses a serious threat to crop growth. The selection of appropriate soil modifiers and water-soluble fertilizers for saline soils represents a crucial method for enhancing crop yields. The modifiers and medium-element water-soluble fertilizers were prepared using hydrochar derived from rice straw. Two distinct experiments were designed to study the effect of modifiers and water-soluble fertilizers on saline soils. The first experiment, designated as the "Soil Cultivation Experiment" , sought to investigate the impact of various modifiers on soil quality. The second experiment, designated as the "Method of Field Micro-Area Experiment", aimed to assess the influence of water-soluble fertilizers on saline soils. The results showed that the application of modifiers and water-soluble fertilizers significantly enhanced comprehensive soil physical and chemical properties, crop growth, soil enzyme activity, and other key indicators in saline and alkaline soils. The optimal dosage of the modifier was 20 g/kg, which reduced the pH value from 8.62 to 8.21 and the decreased alkalinity by 8.26%. Furthermore, their application effectively boosted nutrient levels, including organic matter, and increased soil enzyme activity. The biomass of alfalfa showed enhancements of 63.01% and 20.87% and the biomass of leymus chinensis increased by 29.39% and 9.02% for the two batches, respectively. Notably, the application of water-soluble fertilizer yielded achieved superior results. This study also provided a theoretical basis for their future application in soda saline-alkali soil., (© 2024. The Author(s).)

Mg-K homeostasis is essential for plant response to abiotic stress, but its regulation remains largely unknown. MsWRKY44 cloned from alfalfa was highly expressed in leaves and petioles. Overexpression of it inhibited alfalfa growth, and promoted leaf senescence and alfalfa sensitivities to acid and Al stresses. The leaf tips, margins and interveins of old leaves occurred yellow spots in MsWRKY44-OE plants under pH4.5 and pH4.5 +Al conditions. Meanwhile, Mg-K homeostasis was substantially changed with reduction of K accumulation and increases of Mg as well as Al accumulation in shoots of MsWRKY44-OE plants. Further, MsWRKY44 was found to directly bind to the promoters of MsMGT7 and MsCIPK23, and positively activated their expression. Transiently overexpressed MsMGT7 and MsCIPK23 in tobacco leaves increased the Mg and Al accumulations but decreased K accumulation. These results revealed a novel regulatory module MsWRKY44-MsMGT7/MsCIPK23, which affects the transport and accumulation of Mg and K in shoots, and promotes alfalfa sensitivities to acid and Al stresses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The crude protein (CP) content is an important determining factor for the quality of alfalfa, and its accurate and rapid evaluation is a challenge for the industry. A model was developed by combining Fourier transform infrared spectroscopy (FTIS) and chemometric analysis. Fourier spectra were collected in the range of 4000~400 cm -1 . Adaptive iteratively reweighted penalized least squares (airPLS) and Savitzky-Golay (SG) were used for preprocessing the spectral data; competitive adaptive reweighted sampling (CARS) and the characteristic peaks of CP functional groups and moieties were used for feature selection; partial least squares regression (PLSR) and random forest regression (RFR) were used for quantitative prediction modelling. By comparing the combined prediction results of CP content, the predictive performance of airPLST-cars-PLSR-CV was the best, with an RP2 of 0.99 and an RMSEP of 0.053, which is suitable for establishing a small-sample prediction model. The research results show that the combination of the PLSR model can achieve an accurate prediction of the crude protein content of alfalfa forage, which can provide a reliable and effective new detection method for the crude protein content of alfalfa forage.

Gastrointestinal nematode (GIN) infection poses the most significant obstacle to the sustainable development of small ruminant (sheep and goat) farming globally. Resistance of GINs to synthetic anthelmintic drugs has led to rising interest in exploring alternative methods for parasite control, such as the utilization of bioactive plants with anti-parasitic properties. In this investigation, black seed (Nigella sativa), a shrub high in secondary antioxidant compounds, and sericea lespedeza (Lespedeza cuneata), a perennial legume high in tannins with anti-parasitic properties were combined to determine if two bioactive plants containing different types of secondary compounds can provide a stronger anti-parasitic effect than sericea lespedeza alone. In a 49-day trial, naturally parasitized 6-7-month-old intact male Spanish goats (n = 15/treatment) were fed pelletized feeds encompassing sericea lespedeza leaf meal (SL), a combination of black seed meal (BS) and sericea lespedeza leaf meal (BS-SL - 75 % SL, 25 % BS), or alfalfa (Medicago sativa, control parasitized; CONP), with an additional group of dewormed kids given the alfalfa pellets (Control treated; CONT). Weekly measurements of animal weights and samples of blood and feces were collected to determine the packed cell volume (PCV), GIN fecal egg counts (FEC), and coccidia fecal oocyte counts (FOC), respectively. All animals were processed at the end of the trial (60 total), with adult Haemonchus contortus worms recovered from the abomasum of each goat for counting and sex determination. Carcass weights were recorded after processing. Goats given the SL and BS-SL treatments had lower FEC (P<0.05) than the parasitized alfalfa (CONP) goats. At the end of the study, the SL and BS-SL groups' FOC values were lower (P < 0.05) than the CONT and CONP groups. A rise in PCV values was seen over time for all groups; SL, BS-SL, and CONT animals exhibited higher PCV values (P < 0.05) in comparison to the CONP goats. The parasitized goats fed SL-only pellets showed greater feed intake and animal body weights (P < 0.05) compared to goats fed BS-SL or alfalfa pellets. However, the treatments had no effect on the weight of the goats' carcasses. Although the H. contortus adult worm counts in the CONT goats (alfalfa-dewormed) were lower (P < 0.05) than in the CONP goats (alfalfa-parasitized), they did not differ from the SL or BS-SL animals. This study indicates that sericea lespedeza leaf meal pellet diet, either by itself or in combination with black seed meal, showed promising results as an anthelmintic and may prove to be an effective alternative to exclusive use of conventional deworming drugs. The addition of black seed did not appear to enhance the effectiveness of sericea lespedeza in this study., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Lucerne ( Medicago sativa ), is a cornerstone of China's livestock industry, however, due to the backward agronomic strategies and technology, lucerne in China faces cultivation challenges that result in lower productivity and quality than global standards. Therefore, we undertook a meta-analysis to evaluate the impact of five distinct fertilization types on lucerne yield and nutritional quality in various locations in China. The fertilizer practices included manure application, combined mineral fertilizer and manure application (FM), biological fertilizer application, unbalanced application of two or more mineral fertilizer types, and balanced mineral fertilizer application. Furthermore, we investigate influential factors of yield and quality of lucerne under fertilization, including climatic variables (mean annual precipitation, mean annual temperature), initial soil properties (soil organic carbon; total nitrogen, pH), and agronomic factors (seeding rate, harvest frequency, and lucerne stand age)., Methods: Our study analyzed 53 published papers to discern the most beneficial fertilizer for enhancing lucerne yield and nutritional quality., Results and Discussion: The results showed that the fertilizer practices, on average, significantly improved yield by 31.72% and crude protein content by 11.29%, with FM emerging as the most effective, this is because mineral fertilizers provide available nutrients for lucerne, manure provides essential organic matter for microorganisms and improve soil properties. In addition, the fertilizer practices significantly reduced neutral and acid detergent fiber contents by 6.28% and 8.50%, respectively, while increasing ash content and relative feeding value. Furthermore, climatic variables, soil properties, and planting system factors such as sowing date and harvest frequency significantly affected yield and nutritional quality. The practical implications of our results emphasize the need for balanced and strategic fertilizer application to optimize lucerne production and highlight the potential to adjust cultivation practices according to environmental conditions. Balanced and strategic fertilizer application can simultaneously improve soil properties, enhance soil carbon sequestration, and reduce the emission of greenhouse gases from the soil, which is a vital measure for realizing sustainable agricultural development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Mao, Wang, Luo, Cao, Siddique, Mirzaei, Saunders, Aghamir, Radicetti, Xiang, Zhang, Li and Shen.)

Objective: This study aimed to investigate the regulatory effects of exogenous hydrogen sulfide (H 2 S) on seed germination, seedling growth, and reactive oxygen species (ROS) homeostasis in alfalfa under chromium (Cr) ion (III) stress., Methods: The effects of 0-4 mM Cr(III) on the germination and seedling growth of alfalfa were first assessed. Subsequently, following seed NaHS immersion, the influence of H 2 S on alfalfa seed germination and seedling growth under 2 mM Cr(III) stress was investigated, and the substance contents and enzyme activities associated with ROS metabolism were quantified., Results: Compared to the control group, alfalfa plant germination was delayed under 2 mM Cr(III) stress for up to 48 h ( p  < 0.05). At 120 h, the total seedling length was approximately halved, and the root length was roughly one-third of the control. Treatment with 0.02-0.1 mM NaHS alleviated the delay in germination and root growth inhibition caused by 2 mM Cr(III) stress, resulting in an increased ratio of root length to hypocotyl length from 0.57 to 1 above. Additionally, immersion in 0.05 mM NaHS reduced hydrogen peroxide (H 2 O 2 ) and oxygen-free radicals (O 2 · - ) levels ( p  < 0.05), boosted glutathione (GSH) levels ( p  < 0.05), and notably enhanced catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) activities ( p  < 0.05) compared to the 2 mM Cr(III) stress treatment group., Conclusion: Seed immersion in NaHS mitigated the delay in germination and inhibition of root elongation under 2 mM Cr(III) stress. This effect is likely attributed to the regulation of intracellular ROS homeostasis and redox balance through enzymatic and non-enzymatic systems; thus, providing a potential mechanism for combating oxidative stress.

Low temperature is the most common abiotic factor that usually occurs during the seed germination of alfalfa ( Medicago sativa L.). However, the potential regulatory mechanisms involved in alfalfa seed germination under low temperature stress are still ambiguous. Therefore, to determine the relevant key genes and pathways, the phenotypic and transcriptomic analyses of low-temperature sensitive (Instict) and low-temperature tolerant (Sardi10) alfalfa were conducted at 6 and 15 h of seed germination under normal (20 °C) and low (10 °C) temperature conditions. Germination phenotypic results showed that Sardi10 had the strongest germination ability under low temperatures, which was manifested by the higher germination-related indicators. Further transcriptome analysis indicated that differentially expressed genes were mainly enriched in galactose metabolism and carbon metabolism pathways, which were the most commonly enriched in two alfalfa genotypes. Additionally, fatty acid metabolism and glutathione metabolism pathways were preferably enriched in Sardi10 alfalfa. The Weighted Gene Co-Expression Network Analysis (WGCNA) suggested that genes were closely related to galactose metabolism, fatty acid metabolism, and glutathione metabolism in Sardi10 alfalfa at the module with the highest correlation (6 h of germination under low temperature). Finally, qRT-PCR analysis further validated the related genes involved in the above pathways, which might play crucial roles in regulating seed germination of alfalfa under low temperature conditions. These findings provide new insights into the molecular mechanisms of seed germination underlying the low temperature stress in alfalfa.

The first tri-segmented viruses in the family Rhabdoviridae were recently discovered by exploring publicly available plant datasets in several hosts, including alfalfa (Medicago sativa L.) (Bejerman et al. 2023). They were classified in a novel genus "Trirhavirus" within the family Rhabdoviridae. The trirhavirus identified in alfalfa was named Medicago trirhavirus 1 (MeTRV1). Here we report the first confirmation of MeTRV1 in commercial alfalfa fields in Washington State, USA. Samples were collected in 2019-2021 in Benton and Grant Counties, WA. The alfalfa leaves in which the virus was detected displayed irregular chlorotic spotting (Fig.1). Total RNA extraction, library preparation, high throughput sequencing, and bioinformatics analysis were performed as described in Nemchinov et al (2023). Raw reads were trimmed with Trimmomatic 0.39 (Bolger at al. 2014). SPAdes 3.15.5 (Bankevich et al. 2012) was used for assembly. MeTRV1 was identified in four plants out of 100 tested and three complete RNA segments were recovered from one of them. For clarity, the virus found in the alfalfa field samples was designated MeTRV1-Wa. De novo assembly resulted in three contigs, which, when subjected to BLASTn analyses, aligned to the respective RNA segments of MeTRV1. The first contig was 6,498 nucleotides (nts)-long, 99.4% identical to RNA1 of MeTRV1 (BK064256.1), and 5,922 reads mapped to it (coverage 125x). RNA1 of MeTRV1-Wa encoded a protein 2,040 amino acid (aa)-long that aligned with protein L of MeTRV1 (DBA36559.1, 99.8%). The second contig was 4,014 nts-long and 95.2% identical to the RNA2 of MetRV1 (BK064257.1) with 1,751 reads mapping (coverage 59x). It contained four open reading frames (ORFs) encoding proteins N (445 aa, 99.8%, DBA36560.1); P2 (343 aa, 99.4%, DBA36561.1); P3 (183 aa, 99.4%, DBA36562.1); and P4 (72 aa, 98.6%, DBA36563.1). Altogether, 4,653 reads mapped to the third contig (coverage 131x) that was 4,889 nts-long and 99.1% identical to the RNA 3 segment of MeTRV1 (BK064258.1). RNA3 of MeTRV1-Wa encoded four proteins: P6 (274 aa, 100%, DBA36565.1); P7 (189 aa, 99.5%, DBA36566.1); P8 (514 aa, 99 %, DBA36567.1); and P5 (303 aa, 99.7%, DBA36564.1). The 5' trailer of each RNA segment had a nearly identical 24 nts at the end. Genomic organization of the MeTRV1-Wa and the locations of its ORFs are shown in Fig.2. To confirm the virus's presence, two sets of primers were designed based on the predicted sequence of the viral RNA 3 segment. The correct-size products were amplified in RT-PCR assays with RNA extracted from infected plants (Fig.3) and verified by Sanger sequencing. Besides MeTRV1-Wa, sequences of the following viruses known to cause symptoms in alfalfa were identified in the same library: alfalfa mosaic virus, bean leafroll virus, lucerne transient streak virus, and pea streak virus. Thus, the observed symptomatology may not be clearly attributed to MeTRV1-Wa due to coinfecting organisms. However, a possible association of the disease symptoms with the virus presence could be suggested based on comparison with both asymptomatic and symptomatic plants negative for MeTRV1-Wa (Fig.1). Since plant rhabdoviruses are recognized as a cause of economic losses in alfalfa and other major crops and are transmitted by insects (Bejerman et al. 2011, 2015; Jackson et al. 2005; Man and Dietzgen 2014), this first experimental confirmation of the occurrence of the new virus in the U.S. alfalfa is important for understanding its origin, distribution, and pathogenic potential.

Rhizosphere iron plaques derived from Fe-based nanomaterials (NMs) are a promising tool for sustainable agriculture. However, the requirement for flooded conditions to generate iron plaque limits the scope of the NM application. In this study, we achieved in situ Fenton oxidation of a highly chlorinated persistent organic pollutant (2,2',4,5,5'-pentachlorobiphenyl, PCB101) through iron plaque mediated by the interaction between α-Fe 2 O 3 NMs and plant-rhizobacteria symbionts under dryland conditions. Mechanistically, the coexistence of α-Fe 2 O 3 NMs and Pseudomonas chlororaphis JD37 stimulated alfalfa roots to secrete acidic and reductive agents as well as H 2 O 2 , which together mediated the rhizosphere Fenton reaction and converted α-Fe 2 O 3 NMs into iron plaque rich in Fe(II)-silicate. Further verifications reproduced the Fenton reaction in vitro using α-Fe 2 O 3 NMs and rhizosphere compounds, confirming the critical role of •OH in the oxidative degradation of PCB101. Significant reductions in PCB101 content by 18.6%, 42.9%, and 23.2% were respectively found in stem, leaf, and soil after a 120-d treatment, proving the effectiveness of this NMs-plant-rhizobacteria technique for simultaneously safe crop production and soil remediation. These findings can help expand the potential applications of nanobio interaction and its mediated iron plaque generation for both agricultural practice and soil remediation.

Alfalfa spring black stem and leaf spot disease (ASBS) is a cosmopolitan soil-borne and seed-borne disease caused by Phoma medicaginis , which adversely affects the yield, and nutritive value and can stimulate production of phyto-oestrogenic compounds at levels that may adversely affect ovulation rates in animals. This review summarizes the host range, damage, and symptoms of this disease, and general features of the infection cycle, epidemic occurrence, and disease management. ASBS has been reported from over 40 countries, and often causes severe yield loss. Under greenhouse conditions, reported yield loss was 31-82% for roots, 32-80% for leaves, 21% for stems and 26-28% for seedlings. In field conditions, the forage yield loss is up to 56%, indicating that a single-cut yield of 5302 kg/ha would be reduced to 2347 kg/ha. P. medicaginis can infect up to 50 species of plants, including the genera Medicago , Trifolium , Melilotus , and Vicia . ASBS is more severe during warm spring conditions before the first harvest than in hot summer and cooler winter conditions, and can infect alfalfa roots, stems, leaves, flowers, pods, and seeds, with leaf spot and/or black stem being the most typical symptoms. The primary infection is caused by the overwintering spores and mycelia in the soil, and on seeds and the cortex of dead and dry stems. The use of resistant cultivars is the most economical and effective strategy for the control of ASBS. Although biological control has been studied in the glasshouse and is promising, chemical control is the main control method in agriculture.

Background: Selenium is essential for livestock and human health. The traditional way of adding selenium to livestock diets has limitations, and there is a growing trend to provide livestock with a safe and efficient source of selenium through selenium-enriched pasture. Therefore, this study was conducted to investigate the effects of selenium enrichment on fermentation characteristics, selenium content, selenium morphology, microbial community and in vitro digestion of silage alfalfa by using unenriched (CK) and selenium-enriched (Se) alfalfa as raw material for silage., Results: In this study, selenium enrichment significantly increased crude protein, soluble carbohydrate, total selenium, and organic selenium contents of alfalfa silage fresh and post-silage samples, and it significantly decreased neutral detergent fiber and acid detergent fiber contents (p < 0.05). Selenium enrichment altered the form of selenium in plants, mainly in the form of SeMet and SeMeCys, which were significantly higher than that of CK (p < 0.05). Selenium enrichment could significantly increase the lactic acid content, reduce the pH value, change the diversity of bacterial community, promote the growth of beneficial bacteria such as Lactiplantibacillus and inhibit the growth of harmful bacteria such as Pantoea, so as to improve the fermentation quality of silage. The in vitro digestibility of dry matter (IVDMD), in vitro digestibility of acid detergent fibers (IVADFD) and in vitro digestibility of acid detergent fibers (IVNDFD) of silage after selenium enrichment were significantly higher than those of CK (p < 0.05)., Conclusion: This study showed that the presence of selenium could regulate the structure of the alfalfa silage bacterial community and improve alfalfa silage fermentation quality. Selenium enrichment measures can change the morphology of selenium in alfalfa silage products, thus promoting the conversion of organic selenium., (© 2024. The Author(s).)

The morphological architecture of inflorescence influences seed production. The regulatory mechanisms underlying alfalfa ( Medicago sativa ) inflorescence elongation remain unclear. Therefore, in this study, we conducted a comparative analysis of the transcriptome, proteome, and metabolome of two extreme materials at three developmental stages to explore the mechanisms underlying inflorescence elongation in alfalfa. We observed the developmental processes of long and short inflorescences and found that the elongation capacity of alfalfa with long inflorescence was stronger than that of alfalfa with short inflorescences. Furthermore, integrative analysis of the transcriptome and proteome indicated that the phenylpropanoid biosynthesis pathway was closely correlated with the structural formation of the inflorescence. Additionally, we identified key genes and proteins associated with lignin biosynthesis based on the differential expressed genes and proteins (DEGs and DEPs) involved in phenylpropanoid biosynthesis. Moreover, targeted hormone metabolome analysis revealed that IAA, GA, and CK play an important role in the peduncle elongation of alfalfa inflorescences. Based on omics analysis, we detected key genes and proteins related to plant hormone biosynthesis and signal transduction. From the WGCNA and WPCNA results, we furthermore screened 28 candidate genes and six key proteins that were correlated with lignin biosynthesis, plant hormone biosynthesis, and signaling pathways. In addition, 19 crucial transcription factors were discovered using correlation analysis that might play a role in regulating candidate genes. This study provides insight into the molecular mechanism of inflorescence elongation in alfalfa and establishes a theoretical foundation for improving alfalfa seed production.

Key Message: MsTFL1A is an important gene involved in flowering repression in alfalfa (Medicago sativa) which conditions not only above-ground plant shoot architecture but also root development and growth. Delayed flowering is an important trait for forage species, as it allows harvesting of high-quality forage for a longer time before nutritional values decline due to plant architecture changes related to flowering onset. Despite the relevance of delayed flowering, this trait has not yet been thoroughly exploited in alfalfa. This is mainly due to its complex genetics, sensitivity to inbreeding and to the fact that delayed flowering would be only advantageous if it allowed increased forage quality without compromising seed production. To develop new delayed-flowering varieties, we have characterized the three TERMINAL FLOWERING 1 (TFL1) family of genes in alfalfa: MsTFL1A, MsTFL1B and MsTFL1C. Constitutive expression of MsTFL1A in Arabidopsis caused late flowering and changes in inflorescence architecture, indicating that MsTFL1A is the ortholog of Arabidopsis TFL1. Overexpression of MsTFL1A in alfalfa consistently led to delayed flowering in both controlled and natural field conditions, coupled to an increase in leaf/stem ratio, a common indicator of forage quality. Additionally, overexpression of MsTFL1A reduced root development, reinforcing the role of MsTFL1A not only as a flowering repressor but also as a regulator of root development.We conclude that the precise manipulation of MsTFL1A gene expression may represent a powerful tool to improve alfalfa forage quality., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world, including autotetraploid (M. sativa ssp. sativa) and diploid alfalfa (M. sativa ssp. caerulea, progenitor of autotetraploid alfalfa). Here, we reported a high-quality genome of ZW0012 (diploid alfalfa, 769 Mb, contig N50 = 5.5 Mb), which was grouped into the Northern group in population structure analysis, suggesting that our genome assembly filled a major gap among the members of M. sativa complex. During polyploidization, large phenotypic differences occurred between diploids and tetraploids, and the genetic information underlying its massive phenotypic variations remains largely unexplored. Extensive structural variations (SVs) were identified between ZW0012 and XinJiangDaYe (an autotetraploid alfalfa with released genome). We identified 71 ZW0012-specific PAV genes and 1296 XinJiangDaYe-specific PAV genes, mainly involved in defence response, cell growth, and photosynthesis. We have verified the positive roles of MsNCR1 (a XinJiangDaYe-specific PAV gene) in nodulation using an Agrobacterium rhizobia-mediated transgenic method. We also demonstrated that MsSKIP23&#95;1 and MsFBL23&#95;1 (two XinJiangDaYe-specific PAV genes) regulated leaf size by transient overexpression and virus-induced gene silencing analysis. Our study provides a high-quality reference genome of an important diploid alfalfa germplasm and a valuable resource of variation landscape between diploid and autotetraploid, which will facilitate the functional gene discovery and molecular-based breeding for the cultivars in the future., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Alfalfa ( Medicago sativa L .) is one of the most important forage crops in the world. Drought is recognized as a major challenge limiting alfalfa production and threatening food security. Although some literature reviews have been conducted in this area, bibliometric reviews based on large amounts of published data are still lacking. In this paper, a bibliometric analysis of alfalfa drought stress from 1998-2023 was conducted using the Web of Science Core Collection database in order to assess global trends in alfalfa drought stress research and to provide new directions for future research. The results showed that the annual publication output maintained an increase in most years, with China and the United States contributing significantly to the field. Most of the journals published are specialized journals in botany, environmental science, soil science and crop science, as well as related agribusiness journals. "plant growth" and "yield" were the most frequently used keywords, reflecting the important purpose of research in this field. And two main research directions were identified: research on drought response mechanism of alfalfa and exploration of drought-resistant technology. In addition, physiological, biochemical, and molecular responses of drought tolerance and high yield in alfalfa, transgenics, and microbial fertilizer research have been hot research topics in recent years and may continue in the future. The ultimate goal of this paper is to provide a foundational reference for future research on alfalfa's drought resistance and yield optimization mechanisms, thereby enhancing the crop's application in agricultural production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhou, Li, Gao, Wang, Wang, Huang, Zhang, Zhao and Wang.)

BBX protein is a class of zinc finger transcription factors that have B-box domains at the N-terminus, and some of these proteins contain a CCT domain at the C-terminus. It plays an important role in plant growth, development, and metabolism. However, the expression pattern of BBX genes in alfalfa under hormonal and salt stresses is still unclear. In this study, we identified a total of 125 BBX gene family members by the available Medicago reference genome in diploid alfalfa ( Medicago sativa spp. Caerulea ), a model plant ( M. truncatula ), and tetraploid alfalfa ( M . sativa ), and divided these members into five subfamilies. We found that the conserved motifs of BBXs of the same subfamily reveal similarities. We analyzed the collinearity relationship and duplication mode of these BBX genes and found that the expression pattern of BBX genes is specific in different tissues. Analysis of the available transcriptome data suggests that some members of the BBX gene family are involved in multiple abiotic stress responses, and the highly expressed genes are often clustered together. Furthermore, we identified different expression patterns of some BBX genes under salt, ethylene, salt and ethylene, salicylic acid, and salt and salicylic acid treatments, verified by qRT-PCR, and analyzed the subcellular localization of MsBBX2 , MsBBX17 , and MsBBX32 using transient expression in tobacco. The results showed that BBX genes were localized in the nucleus. This study systematically analyzed the BBX gene family in Medicago plants, which provides a basis for the study of BBX gene family tolerance to abiotic stresses.

Background: Alfalfa (Medicago sativa L.) is the most cultivated forage legume around the world. Under a variety of growing conditions, forage yield in alfalfa is stymied by biotic and abiotic stresses including heat, salt, drought, and disease. Given the sessile nature of plants, they use strategies including, but not limited to, differential gene expression to respond to environmental cues. Transcription factors control the expression of genes that contribute to or enable tolerance and survival during periods of stress. Basic-leucine zipper (bZIP) transcription factors have been demonstrated to play a critical role in regulating plant growth and development as well as mediate the responses to abiotic stress in several species, including Arabidopsis thaliana, Oryza sativa, Lotus japonicus and Medicago truncatula. However, there is little information about bZIP transcription factors in cultivated alfalfa., Result: In the present study, 237 bZIP genes were identified in alfalfa from publicly available sequencing data. Multiple sequence alignments showed the presence of intact bZIP motifs in the identified sequences. Based on previous phylogenetic analyses in A. thaliana, alfalfa bZIPs were similarly divided and fell into 10 groups. The physico-chemical properties, motif analysis and phylogenetic study of the alfalfa bZIPs revealed high specificity within groups. The differential expression of alfalfa bZIPs in a suite of tissues indicates that bZIP genes are specifically expressed at different developmental stages in alfalfa. Similarly, expression analysis in response to ABA, cold, drought and salt stresses, indicates that a subset of bZIP genes are also differentially expressed and likely play a role in abiotic stress signaling and/or tolerance. RT-qPCR analysis on selected genes further verified these differential expression patterns., Conclusions: Taken together, this work provides a framework for the future study of bZIPs in alfalfa and presents candidate bZIPs involved in stress-response signaling., (© 2024. The Author(s).)

Introduction: This study used Bacillus amyloliquefaciens DGL1 isolated from the arid sandy land of the Qinghai-Tibetan Plateau as the research strain and investigated the effects of DGL1 on the biomass, physiology, and metabolites of Medicago sativa under different intensities of drought stress to provide a high-quality bacterial source and a theoretical basis for the research and development of biological fertilizer suitable for arid areas., Methods: The exopolysaccharides (EPS), 1-Aminocyclopropane-1-carboxylate deaminase (ACC), and phosphorus solubilizing capacity of DGL1 were determined. The effects of a DGL1 suspension on alfalfa biomass, physiological indexes, degree of peroxidation of cell membranes, and activity of antioxidant enzymes were determined after irrigating roots under drought stress. The effects on soil physicochemical properties were also evaluated, and metabolomics analysis was performed to explore the effect of DGL1 on the metabolites of alfalfa under drought stress., Results: Strain DGL1 produced extracellular polysaccharide EPS and ACC deaminase and was capable of phosphorus solubilization. Treatment with DGL1 increased the biomass of alfalfa under different degrees of drought stress, significantly increased the activities of alfalfa antioxidant enzymes Super Oxide Dismutase (SOD), Peroxidase (POD), and catalase (CAT), reduced the content of MDA and H 2 O 2 , and increased the content of quick-acting phosphorus, quick-acting potassium, ammonium nitrogen, and nitrate nitrogen in the soil, thus improving soil fertility. Through metabolomics analysis, DGL1 was shown to affect amino acid metabolic pathways, such as arginine, leucine, glutamate, and tyrosine, as well as the levels of energy-providing polysaccharides and lipids, in alfalfa under 15% PEG-6000 drought stress, enhancing alfalfa's capacity to resist drought stress., Discussion: Strain DGL1 enhances the drought suitability of alfalfa and has the potential for dryland development as a biological agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Xie, Qiao, Chang, Wang, Li, Wu, Li and Gao.)

Introduction: Phosphorus (P) fertilizer is critical to maintain a high yield and quality of alfalfa (Medicago sativa L.). There are several fertilizer types and soil types in China, and the application of a single type of P fertilizer may not be suitable for present-day alfalfa production., Methods: In order to select the optimal combination of alfalfa and soil type and fertilizer type for improving P utilization efficiency. We conducted a greenhouse pot experiment, calcium superphosphate (SSP), diammonium phosphate (DAP), ammonium polyphosphate (APP), potassium dihydrogen phosphate (KP), and no-fertilizer control treatments were applied to alfalfa in sandy and saline-alkali soils. The response of alfalfa root morphology and rhizosphere processes to different P fertilizers was investigated., Results and Discussion: The results showed that shoot biomass of alfalfa was slightly higher in sandy soil than in saline-alkali soil. Shoot biomass of alfalfa increased by 223%-354% in sandy soil under P treatments compared with the control, and total root length increased significantly by 74% and 53% in DAP and SSP treatments, respectively. In saline-alkali soil, alfalfa shoot biomass was significantly increased by 229% and 275% in KP and DAP treatments, and total root length was increased by 109% only in DAP treatment. Net P uptake of alfalfa in DAP treatment was the highest in both soils, which were 0.73 and 0.54 mg plant -1 , respectively. Alfalfa shoot P concentration was significantly positively correlated with shoot and root biomass ( P < 0.05, 0.01 or 0.001) whereas negatively correlated with acid phosphatase concentration ( P < 0.05). Improvement of plant growth and P uptake induced by P fertilizer application was greater in sandy soil than in saline-alkali soil. DAP and KP was the most efficient P fertilizers in both sandy soil and saline-alkali soil., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Zhang, Wan, Sun and Li.)

Salinity and Phoma medicaginis infection represent significant challenges for alfalfa cultivation in South Africa, Europe, Australia, and, particularly, Tunisia. These constraints have a severe impact on both yield and quality. The primary aim of this study was to establish the genetic basis of traits associated with biomass and growth of 129 Medicago sativa genotypes through genome-wide association studies (GWAS) under combined salt and P. medicaginis infection stresses. The results of the analysis of variance (ANOVA) indicated that the variation in these traits could be primarily attributed to genotype effects. Among the test genotypes, the length of the main stem, the number of ramifications, the number of chlorotic leaves, and the aerial fresh weight exhibited the most significant variation. The broad-sense heritability ( H ²) was relatively high for most of the assessed traits, primarily due to genetic factors. Cluster analysis, applied to morpho-physiological traits under the combined stresses, revealed three major groups of accessions. Subsequently, a GWAS analysis was conducted to validate significant associations between 54,866 SNP-filtered single-nucleotide polymorphisms (SNPs) and seven traits. The study identified 27 SNPs that were significantly associated with the following traits: number of healthy leaves (two SNPs), number of chlorotic leaves (five SNPs), number of infected necrotic leaves (three SNPs), aerial fresh weight (six SNPs), aerial dry weight (nine SNPs), number of ramifications (one SNP), and length of the main stem (one SNP). Some of these markers are related to the ionic transporters, cell membrane rigidity (related to salinity tolerance), and the NBS&#95;LRR gene family (associated with disease resistance). These findings underscore the potential for selecting alfalfa genotypes with tolerance to the combined constraints of salinity and P. medicaginis infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mnafgui, Jabri, Jihnaoui, Maiza, Guerchi, Zaidi, Basson, Keyster, Djébali, Pecetti, Hanana, Annicchiarico, Sakiroglu, Ludidi and Badri.)

The inclination toward natural products has led to the onset of the discovery of new bioactive metabolites that could be targeted for specific therapeutic or agronomic applications. Despite increasing knowledge coming to light of plant-derived materials as leads for new herbicides, relatively little is known about the mode of action on herbicide-resistant weeds. Cyanamide (CA) is a naturally occurring herbicide synthesized by hairy vetch (Vicia villosa Roth.). However, it has not been experimentally verified whether CA suppresses target plants via sustained discharge at low concentrations, as is often the case with most plant-derived materials. This study aimed to detect the toxicity and the mode of action of CA to alfalfa (Medicago sativa L.) and redroot pigweed (Amaranthus retroflexus L.). The toxicity of CA toward the alfalfa and redroot pigweed by three different exposure patterns was compared: low-concentration repeated exposure with 0.3 g/L CA (LRE), high-concentration single exposure with 1.2 g/L CA (HSE), and distilled water spray as control. The results showed that CA had a stronger inhibitory effect on redroot pigweed growth compared to alfalfa under both LRE and HSE exposure modes, with leaves gradually turning yellow and finally wilting. Beyond that, field trials were conducted to corroborate the toxicity of CA to alfalfa and redroot pigweed. The results have also shown that CA could inhibit the growth of redroot pigweed without significant adverse effects on alfalfa. The outcomes concerning electrolyte permeability, root activity, and malondialdehyde (MDA) content indicated that CA suppressed the growth of redroot pigweed by interfering with the structure of the cell membrane and impacting cellular osmotic potential. CA could destroy the cell membrane structure to inhibit the growth of the redroot pigweed by both LRE and HSE exposure modes, which provides a theoretical basis for preventing and controlling redroot pigweed in alfalfa fields., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Root rot is a common disease, that severely affects the yield and quality of alfalfa. Biocontrol is widely used to control plant diseases caused by pathogenic fungi, however, biocontrol strains for alfalfa root rot are very limited. In this study, a Bacillus subtilis CG-6 strain with a significant biocontrol effect on alfalfa root rot was isolated. CG-6 secretes antibacterial enzymes and siderophore, phosphate solubilization and indoleacetic acid (IAA). The inhibition rate of strain CG-6 against Fusarium oxysporum was 87.33%, and it showed broad-spectrum antifungal activity. Inoculation with CG-6 significantly reduced the incidence of alfalfa root rot, the control effect of greenhouse cultivation reached 58.12%, and CG-6 treatment significantly increased alfalfa plant height, root length, fresh weight, and dry weight. The treatment with CG-6 significantly increased the levels of antioxidant enzymes (catalase, peroxidase, superoxide dismutase, and lipoxygenase) in alfalfa leaves by 15.52%-34.03%. Defensive enzymes (chitinase and β-1,3-glucanase) increased by 24.37% and 28.08%, respectively. The expression levels of regulatory enzyme genes (MsCAT, MsPOD, MsCu, Zn-SOD1, MsCu, Zn-SOD2, MsCu, Zn-SOD3, and MsLOX2) and systemic resistance genes (MsPR1, MsPDF1.2, and MsVSP2) increased by 0.50-2.85 fold, which were higher than those in the pathogen treatment group. Therefore, CG-6 could be used as a potential strain to develop biopesticides against alfalfa root rot., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Lignin provides structural support to plants; however, it reduces their utilization rate. According to our previous studies, selenium (Se) reduces lignin accumulation in alfalfa, but the specific mechanism involved remains unclear. Therefore, at the seedling stage, four root irrigation treatments using 2.5, 50, and 5 μmol/L sodium selenite (S-RI), selenomethionine (SS-RI), Se nanoparticles (SSS-RI), and deionized water (CK-RI) were performed. At the branching stage, four treatments of foliar spraying with the three Se fertilizers described above at a concentration of 0.5 mmol/L (S-FS, SS-FS, and SSS-FS) and deionized water (CK-FS) were administered. The results revealed that all Se treatments chiefly reduced the level of deposition of syringyl (S) lignin in the first internode of alfalfa stems. SS-FS and SSS-FS treatments mainly reduced the deposition of S and guaiacyl (G) lignins in the sixth internode of alfalfa stems, respectively, while S-FS treatment only slightly reduced the deposition of G lignin. S, SS, and SSS-RI treatments reduced the level of deposition of S and G lignins in the sixth internode of alfalfa stems. Se application increased plant height, stem diameter, epidermis (cortex) thickness, primary xylem vessel number (diameter), and pith diameter of alfalfa but decreased primary xylem area and pith parenchyma cell wall thickness of the first internode, and SS(SSS)-FS treatment reduced the mechanical strength of alfalfa stems. Therefore, Se application could decrease lignin accumulation by regulating the organizational structure parameters of alfalfa stems and the deposition pattern of the lignin monomers.

DREB has been reported to be involved in plant growth and response to environmental factors. However, the function of DREB in growth and development has not been elucidated in alfalfa ( Medicago sativa L.), a perennial tetraploid forage cultivated worldwide. In this study, an ortholog of MtDREB1C was characterized from alfalfa and named MsDREB1C accordingly. MsDREB1C was significantly induced by abiotic stress. The transcription factor MsDREB1C resided in the nucleus and had self-transactivation activity. The MsDREB1C overexpression (OE) alfalfa displayed growth retardation under both long-day and short-day conditions, which was supported by decreased MsGA20ox and upregulated MsGA2ox in the OE lines. Consistently, a decrease in active gibberellin (GA) was detected, suggesting a negative effect of MsDREB1C on GA accumulation in alfalfa. Interestingly, the forage quality of the OE lines was better than that of WT lines, with higher crude protein and lower lignin content, which was supported by an increase in the leaf-stem ratio (LSR) and repression of several lignin-synthesis genes ( MsNST , MsPAL1 , MsC4H , and Ms4CL ). Therefore, this study revealed the effects of MsDREB1C overexpression on growth and forage quality via modifying GA accumulation and lignin synthesis, respectively. Our findings provide a valuable candidate for improving the critical agronomic traits of alfalfa, such as overwintering and feeding value of the forage.

Expansins, a class of cell-wall-loosening proteins that regulate plant growth and stress resistance, have been studied in a variety of plant species. However, little is known about the Expansins present in alfalfa ( Medicago sativa L.) due to the complexity of its tetraploidy. Based on the alfalfa (cultivar "XinjiangDaye") reference genome, we identified 168 Expansin members ( MsEXPs ). Phylogenetic analysis showed that MsEXPs consist of four subfamilies: MsEXPAs (123), MsEXPBs (25), MsEXLAs (2), and MsEXLBs (18). MsEXPAs, which account for 73.2% of MsEXPs, and are divided into twelve groups (EXPA-I-EXPA-XII). Of these, EXPA-XI members are specific to Medicago trunctula and alfalfa. Gene composition analysis revealed that the members of each individual subfamily shared a similar structure. Interestingly, about 56.3% of the cis -acting elements were predicted to be associated with abiotic stress, and the majority were MYB- and MYC-binding motifs, accounting for 33.9% and 36.0%, respectively. Our short-term treatment (≤24 h) with NaCl (200 mM) or PEG (polyethylene glycol, 15%) showed that the transcriptional levels of 12 MsEXPs in seedlings were significantly altered at the tested time point(s), indicating that MsEXPs are osmotic-responsive. These findings imply the potential functions of MsEXPs in alfalfa adaptation to high salinity and/or drought. Future studies on MsEXP expression profiles under long-term (>24 h) stress treatment would provide valuable information on their involvement in the response of alfalfa to abiotic stress.

Non-thermal plasma (NTP) has proven to be a green method in the agricultural field for the stimulation of germination, growth, and production of nutraceutical compounds in some cases. However, the process is far from being fully understood and depends on the targeted plant species and the NTP used. In this work, we focus on the production of alfalfa sprouts from NTP-treated seeds under different voltage conditions. A flexible electrode configuration was used to produce the NTP, which can also be placed on packages for in-package treatments. The surface of the seeds was analyzed, indicating that the microstructure was strongly affected by NTP treatment. Biometric measurements evidenced the possibility of stimulating the sprout growth in some conditions by up to 50% compared to the sprouts obtained from untreated seeds. Biochemical traits for the sprouts obtained in different processing conditions were also studied, such as the concentrations of chlorophyll pigments, flavonoids and polyphenols, and antioxidant activity. Most NTP treatments led to inhibitory effects, proving the strong dependence between NTP treatment and targeted plant species.

Plant growth-promoting rhizobacteria have been shown to enhance plant tolerance to drought stress through various mechanisms. However, there is limited research on improving drought resistance in alfalfa by genetically modifying PGPR to produce increased levels of cytokinins. Herein, we employed synthetic biology approaches to engineer two novel strains of Sinorhizobium meliloti capable of overproducing trans-Zeatin and investigated their potential in enhancing drought tolerance in alfalfa. Our results demonstrate that alfalfa plants inoculated with these engineered S. meliloti strains exhibited reduced wilting and yellowing while maintaining higher relative water content under drought conditions. The engineered S. meliloti -induced tZ activated the activity of antioxidant enzymes and the accumulation of osmolytes. Additionally, the increased endogenous tZ content in plants alleviated the impact of drought stress on the alfalfa photosynthetic rate. However, under nondrought conditions, inoculation with the engineered S. meliloti strains had no significant effect on alfalfa biomass and nodule formation.

Alfalfa ( Medicago sativa L.) is an important forage legume and soil salinization seriously affects its growth and yield. In a previous study, we identified a salt-tolerant variety 'Gongnong NO.1' and a salt-sensitive variety 'Sibeide'. To unravel the molecular mechanism involved in salt stress, we conducted transcriptomic analysis on these two cultivars grown under 0 and 250 mM NaCl treatments for 0, 12, and 24 h. Totals of 336, and 548 differentially expressed genes (DEGs) in response to NaCl were, respectively, identified in the 'Gongnong NO.1' and 'Sibeide' varieties. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analysis showed that the DEGs were classified in carbohydrate metabolism, energy production, transcription factor, and stress-associated pathway. Expression of MsHPCA1 , encoding a putative H 2 O 2 receptor, was responsive to both NaCl and H 2 O 2 treatment. MsHPCA1 was localized in cell membrane and overexpression of MsHPCA1 in alfalfa increased salt tolerance and H 2 O 2 content. This study will provide new gene resources for the improvement in salt tolerance in alfalfa and legume crops, which has important theoretical significance and potential application value.

We have previously reported on the detection of an unknown picorna-like virus in alfalfa samples. The exact host of the virus was unclear due to its similarity to the members of Iflaviridae family, which typically infect arthropods. The virus was provisionally named alfalfa-associated picorna-like virus 2. Here, we report a complete genomic sequence of the virus., Competing Interests: The authors declare no conflict of interest.

Background: Alfalfa, the most economically important forage legume worldwide, features modest genetic progress due to long selection cycles and the extent of the non-additive genetic variance associated with its autotetraploid genome., Methods: To improve the efficiency of genomic selection in alfalfa, we explored the effects of genome parametrization (as tetraploid and diploid dosages, plus allele ratios) and SNP marker subsetting (all available SNPs, only genic regions, and only non-genic regions) on genomic regressions, together with various levels of filtering on reading depth and missing rates. We used genotyping by sequencing-generated data and focused on traits of different genetic complexity, i.e., dry biomass yield in moisture-favorable (FE) and drought stress (SE) environments, leaf size, and the onset of flowering, which were assessed in 143 genotyped plants from a genetically broad European reference population and their phenotyped half-sib progenies., Results: On average, the allele ratio improved the predictive ability compared with other genome parametrizations (+7.9% vs. tetraploid dosage, +12.6% vs. diploid dosage), while using all the SNPs offered an advantage compared with any specific SNP subsetting (+3.7% vs. genic regions, +7.6% vs. non-genic regions). However, when focusing on specific traits, different combinations of genome parametrization and subsetting achieved better performances. We also released Legpipe2 , an SNP calling pipeline tailored for reduced representation (GBS, RAD) in medium-sized genotyping experiments.

The epiphytic microbiota source on plants plays a crucial role in the production of high-quality silage. To gain a better understanding of its contribution, the microbiota of alfalfa (M1C0), corn (M0C1) and the resulting mixture (M1C1) was applied in alfalfa-corn mixed silage production system. M1C0 decreased ammonia-N levels in terms of total nitrogen (57.59-118.23 g/kg TN) and pH (3.59-4.40) values ( p  < 0.01), which increased lactic acid (33.73-61.89 g/kg DM) content ( p  < 0.01). Consequently, this resulted in higher residual water-soluble carbohydrate (29.13-41.76 g/kg DM) and crude protein (152.54-167.91 g/kg DM) contents, as well as lower NDF (427.27 g/kg DM) and ADF (269.53 g/kg DM) contents in the silage compared to M1C1- and M0C1-treated samples. Moreover, M1C0 silage showed significantly higher bacterial alpha diversity indices ( p  < 0.05), including the number of observed species and Chao1 and Shannon diversity indices, at the later stages of ensiling. Lactobacillus, Kosakonia and Enterobacter were the dominant bacterial species in silages, with a relative abundance of >80%. However, the abundance of Lactobacillus amylovorus in M0C1- and M1C1-treated silage increased ( p  < 0.01) in the late stages of ensiling. These findings confirmed that the epiphytic microbiota source exerts competitive effects during anaerobic storage of alfalfa-corn mixed silage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tang, Liao, Huang, Chen, Xu and Chen.)

Drought is a major threat to alfalfa (Medicago sativa L.) production. The discovery of important alfalfa genes regulating drought response will facilitate breeding for drought-resistant alfalfa cultivars. Here, we report a genome-wide association study of drought resistance in alfalfa. We identified and functionally characterized an MYB-like transcription factor gene (MsMYBH), which increases the drought resistance in alfalfa. Compared with the wild-types, the biomass and forage quality were enhanced in MsMYBH overexpressed plants. Combined RNA-seq, proteomics and chromatin immunoprecipitation analysis showed that MsMYBH can directly bind to the promoters of MsMCP1, MsMCP2, MsPRX1A and MsCARCAB to improve their expression. The outcomes of such interactions include better water balance, high photosynthetic efficiency and scavenge excess H 2 O 2 in response to drought. Furthermore, an E3 ubiquitin ligase (MsWAV3) was found to induce MsMYBH degradation under long-term drought, via the 26S proteasome pathway. Furthermore, variable-number tandem repeats in MsMYBH promoter were characterized among a collection of germplasms, and the variation is associated with promoter activity. Collectively, our findings shed light on the functions of MsMYBH and provide a pivotal gene that could be leveraged for breeding drought-resistant alfalfa. This discovery also offers new insights into the mechanisms of drought resistance in alfalfa., (© 2024 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)