The Extraordinary Alkali Bee, Nomia melanderi (Halictidae), the World's Only Intensively Managed Ground-Nesting Bee.

Among the ground-nesting bees are several proven crop pollinators, but only the alkali bee (Nomia melanderi) has been successfully managed. In <80 years, it has become the world's most intensely studied ground-nesting solitary bee. In many ways, the bee seems paradoxical. It nests during the torrid, parched midsummer amid arid valleys and basins of the western United States, yet it wants damp nesting soil. In these basins, extensive monocultures of an irrigated Eurasian crop plant, alfalfa (lucerne), subsidize millions of alkali bees. Elsewhere, its polylectic habits and long foraging range allow it to stray into neighboring crops contaminated with insecticides. Primary wild floral hosts are either non-native or poorly known. Kleptoparasitic bees plague most ground nesters, but not alkali bees, which do, however, host other well-studied parasitoids. Building effective nesting beds requires understanding the hydraulic conductivity of silty nesting soils and its important interplay with specific soil mineral salts. Surprisingly, some isolated populations endure inhospitably cold climates by nesting amid hot springs. Despite the peculiarities and challenges associated with its management, the alkali bee remains the second most valuable managed solitary bee for US agriculture and perhaps the world.

Mechanism of action of microRNA166 on nitric oxide in alfalfa (Medicago sativa L.) under drought stress.

BACKGROUND: Alfalfa is a perennial forage crop of high importance, but its cultivation is often affected by drought stress. Currently, the investigation of drought-related small RNAs is a popular research topic to uncover plant drought resistance mechanisms. Among these small RNAs, microRNA166 (miR166) is associated with drought in numerous plant species. Initial small RNA sequencing studies have shown that miR166 is highly responsive to exogenous nitric oxide (NO) and drought. Therefore, analyzing the expression of Msa-miR166 under nitric oxide and drought treatment is significant. RESULT: Bioinformatics analysis revealed that the miR166 family is widely distributed among plants, ranging from mosses to eudicots, with significant distribution differences between species. The evolutionary degree of Msa-miR166s is highly similar to that of Barrel medic (Medicago truncatula) and Soybean (Glycine max), but significantly different from the model plant Arabidopsis (Arabidopsis thaliana). It is suggested that there are no significant differences in miR166s within the species, and members of Msa-miR166s can form a typical stem-loop. The lowest level of exogenous nitric oxide was observed in Msa-miR166s under drought stress, followed by individual drought, and the highest level was observed after removing endogenous nitric oxide. CONCLUSION: In response to short-term drought, Msa-miR166s down-regulate expression in alfalfa (Medicago sativa L.). Exogenous nitric oxide can reduce the expression of Msa-miR166s in response to short-term drought. These findings suggest that Msa-miR166e-5p is responsive to environmental changes. The expression levels of target genes showed an opposite trend to Msa-miR166s, verifying the accuracy of Degradome sequencing in the early stage. This suggests that alfalfa experiences drought stress when regulated by exogenous nitric oxide, targeting HD ZIP-III, FRI, and CoA ligase genes. Additionally, the expression of Msa-miR166s in response to drought stress varies between leaves and roots, indicating spatiotemporal specificity.

Integrated physiological, metabolomic, and transcriptomic analyses elucidate the regulation mechanisms of lignin synthesis under osmotic stress in alfalfa leaf (Medicago sativa L.).

Alfalfa, an essential forage crop known for its high yield, nutritional value, and strong adaptability, has been widely cultivated worldwide. The yield and quality of alfalfa are frequently jeopardized due to environmental degradation. Lignin, a constituent of the cell wall, enhances plant resistance to abiotic stress, which often causes osmotic stress in plant cells. However, how lignin responds to osmotic stress in leaves remains unclear. This study explored the effects of osmotic stress on lignin accumulation and the contents of intermediate metabolites involved in lignin synthesis in alfalfa leaves. Osmotic stress caused an increase in lignin accumulation and the alteration of core enzyme activities and gene expression in the phenylpropanoid pathway. We identified five hub genes (CSE, CCR, CADa, CADb, and POD) and thirty edge genes (including WRKYs, MYBs, and UBPs) by integrating transcriptome and metabolome analyses. In addition, ABA and ethylene signaling induced by osmotic stress regulated lignin biosynthesis in a contradictory way. These findings contribute to a new theoretical foundation for the breeding of high-quality and resistant alfalfa varieties.

Genome-wide identification of JAZ gene family members in autotetraploid cultivated alfalfa (Medicago sativa subsp. sativa) and expression analysis under salt stress.

BACKGROUND: Jasmonate ZIM-domain (JAZ) proteins, which act as negative regulators in the jasmonic acid (JA) signalling pathway, have significant implications for plant development and response to abiotic stress. RESULTS: Through a comprehensive genome-wide analysis, a total of 20 members of the JAZ gene family specific to alfalfa were identified in its genome. Phylogenetic analysis divided these 20 MsJAZ genes into five subgroups. Gene structure analysis, protein motif analysis, and 3D protein structure analysis revealed that alfalfa JAZ genes in the same evolutionary branch share similar exon‒intron, motif, and 3D structure compositions. Eight segmental duplication events were identified among these 20 MsJAZ genes through collinearity analysis. Among the 32 chromosomes of the autotetraploid cultivated alfalfa, there were 20 MsJAZ genes distributed on 17 chromosomes. Extensive stress-related cis-acting elements were detected in the upstream sequences of MsJAZ genes, suggesting that their response to stress has an underlying function. Furthermore, the expression levels of MsJAZ genes were examined across various tissues and under the influence of salt stress conditions, revealing tissue-specific expression and regulation by salt stress. Through RT‒qPCR experiments, it was discovered that the relative expression levels of these six MsJAZ genes increased under salt stress. CONCLUSIONS: In summary, our study represents the first comprehensive identification and analysis of the JAZ gene family in alfalfa. These results provide important information for exploring the mechanism of JAZ genes in alfalfa salt tolerance and identifying candidate genes for improving the salt tolerance of autotetraploid cultivated alfalfa via genetic engineering in the future.

In silico analysis identified bZIP transcription factors genes responsive to abiotic stress in Alfalfa (Medicago sativa L.).

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.

Regulation of endogenous hormone and miRNA in leaves of alfalfa (Medicago sativa L.) seedlings under drought stress by endogenous nitric oxide.

BACKGROUND: Alfalfa (Medicago sativa. L) is one of the best leguminous herbage in China and even in the world, with high nutritional and ecological value. However, one of the drawbacks of alfalfa is its sensitivity to dry conditions, which is a global agricultural problem. The objective of this study was to investigate the regulatory effects of endogenous nitric oxide (NO) on endogenous hormones and related miRNAs in alfalfa seedling leaves under drought stress. The effects of endogenous NO on endogenous hormones such as ABA, GA3, SA, and IAA in alfalfa leaves under drought stress were studied. In addition, high-throughput sequencing technology was used to identify drought-related miRNAs and endogenous NO-responsive miRNAs in alfalfa seedling leaves under drought stress. RESULT: By measuring the contents of four endogenous hormones in alfalfa leaves, it was found that endogenous NO could regulate plant growth and stress resistance by inducing the metabolism levels of IAA, ABA, GA3, and SA in alfalfa, especially ABA and SA in alfalfa. In addition, small RNA sequencing technology and bioinformatics methods were used to analyze endogenous NO-responsive miRNAs under drought stress. It was found that most miRNAs were enriched in biological pathways and molecular functions related to hormones (ABA, ETH, and JA), phenylpropane metabolism, and plant stress tolerance. CONCLUSION: In this study, the analysis of endogenous hormone signals and miRNAs in alfalfa leaves under PEG and PEG + cPTIO conditions provided an important basis for endogenous NO to improve the drought resistance of alfalfa at the physiological and molecular levels. It has important scientific value and practical significance for endogenous NO to improve plant drought resistance.

Genome-wide identification and expression analysis of the glycosyl hydrolase family 1 genes in Medicago sativa revealed their potential roles in response to multiple abiotic stresses.

Glycoside hydrolase family 1 (GH1) ß-glucosidases (BGLUs), are encoded by a large number of genes, which participate in the development and stress response of plants, particularly under biotic and abiotic stresses through the activation of phytohormones. However, there are few studies systematically analyzing stress or hormone-responsive BGLU genes in alfalfa. In this study, a total of 179 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of alfalfa, and then were classified into five distinct clusters. Sequence alignments revealed several conserved and unique motifs among these MsBGLU proteins. Many cis-acting elements related to abiotic stresses and phytohormones were identified in the promoter of some MsBGLUs. Moreover, RNA-seq and RT-qPCR analyses showed that these MsBGLU genes exhibited distinct expression patterns in response to different abiotic stress and hormonal treatments. In summary, this study suggests that MsBGLU genes play crucial roles in response to various abiotic stresses and hormonal responses, and provides candidate genes for stress tolerance breeding in alfalfa.

Genome-wide identification, characterization, and expression analysis of m6A readers-YTH domain-containing genes in alfalfa.

Eukaryotic messenger RNAs (mRNAs) are often modified with methyl groups at the N6 position of adenosine (m6A), and these changes are interpreted by YTH domain-containing proteins to regulate the metabolism of m6A-modified mRNAs. Although alfalfa (Medicago sativa) is an established model organism for forage development, the understanding of YTH proteins in alfalfa is still limited. In the present investigation, 53 putative YTH genes, each encoding a YT521 domain-containing protein, were identified within the alfalfa genome. These genes were categorized into two subfamilies: YTHDF (49 members) and YTHDC (four members). Each subfamily demonstrates analogous motif distributions and domain architectures. Specifically, proteins encoded by MsYTHDF genes incorporate a single domain structure, while those corresponding to MsYTH5, 8, 12, 16 who are identified as members of the MsYTHDC subfamily, exhibit CCCH-type zinc finger repeats at their N-termini. It is also observed that the predicted aromatic cage pocket that binds the m6A residue of MsYTHDC consists of a sequence of two tryptophan residues and one tyrosine residue (WWY). Conversely, in MsYTHDF, the binding pocket comprises two highly conserved tryptophan residues and either one tryptophan residue (WWW) or tyrosine residue (WWY) in MsYTHDF.Through comparative analysis of qRT-PCR data, we observed distinct expression patterns in specific genes under abiotic stress, indicating their potential regulatory roles. Notably, five genes (MsYTH2, 14, 26, 27, 48) consistently exhibit upregulation, and two genes (MsYTH33, 35) are downregulated in response to both cold and salt stress. This suggests a common mechanism among these YTH proteins in response to various abiotic stressors in alfalfa. Further, integrating qRT-PCR with RNA-seq data revealed that MsYTH2, MsYTH14, and MsYTH16 are highly expressed in leaves at various development stages, underscoring their potential roles in regulating the growth of these plant parts. The obtained findings shed further light on the biological functions of MsYTH genes and may aid in the selection of suitable candidate genes for future genetic enhancement endeavors aimed at improving salt and cold tolerance in alfalfa.

Genome-wide identification of B-box zinc finger (BBX) gene family in Medicago sativa and their roles in abiotic stress responses.

BACKGROUND: B-box (BBX) family is a class of zinc finger transcription factors (TFs) that play essential roles in regulating plant growth, development, as well as abiotic stress. However, no systematic analysis of BBX genes has yet been conducted in alfalfa (Medica go sativa L.), and their functions have not been elucidated up to now. RESULTS: In this study, 28 MsBBX genes were identified from the alfalfa genome, which were clustered into 4 subfamilies according to an evolutionary tree of BBX proteins. Exon-intron structure and conserved motif analysis reflected the evolutionary conservation of MsBBXs in alfalfa. Collinearity analysis showed that segmental duplication promoted the expansion of the MsBBX family. Analysis of cis-regulatory elements suggested that the MsBBX genes possessed many growth/development-, light-, phytohormone-, and abiotic stress-related elements. MsBBX genes were differentially expressed in leaves, flowers, pre-elongated stems, elongated stems, roots and nodules, and most MsBBXs were remarkably induced by drought, salt and various plant growth regulators (ABA, JA, and SA). Further functional verification demonstrated that overexpressing of the MsBBX11 gene clearly promoted salt tolerance in transgenic Arabidopsis by regulating growth and physiological processes of seedlings. CONCLUSIONS: This research provides insights into further functional research and regulatory mechanisms of MsBBX family genes under abiotic stress of alfalfa.

Effects of selenium enrichment on fermentation characteristics, selenium content and microbial community of alfalfa silage.

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.

A genome-wide association study reveals novel loci and candidate genes associated with plant height variation in Medicago sativa.

BACKGROUND: Plant height (PH) is an important agronomic trait influenced by a complex genetic network. However, the genetic basis for the variation in PH in Medicago sativa remains largely unknown. In this study, a comprehensive genome-wide association analysis was performed to identify genomic regions associated with PH using a diverse panel of 220 accessions of M. sativa worldwide. RESULTS: Our study identified eight novel single nucleotide polymorphisms (SNPs) significantly associated with PH evaluated in five environments, explaining 8.59-12.27% of the phenotypic variance. Among these SNPs, the favorable genotype of chr6__31716285 had a low frequency of 16.4%. Msa0882400, located proximal to this SNP, was annotated as phosphate transporter 3;1, and its role in regulating alfalfa PH was supported by transcriptome and candidate gene association analysis. In addition, 21 candidate genes were annotated within the associated regions that are involved in various biological processes related to plant growth and development. CONCLUSIONS: Our findings provide new molecular markers for marker-assisted selection in M. sativa breeding programs. Furthermore, this study enhances our understanding of the underlying genetic and molecular mechanisms governing PH variations in M. sativa.

The chromosome-level assembly of the wild diploid alfalfa genome provides insights into the full landscape of genomic variations between cultivated and wild alfalfa.

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_1 and MsFBL23_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.

Biological nitrogen fixation maintains carbon/nitrogen balance and photosynthesis at elevated CO2.

Understanding crop responses to elevated CO2 is necessary to meet increasing agricultural demands. Crops may not achieve maximum potential yields at high CO2 due to photosynthetic downregulation, often associated with nitrogen limitation. Legumes have been proposed to have an advantage at elevated CO2 due to their ability to exchange carbon for nitrogen. Here, the effects of biological nitrogen fixation (BNF) on the physiological and gene expression responses to elevated CO2 were examined at multiple nitrogen levels by comparing alfalfa mutants incapable of nitrogen fixation to wild-type. Elemental analysis revealed a role for BNF in maintaining shoot carbon/nitrogen (C/N) balance under all nitrogen treatments at elevated CO2, whereas the effect of BNF on biomass was only observed at elevated CO2 and the lowest nitrogen dose. Lower photosynthetic rates at were associated with the imbalance in shoot C/N. Genome-wide transcriptional responses were used to identify carbon and nitrogen metabolism genes underlying the traits. Transcription factors important to C/N signalling were identified from inferred regulatory networks. This work supports the hypothesis that maintenance of C/N homoeostasis at elevated CO2 can be achieved in plants capable of BNF and revealed important regulators in the underlying networks including an alfalfa (Golden2-like) GLK ortholog.

Antagonistic activity and mechanism of Bacillus subtilis CG-6 suppression of root rot and growth promotion in Alfalfa.

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 ß-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.

Transcriptional control of a metabolic switch regulating cellular methylation reactions is part of a common response to stress in divergent bee species.

Recent global declines in bee health have elevated the need for a more complete understanding of the cellular stress mechanisms employed by diverse bee species. We recently uncovered the biomarker lethal (2) essential for life [l(2)efl] genes as part of a shared transcriptional program in response to a number of cell stressors in the western honey bee (Apis mellifera). Here, we describe another shared stress-responsive gene, glycine N-methyltransferase (Gnmt), which is known as a key metabolic switch controlling cellular methylation reactions. We observed Gnmt induction by both abiotic and biotic stressors. We also found increased levels of the GNMT reaction product sarcosine in the midgut after stress, linking metabolic changes with the observed changes in gene regulation. Prior to this study, Gnmt upregulation had not been associated with cellular stress responses in other organisms. To determine whether this novel stress-responsive gene would behave similarly in other bee species, we first characterized the cellular response to endoplasmic reticulum (ER) stress in lab-reared adults of the solitary alfalfa leafcutting bee (Megachile rotundata) and compared this with age-matched honey bees. The novel stress gene Gnmt was induced in addition to a number of canonical gene targets induced in both bee species upon unfolded protein response (UPR) activation, suggesting that stress-induced regulation of cellular methylation reactions is a common feature of bees. Therefore, this study suggests that the honey bee can serve as an important model for bee biology more broadly, although studies on diverse bee species will be required to fully understand global declines in bee populations.

Ferric Oxide Nanomaterials and Plant-Rhizobacteria Symbionts Cogenerate Iron Plaque for Removing Highly Chlorinated Contaminants in Dryland Soils.

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-Fe2O3 NMs and plant-rhizobacteria symbionts under dryland conditions. Mechanistically, the coexistence of α-Fe2O3 NMs and Pseudomonas chlororaphis JD37 stimulated alfalfa roots to secrete acidic and reductive agents as well as H2O2, which together mediated the rhizosphere Fenton reaction and converted α-Fe2O3 NMs into iron plaque rich in Fe(II)-silicate. Further verifications reproduced the Fenton reaction in vitro using α-Fe2O3 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.

Synthetic Alfalfa Infusion Odour Attracts Gravid Culex quinquefasciatus Under Laboratory Conditions.

Gravid culicine mosquitoes rely on olfactory cues for selecting breeding sites containing organic detritus. While this capacity of the mosquitoes is used for surveillance and control, the current methodology is unwieldy, unreliable and expensive in time and labour. This study evaluated the dose-dependent attraction and oviposition response of gravid Culex quinquefasciatus to alfalfa infusions. Through combined chemical and electrophysiological analyses, bioactive volatile organic compounds (VOCs) in the headspace of alfalfa infusions, eliciting attraction, were identified. While phenolic and indolic compounds were the most abundant bioactive VOCs, additional VOCs, including a monoterpene, were required to elicit a significant behavioural response to the synthetic odour blend of alfalfa infusions. Comparative analysis with the commercially available mosquito oviposition pheromone (MOP) was also conducted demonstrating that this standardised synthetic alfalfa infusion odour blend offers a promising lure for targeted surveillance and control of Culex mosquitoes, which may contribute to disease prevention and public health protection.

MsSPL12 is a positive regulator in alfalfa (Medicago sativa L.) salt tolerance.

KEY MESSAGE: Over expression of MsSPL12 improved alfalfa salt tolerance by reducing Na+ accumulation and increasing antioxidant enzyme activity and regulating down-stream gene expression. Improvement of salt tolerance is one of the major goals in alfalfa breeding. Here, we demonstrated that MsSPL12, an alfalfa transcription factor gene highly expressed in the stem cells, plays a positive role in alfalfa salt tolerance. MsSPL12 is localized in the nucleus and shows transcriptional activity in the presence of its C-terminus. To investigate MsSPL12 function in plant response to salt stress, we generated transgenic plants overexpressing either MsSPL12 or a chimeric MsSPL12-SRDX gene that represses the function of MsSPL12 by using the Chimeric REpressor gene-Silencing Technology (CRES-T), and observed that overexpression of MsSPL12 increased the salt tolerance of alfalfa transgenic plants associated with an increase in K+/Na+ ratio and relative water content (RWC) under salt stress treatment, but a reduction in electrolyte leakage (EL), reactive oxygen species (ROS), malondialdehyde (MDA), and proline (Pro) compared to wild type (WT) plants. However, transgenic plants overexpressing MsSPL12-SRDX showed an inhibited plant growth and a reduced salt tolerance. RNA-sequencing and quantitative real-time PCR analyses revealed that MsSPL12 affected the expression of plant abiotic resistance-related genes in multiple physiological pathways. The potential MsSPL12-mediated regulatory pathways based on the differentially expressed genes between the MsSPL12 overexpression transgenics and WT controls were predicted. In summary, our study proves that MsSPL12 is a positive regulator in alfalfa salt tolerance and can be used as a new candidate for manipulation to develop forage crops with enhanced salt tolerance.

Effect of alfalfa supplementary change dietary non-fibrous carbohydrate (NFC) to neutral detergent fiber (NDF) ratio on rumen fermentation and microbial function in Gansu alpine fine wool sheep (Ovis aries).

Bodyweight loss and rumen microbial dysfunction of grazing sheep was a challenge for the sheep production industry during cold season, which were considered to correlated with under-roughage-feeding. Alfalfa is a good roughage supplementary for ruminants, which can improve grazing sheep bodyweight-loss and rumen microbial dysfunction during grass-withering period. This study evaluated the effects of alfalfa hay supplementary change dietary non-fibrous carbohydrate/neutral detergent fiber (NFC/NDF) ratios on rumen fermentation and microbial function of Gansu alpine fine wool sheep during extreme cold season. 120 ewes (3-4 yrs) with an average body weight of 28.71 ± 1.22 kg were allocated randomly into three treatments, and fed NFC/NDF of 1.92 (H group), 1.11 (M group), and 0.68 (L group), respectively. This study was conducted for 107 d, including 7 d of adaption to the diets. The rumen fermentation parameters and microbial characteristics were measured after the end of feeding trials. The results showed that the concentrations of sheep body weight, nitrogen components (Total-N, Soluble protein-N and Ammonia-N), blood biochemical indices (LDH, BUN and CHO) and ruminal volatile fatty acids (TVFA and propionate) significantly increased with an increase in the proportion of NFC/NDF ratios (p < .05), and the acetate and acetate/propionat ratio presented a contrary decreasing trend (p < .05). A total of 1018 OTUs were obtained with 97% consistency. Ruminococcus, Ruminococcaceae and Prevotella were observed as the predominant phyla in ruminal fluid microbiota. Higher NFC/NDF ratios with Alfalfa supplementary increased the richness and diversity of ruminal fluid microbiota, and decreased ruminal fluid microbiota beta-diversity. Using clusters of orthologous groups (COG), the ruminal fluid microbiota of alfalfa supplementary feeding showed low immune pathway and high carbohydrate metabolism pathway. In summary, the study suggested that there was an increasing tendency in dietary NFC/NDF ratio of 1.92 in body weight, ruminal fermentation, microbial community composition and fermentation characteristics through developing alfalfa supplementary system.

Di-(2-ethylhexyl) phthalate (DEHP) contamination suppressed soil microbial biomass carbon and mitigated CO2 emissions against the background of alfalfa from different soils.

Plastic mulching and organic amendments are prevalent agricultural practices worldwide. Plastic mulching has long been suspected as a significant source of DEHP contamination in terrestrial ecosystems. However, effects of DEHP contamination on greenhouse gas emissions and microbial biomass carbon (MBC) remain unclear. Here, a microcosm experiment was set up to assess the impact of DEHP exposure on MBC and carbon dioxide (CO2) emission in two different soils (acidic and alkaline) with the inclusion of alfalfa straw. The treatment includes: (i) control with no amendment (T1); (ii) alfalfa straw addition (20 g kg-1) (T2); (iii) DEHP (10 mg kg-1) + alfalfa straw (T3); and (iv) DEHP (100 mg kg-1) + alfalfa straw (T4). Against the background of alfalfa inclusion, DEHP exposure led to a potential reduction in cumulative CO2 emissions by 16.35 % and 6.91 % in alkaline soil and 12.27 % and 13.65 % in acidic soil for T3 and T4, respectively. The addition of DEHP triggered CO2 emissions and manifested a detrimental negative priming effect in both soil types. In both soils, average CO2 emission fluxes were highest for the T2 treatment. The MBC fluctuated at around 80 mg kg-1 for the control group, alfalfa straw alone (T2) treatment considerably enhanced MBC contents, whereas DEHP contamination in T3 and T4 treatments suppressed the stimulatory effect of alfalfa on MBC in both alkaline and acidic soils. Furthermore, a positive relationship was observed between soil CO2 emissions and MBC in both soils. Overall, these findings highlight the toxic impact of DEHP on MBC and its role in mitigating CO2 emissions in diverse soils. DEHP exposure counters the CO2 emissions induced by alfalfa straw. In addition, the inhibitory effect of DEHP on CO2 fluxes in alkaline soil is less pronounced than in acidic soil. Therefore, further cutting-edge research is crucial since DEHP contamination poses serious ecological threats to agroecosystems.