Alfalfa Leaf Research Articles

Response of Alfalfa Leaf Traits and Rhizosphere Fungal Communities to Compost Application in Saline-Sodic Soil.

Soil salinization is considered a major global environmental problem due to its adverse effects on agricultural sustainability and production. Compost is an environmentally friendly and sustainable measure used for reclaiming saline-sodic soil. However, the responses of the physiological characteristics of alfalfa and the structure and function of rhizosphere fungal communities after compost application in saline-sodic soil remain elusive. Here, a pot experiment was conducted to explore the effect of different compost application rates on soil properties, plant physiological traits, and rhizosphere fungal community characteristics. The results showed that compost significantly increased soil nutrients and corresponding soil enzyme activities, enhanced leaf photosynthesis traits, and ion homeostasis compared with the control treatment. We further found that the rhizosphere fungal communities were dominated by Sodiomyces at the genus level, and the relative abundance of pathogenic fungi, such as Botryotrichum, Plectosphaerella, Pseudogymnoascus, and Fusarium, declined after compost application. Moreover, the α-diversity indexes of the fungal community under compost application rates of 15% and 25% significantly decreased in comparison to the control treatment. The soil SOC, pH, TP, and TN were the main environmental factors affecting fungal community composition. The leaf photosynthetic traits and metal ion contents showed significantly positive correlations with Sodiomyces and Aspergillus. The fungal trophic mode was dominated by Pathotroph-Saprotroph-Symbiotroph and Saprotroph. Overall, our findings provide an important basis for the future application of microbial-based strategies to improve plant tolerance to saline-alkali stress.

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

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

Alfalfa leaf weevil larvae and adults feeding induces physiological change in defensive enzymes of alfalfa.

When insects harm plants, they activate relevant enzyme systems for defense, and changes in enzyme activity, to a certain extent, reflect the host plant's ability to resist insect damage. Alfalfa leaf weevils (Hypera postica Gyllenhal) are the main economic insect pest of alfalfa, which seriously affect its yield and quality. To clarify the effects of feeding induction by alfalfa leaf weevil larvae and adults on defense enzymes in alfalfa, 'Zhongmu No. 1' variety was used as the experimental material. Comprehensive correlation analysis and principal component analysis were used to evaluate the corresponding patterns of 12 physiological indicators of alfalfa induced by insect feeding of different densities. Results showed that after feeding induction by adult and larval alfalfa leaf weevils, total antioxidant capacity (T-AOC), malondialdehyde (MDA), phenylalanine ammonia-lyase (PAL), tyrosine ammonia lyase (TAL), lipoxygenase (LOX), chymotrypsin inhibitors (CI), trypsin inhibitor (TI), and jasmonic acid (JA) in the alfalfa leaves increased with increasing feeding time. However, activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and polyphenolic oxidase (PPO) in alfalfa leaves first increased and then decreased, showing a downward trend.

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters-such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride-were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Biocontrol of Hemileia vastatrix, the Causal Agent of Coffee Leaf Rust, by Paenibacillus sp. NMA1017.

Coffee leaf rust (CLR), caused by Hemileia vastatrix, is considered a highly important phytosanitary problem in Mexico. Currently, there are few microorganisms used as biocontrol alternatives to chemical control of CLR in organic coffee fields in Mexico. This study evaluates the use of Paenibacillus sp. NMA1017 as a biocontrol agent to inhibit the development of H. vastatrix in in vitro and in vivo (greenhouse) experiments. Hemileia vastatrix urediniospores were placed on water agar plates, and then Paenibacillus sp. NMA1017 was inoculated simultaneously or 8 h later. Urediniospores germination rate was reduced by 94% when the NMA1017 strain was inoculated simultaneously with the urediniospores and reduced by 38% when NMA1017 was inoculated 8 h later. Experiments with 8-month-old Bourbon coffee plants that were infected with H. vastatrix showed that disease incidence was reduced by 38, 90, and 50% when NMA1017 was applied 8 days before, simultaneously, or 8 days after the application of H. vastatrix, respectively. Paenibacillus sp. NMA1017 also reduced the severity of CLR on the leaves by up to 62%. The germination urediniospores of other rust pathogens such as Puccinia sorghi (maize leaf rust), Puccinia triticina (wheat leaf rust), Puccinia graminis f. sp. tritici (black stem rust of wheat), Uromyces striatus (alfalfa leaf rust), and Phragmidium sp. (rosebush leaf rust) were also inhibited. Use of the potential biocontrol agent Paenibacillus sp. NMA1017 might help reduce the application of chemical fungicides for the control of CLR, making coffee a more sustainable crop and providing management options for organic coffee growers.

Transcriptome-Wide Identification of Dark- and Salt-Induced Senescence-Related NAC Gene Family Members in Alfalfa.

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.

Alfalfa Spring Black Stem and Leaf Spot Disease Caused by Phoma medicaginis: Epidemic Occurrence and Impacts.

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.

Effects of Particle Size Distribution on the Physicochemical, Functional, and Structural Properties of Alfalfa Leaf Powder

To explore the effects of particle size distribution on its physicochemical, functional, and structural properties, alfalfa leaf powders with mean particle sizes (D50) of 506.1, 246.3, 209.8, 92.01, and 20.68 μm were prepared by sieving. The physicochemical, functional, and structural properties of alfalfa were compared, and correlation and principal component analyses were conducted. As the D50 of alfalfa leaf decreased, the bulk density, tap density, and the swelling capacity increased first and then decreased, but the compressibility, transition temperature, and melting temperature exhibited an opposite trend. The solubility, lightness, and inhibition of angiotensin-converting enzymes and tyrosinase were enhanced. Specifically, the alfalfa leaf with a D50 of 209.8 μm exhibited a higher bulk density and swelling capacity and a lower compressibility, transition temperature, and melting temperature. The alfalfa leaf with a D50 of 20.68 μm presented better solubility, lightness, and inhibition of angiotensin-converting enzymes and tyrosinase. Additionally, the surface roughness and the number of surface hydroxyls improved and the crystallinity index decreased, but the type of surface functional groups was unchanged. These changes in microstructure can provide an explanation for the trend of the physicochemical and functional properties. Moreover, based on the results of the correlation analysis and principal component analysis, it can be concluded that there are strong correlations among the particle size, physicochemical properties, and functional properties of alfalfa leaf. Overall, this conclusion can help determine the appropriate grinding particle size range for alfalfa leaf in different functional food products.

Evaluation of Medicago sativa ethanol leaf extract for antidiarrheal activity in Wistar rats

Aim: Medicago sativa leaf is traditionally used for the treatment of gastrointestinal disorders in Nigeria. This research investigates the in vivo antidiarrheal activity of M. sativa (Alfalfa) leaf extract in Wistar rats. Method: The study employed three models: castor oil-induced diarrhea, gastrointestinal motility (charcoal meal), and castor oil-induced intestinal fluid accumulation. The phytochemical analysis as well acute toxicity tests were carried out in the leaf extract. Results: The results demonstrate a dose-dependent and significant antidiarrheal effect of M. sativa leaf extract. In the castor oil-induced diarrhea model, the extract reduced fecal frequency, delayed onset, and lowered severity, with the highest effect at 600 mg/kg compared to a positive control (Loperamide). Gastrointestinal motility was inhibited by the extract in a dose-dependent manner, achieving maximum effect at 600 mg/kg, comparable to atropine sulfate. The castor oil-induced intestinal fluid accumulation model revealed a significant decrease in fluid volume at 600 mg/kg, exhibiting a potent inhibitory effect. The oral LD50 values obtained were greater than 5000 mg/kg in rats Conclusion: This study provides compelling evidence of Medicago sativa leaf extract's potential as an antidiarrheal agent in Wistar rats. Further investigations could explore its mechanism of action and safety profile, contributing valuable insights to the development of novel antidiarrheal therapies.

Prediction of Leaf Break Resistance of Green and Dry Alfalfa Leaves by Machine Learning Methods

Alfalfa holds an extremely significant place in animal nutrition when it comes to providing essential nutrients. The leaves of alfalfa specifically boast the highest nutritional value, containing a remarkable 70% of crude protein and an impressive 90% of essential vitamins. Due to this incredible nutritional profile, it becomes exceedingly important to ensure that the harvesting and threshing processes are executed with utmost care to minimize any potential loss of these invaluable nutrients present in the leaves. To minimize losses, it is essential to accurately determine the resistance of the leaves in both their green and dried forms. This study aimed to estimate the breaking resistance of green and dried alfalfa plants using machine learning methods. During the modeling phase, five different popular machine learning methods, Extra Trees (ET), Random Forest (RF), Gradient Boost (GB), Extreme Gradient Boosting (XGB), and CatBoost (CB), were used. The correlation coefficient (R2), root mean square error (RMSE), mean absolute error (MAE) and mean absolute percentage error (MAPE) metrics were used to evaluate the models. The obtained metric results and the graphs obtained from the prediction values of the models revealed that the machine learning methods made successful predictions. The best R2 (0.9853), RMSE (0.0171), MAE (0.0099) and MAPE (0.0969) values for the dry alfalfa plant were obtained from the model established with the ET method, while the best RMSE (0.0616) and R2 (0.96) values for the green alfalfa plant were obtained from the model established with the RF method and the best MAE (0.0340) value was obtained from the model established with the ET method. Additionally, the best MAPE (0.1447) value was obtained from the model established with the GB method.

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.

A dataset for estimating alfalfa leaf area and predicting leaf area index.

A dataset for estimating alfalfa leaf area and predicting leaf area index.

Replacing alfalfa hay with a novel alfalfa leaf pellet product (ProLEAF MAX) and/or alfalfa stems (ProFiber Plus) in the diet of developing dairy heifers alters dry matter intake, but does not negatively impact growth or development

Abstract Alfalfa is a commonly grown forage in the Intermountain West region of the United States and is often included in the diet of dairy cattle. Alfalfa provides a variety of different nutrients, but the nutrient content of alfalfa varies depending on factors such as the soil, region, cutting, and climate. However, alfalfa leaves tend to have less variation in their nutrient content than alfalfa stems. Fractionating alfalfa may be one way to improve control of nutrients provided when developing a ration for developing dairy heifers. The purpose of this study was to determine whether including fractionated alfalfa in the diet impacts the growth or conception rates of developing dairy heifers. Heifers were allocated to one of three treatments: a control group fed a typical diet (CON; n = 8), a diet that replaced alfalfa with fractionated alfalfa leaf pellets and alfalfa stems (ProLEAF MAX + ProFiber Plus; PLM + PFP; n = 8), or a diet that replaced alfalfa with alfalfa stems (PFP; n = 8) for 85 d. Heifers were fed individually twice daily and weight, hip height (HH), and wither height (WH) were recorded every 14 d. Additionally, blood was collected every 28 d, and conception rates were recorded at the end of the trial. Heifers receiving the PFP diet consumed less dry matter (P = 0.001) than the CON treatment. Analyses were then conducted to determine nutrient intake and heifers receiving the PFP diet also consumed less neutral detergent fiber (P = 0.02), acid detergent fiber (P = 0.02), crude protein (P = 0.001), and net energy for maintenance (P = 0.001) than heifers consuming the CON diet; however, no differences (P > 0.10) were observed between heifers fed the CON and PLM + PFP diets. Analysis of body weight gain over the feeding period showed no difference (P = 0.52) among heifers consuming the different treatment diets. Additionally, treatment did not affect average daily gain (P = 0.49), gain:feed (P = 0.82), HH gain (P = 0.20), or WH gain (P = 0.44) among heifers receiving different diets. Treatment × time altered (P < 0.001) blood urea nitrogen when analyzed as a repeated measure. Total feed cost was lowest (P < 0.001) for the PFP diet and cost of gain tended (P = 0.09) to be increased for the PLM + PFP diet compared to the CON diet. Overall, these data indicate that including alfalfa stems in a developing heifer diet may decrease dry matter intake, lower input costs, and increase profitability, without negatively impacting growth.

Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions

Alfalfa (Medicago sativa L.) is considered as the most important forage legume with high biomass yield and nutritional quality for ruminants. The alfalfa leaf cell walls are highly digestible, but stem cell walls of alfalfa are not readily digestible. The cell wall component of alfalfa has a large source of dietary energy, but ruminant animals can digest less than half of this component due to the presence of high lignin content. The main goal of this review is to make a summary of existing knowledge of alfalfa cell wall thickening and lignification patterns and suggest future directions for improving alfalfa stem cell wall digestibility. We describe alfalfa cell wall biochemistry, alfalfa stem morphology, stem tissue degradation, and existing methods to improve alfalfa digestibility, and discuss the potential future strategies for improving alfalfa cell wall digestibility. Information on these will help alfalfa breeders and producers identify superior alfalfa cultivars with improved stem cell wall digestibility. Concentrating future efforts on the selection and identification of traits and associated genes that affect cell wall digestibility could improve alfalfa cell wall digestibility.

Enhancing alfalfa photosynthetic performance through arbuscular mycorrhizal fungi inoculation across varied phosphorus application levels

This study evaluated the effects of arbuscular mycorrhizal fungi inoculation on the growth and photosynthetic performance of alfalfa under different phosphorus application levels. This experiment adopts two-factors completely random design, and sets four levels of fungi application: single inoculation with Funneliformis mosseae (Fm, T1), single inoculation with Glomus etunicatum (Ge, T2) and mixed inoculation with Funneliformis mosseae × Glomus etunicatum (Fm×Ge, T3) and treatment uninfected fungus (CK, T0). Four phosphorus application levels were set under the fungi application level: P2O5 0 (P0), 50 (P1), 100 (P2) and 150 (P3) mg·kg-1. There were 16 treatments for fungus phosphorus interaction. The strain was placed 5 cm below the surface of the flowerpot soil, and the phosphate fertilizer was dissolved in water and applied at one time. The results showed that the intercellular CO2 concentration (Ci) of alfalfa decreased at first and then increased with the increase of phosphorus application, except for light use efficiency (LUE) and leaf instantaneous water use efficiency (WUE), other indicators showed the opposite trend. The effect of mixed inoculation (T3) was significantly better than that of non-inoculation (T0) (p < 0.05). Pearson correlation analysis showed that Ci was significantly negatively correlated with alfalfa leaf transpiration rate (Tr) and WUE (p < 0.05), and was extremely significantly negatively correlated with other indicators (p < 0.01). The other indexes were positively correlated (p < 0.05). This may be mainly because the factors affecting plant photosynthesis are non-stomatal factors. Through the comprehensive analysis of membership function, the indexes of alfalfa under different treatments were comprehensively ranked, and the top three were: T3P2>T3P1>T1P2. Therefore, when the phosphorus treatment was 100 mg·kg-1, the mixed inoculation of Funneliformis mosseae and Glomus etunicatum had the best effect, which was conducive to improving the photosynthetic efficiency of alfalfa, increasing the dry matter yield, and improving the economic benefits of local alfalfa in Xinjiang. In future studies, the anatomical structure and photosynthetic performance of alfalfa leaves and stems should be combined to clarify the synergistic mechanism of the anatomical structure and photosynthetic performance of alfalfa.

Alfalfa leaf meal as a new protein feedstuff improves meat quality by modulating lipid metabolism and antioxidant capacity of finishing pigs.

The effects of alfalfa leaf meal (ALM) on the meat quality of finishing pigs are largely unknown. Here, we investigated the effects of ALM diet on meat quality by replacing 0%, 25%, 50%, and 75% of soybean meal in the diet of finishing pigs, respectively. The findings showed that 25% ALM diet increased the IMF, cooked meat rate, a* and antioxidant capacity of longissimus dorsi (LD), improved amino acid composition, increased MUFA content, and increased LD lipid synthesis and mRNA expression of antioxidation-related genes. At the same time, ALM diet altered serum lipid metabolism (TG, FFA). Correlation analysis showed that antioxidant capacity was positively correlated with meat quality. In addition, metabolomic analysis of LD showed that the main metabolites of 25% ALM diet altered stachydrine and l-carnitine were associated with meat quality and antioxidant capacity. In conclusion, ALM replacing 25% soybean meal diet can improve the meat quality of pigs.

Effects of Nozzle Types and Spraying Volume on the Control of Hypera postica Gyllenhal by Using An Unmanned Aerial Vehicle

In the current study, an unmanned aerial vehicle (UAV) was selected for agricultural spraying, with two nozzles, two insecticides and three spraying volumes as the spraying variables; this paper explores the impact of spraying volume on the droplet deposition in alfalfa fields. Furthermore, by comparing the control effect of spraying insecticides on alfalfa leaf weevils and the safety of pasture by UAV, the aim is to establish efficient pesticide spraying techniques for pest control in alfalfa fields, providing guidance for the green control of alfalfa leaf weevils. The effective droplet proportion of the fan-shaped nozzle (SX11001VS) was higher than that of the hollow conical nozzle (TX-VK4), and increasing the spraying volume cannot significantly improve that situation. When the spraying volume increased from 22.5 L/ha to 45.0 L/ha, the average droplet coverage, density and deposition of the two types of nozzles increased with the spraying volume. However, when the spraying volume was 30.0 L/ha and 45.0 L/ha, the average deposition of the two types of nozzles was similar. The control effect of chlorantraniliprole on alfalfa leaf weevils sprayed by using a UAV was higher than that of spinosad. There was a positive correlation between the spraying volume and the control effect, and the prevention effect of the hollow conical nozzle was better than that of the fan-shaped nozzle. The residues of chlorantraniliprole in alfalfa plants after spraying increased with the spraying volume, whether a fan-shaped nozzle or a hollow conical nozzle was used.

Influence of potassium fertilization on alfalfa leaf and stem yield, forage quality, nutrient removal, and plant health

AbstractPotassium (K) is an essential nutrient for plant growth. In K‐deficient soils, fertilization has been shown to increase herbage yield of alfalfa. The purpose of this study was to determine the effects of K fertilization on alfalfa leaf and stem yield, forage quality, nutrient removal, and plant health of a nonlodging experimental germplasm. Five alfalfa rotations had K fertilizer applied at three rates (0, 186, and 372 kg K ha−1) to soils already containing sufficient soil test K (223 mg kg−1). No overall yield differences were found related to K application rates, though K removal in total herbage increased by 30–58% with K fertilization, depending on alfalfa stand age. Leaf:stem ratios were not impacted by K fertilization. The concentrations of B, Ca, and Na decreased with K fertilization, while K concentrations increased, which may have resulted in B deficiencies. Leaf in vitro digestibility decreased significantly with the application of K. There was no benefit to crown rot disease resistance with increased K fertilization. This study supports that the addition of K to already sufficient soils does not lead to any additional economic benefits and may reduce productivity due to the reduction of B uptake and in vitro digestibility.

Multi-omics analyses reveal new insights into nutritional quality changes of alfalfa leaves during the flowering period.

High-quality alfalfa is an indispensable resource for animal husbandry and sustainable development. Its nutritional quality changes dramatically during its life cycle and, at present, no molecular mechanisms for nutrient metabolic variation in alfalfa leaves at different growth stages have been clearly reported. We have used correlation and network analyses of the alfalfa leaf metabolome, proteome, and transcriptome to explore chlorophyll, flavonoid, and amino acid content at two development stages: budding stage (BS) and full-bloom stage (FBS). A high correlation between the expression of biosynthetic genes and their metabolites revealed significant reductions in metabolite content as the plant matured from BS to FBS. l-Glutamate, the first molecule of chlorophyll biosynthesis, decreased, and the expression of HemA, which controls the transformation of glutamyl-tRNA to glutamate 1-semialdehyde, was down-regulated, leading to a reduction in leaf chlorophyll content. Flavonoids also decreased, driven at least in part by increased expression of the gene encoding CYP75B1: flavonoid 3'-monooxygenase, which catalyzes the hydroxylation of dihydroflavonols and flavonols, resulting in degradation of flavonoids. Expression of NITRILASE 2 (NIT2) and Methyltransferase B (metB), which regulate amino acid metabolism and influence the expression of genes of the glycolysis-TCA pathway, were down-regulated, causing amino acid content in alfalfa leaves to decrease at FBS. This study provides new insights into the complex regulatory network governing the content and decrease of chlorophyll, amino acids, flavonoids, and other nutrients in alfalfa leaves during maturation. These results further provide a theoretical basis for the generation of alfalfa varieties exhibiting higher nutritional quality, high-yield cultivation, and a timely harvest.

Photosensitisation and green egg yolks in laying hens caused by the feeding of ensiled alfalfa leaves

ABSTRACT 1. The present study was carried out to determine the effects of feeding ensiled alfalfa leaves (ALS) as an alternative protein source to laying hens under the terms of an organic diet. Due to the occurrence of unexpected negative health effects and undesirable egg yolk pigmentation in the test groups the trial was prematurely stopped and further analysis was conducted to evaluate the responsible substances. 2. Body weights of the test groups decreased significantly already in week 2 of the trial. Performance variables dropped. Olive green pigmented egg yolks were found in groups fed diets containing ALS. Severe comb necrosis occurred in the experimental group receiving the highest level of ALS (20%) combined with the option of free-range access and therefore natural light exposure. 3. The noxious agent found in ALS, blood serum and egg yolk was the photosensitising chlorophyll derivate pheophorbid a (PPBa), deriving from a strong depletion of chlorophyll contained in the alfalfa leaves. PPBa caused the olive-green pigmentation found in yolks and led to photosensitivity in groups with the highest level of ALS in the diet in combination with light exposure. 4. By aiming for high protein and amino acid levels, harvesting and processing have, unintentionally and initially unnoticed, led to a strong accumulation of phototoxic PPBa. From these results it is strongly advised not to include ensiled alfalfa leaves as a protein source in organic laying hen diets.