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Red clover isoflavones, particularly biochanin A and formononetin, are known for their benefits in enhancing feed efficiency and nitrogen utilization in ruminants. However, their specific effects on rumen fermentation and microbial diversity remain insufficiently explored. This study investigated the impacts of red clover isoflavones on rumen function and bacterial diversity in dairy cows, utilizing both in vivo and in vitro methodologies. In the in vivo study, 40 Holstein dairy cows were allocated to four groups, each receiving red clover isoflavones at doses of 0, 0.4, 0.8, and 1.6 g/kg. Rumen fluid was collected for analysis of fermentation parameters, enzyme activity, and microbial composition through shotgun metagenomic sequencing. Concurrently, an in vitro rumen fermentation trial was conducted to evaluate the effects of biochanin A and formononetin on urea hydrolysis. Results from the in vivo experiments showed that red clover isoflavones significantly decreased ammonia nitrogen (NH₃-N) concentrations and urease activity in the rumen ( P  < 0.05). Species level metagenomic analysis indicated a reduced abundance of proteolytic and ureolytic bacteria, such as Prevotella sp 002317355 and Treponema_D bryantii_C , with a corresponding increase in cellulolytic bacteria, including Ruminococcus_D sp 900319075 and Ruminococcus_C sp 000433635 ( P  < 0.05). The in vitro trial further demonstrated that biochanin A and formononetin significantly reduced urea decomposition rates ( P  < 0.05), with biochanin A exerting a more pronounced effect. These findings align with the observed reduction in ureolytic and proteolytic bacteria, along with an increase in cellulolytic bacteria across both trials. In conclusion, biochanin A emerged as the primary active component of red clover isoflavones, modulating urea nitrogen hydrolysis and rumen fermentation. This study substantiates previous findings and highlights the potential of red clover isoflavones for enhancing rumen microbial fermentation, offering a promising strategy for future dairy industry applications. Key points • Red clover isoflavones inhibit urease activity to decrease the abundance of urealytic bacteria. • Biochanin A reduces ammonia nitrogen and urease activity, promoting protein efficiency. • Red clover isoflavones may improve dairy cow rumen health and nitrogen utilization.

Red clover ( Trifolium pratense L.) is known for its large taproot, nitrogen fixation capabilities and production of forage high in protein and digestibility. It has the potential to strengthen temperate pastural systems against future adverse climatic events by providing higher biomass during periods of water deficit. Being outcrossing and self-incompatible, red clover is a highly heterozygous species. If evaluated and utilized correctly, this genetic diversity can be harnessed to develop productive, persistent cultivars. In this study, we selected 92 geographically diverse red clover novel germplasm populations for assessment in multi-location, multi-year field trials and for genetic diversity and genetic relationship characterization using pooled genotyping-by-sequencing (GBS). Through the development of integrated linear mixed models based on genomic, phenotypic, and environmental information we assessed variance components and genotype-by-environment (G x E) interactions for eight physiological and morphological traits. Key interactions between environmental variables and plant performance were also evaluated using a common garden site at Lincoln. We found that the genetic structure of the 92 populations was highly influenced by country of origin. The expected heterozygosity within populations ranged between 0.08 and 0.17 and varied with geographical origin. For the eight physiological and morphological traits measured there was high narrow-sense heritability ( h 2 > 0.70). The influence of environmental variables, such as mean precipitation, temperature and isothermality of the original collection locations, on plant and trait performance in the local field trials was also highlighted. Along with the identification of genes associated with these bioclimatic variables that could be used as genetic markers for selection in future breeding programs. Our study identifies the importance of diverse germplasm when adding genetic variation into breeding programs. It also identifies efficient evaluation methods and key climatic variables that should be considered when developing adaptive red clover cultivars.

This study aimed to optimize the pelleting and coating process for red clover seeds, addressing the issue of low pelleting success rates. Through theoretical analysis and experimental research, coating pan fill rate, powder supply quantity, and pelleting time were identified as key factors influencing the pelleting success rate. Single-factor experiments were conducted to investigate the effects of these parameters on the quality of red clover seed pelleting and coating. Based on these results, orthogonal trials were carried out, and response surface analysis was employed to reveal the influence patterns and interactions of each factor. The research results indicate that the factors affecting the pelleting success rate, ranked in order of importance, are coating pan fill rate, pelleting time, and powder supply quantity. Through mathematical model optimization, the optimal combination of process parameters was determined to be coating pan fill rate of 35.9%, powder supply quantity of 160.2 g, and pelleting time of 6.9 s. Under these conditions, a pelleting success rate of 94.3% was achieved in validation experiments. This study provides a theoretical foundation and practical guidance for optimizing the pelleting and coating process of red clover seeds, which is significant for improving seed coating quality and promoting red clover cultivation.

Red clover casebearer moth ( Coleophora deauratella ) (Leinig and Zeller) (Lepidoptera: Coleophoridae) is an invasive insect pest in red clover ( Trifolium pratense L.) seed production systems in North America and New Zealand. Recent discoveries of C. deauratella in Oregon and New Zealand prompted research investigating the seasonal phenology and population dynamics of C. deauratella to inform management strategies and develop a risk prediction framework to mitigate outbreak severity. We sampled 76 site-years across three geographic regions, including western (Willamette Valley) and eastern Oregon and New Zealand. An attractant-based trap network was deployed across sampled regions using a female sex pheromone to lure male moths in commercial red clover seed production fields. Remotely sensed temperature and landscape composition data were extracted for phenological and geospatial modeling. Nonlinear logistic regression was used to develop regionally explicit phenology models that predict the unimodal timing of C. deauratella flights. Molecular gut-content analyses revealed the dietary history of early-season captures and informed landscape analysis covariate selection. A spatial Bayesian generalized linear mixed model (GLMM) was developed to test landscape-level effects of landscape composition and configuration predictors on C. deauratella abundance. The spatiotemporal dominance of clover and grassland land area was positively associated with Oregon C. deauratella populations. These results can be used to forecast C. deauratella risk across space and time and advise integrated pest management practices.

Isoflavone components extracted from red clover have anti-inflammatory, antioxidant and immune boosting effects. We hypothesize that red clover isoflavones (RCIs) achieve health-promoting effects via altering the gut microbiota. A total of 48 mice (20 ± 2 g) were randomly divided into a control group, low-dose group (0.05% RCIs in feed), middle-dose group (0.1% RCIs in feed), and high-dose group (0.2% RCIs in feed) with 12 mice per group. The feeding period was 20 d. The results showed that RCIs can increase the daily gain and decrease the ratio of feed to gain in mice. The organ indexes and blood biochemical indexes of the mice in each RCI group were in the normal range, indicating that RCIs do not damage liver or kidney function. RCI supplementation increased serum immunity and altered the microbial community structure in the cecum of the mice. RCIs can increase the diversity of beneficial bacteria such as Bacteroidaceae, Muribaculaceae, and Akkermansiaceae, and reduced the pathogenic Staphylococcaceae. Therefore, supplementing the diet with RCIs results in improved growth performance and notable alterations in the cecal microbiota in mice, and has potential applications as a feed additive to improve livestock production.

In order to solve the problem of the low qualification rate of the pilling and coating of small-grain forage seeds, a vibration force field is introduced to the traditional vertical disk coating machine to promote the mixing of materials and improve the qualification rate of the pilling. Using the typical small-grain forage seed red clover as an example, we used the vibration force field after adding seed powder particles to a coating pot for the theoretical analysis of the force situation, using the discrete element software EDEM to construct a red clover seed simulation model with the coefficient of discretization as the evaluation index. We studied the effects of the rotational speed of the coating pot, the vibration frequency of the pot, the amplitude of the vibration of the pot, and the other operating parameters of the pot on the uniformity of the seed powder mixing, with the pelletization of the pass rate as the physical evaluation standard, using a one-way test to study the effect of operating parameters on the pelletization pass rate. We used the qualified rate as the physical evaluation standard, through a single-factor test, to study the influence of the working parameters on the qualification rate of the pilling. The results show that the simulation test evaluation index of the discrete coefficient and the physical test evaluation index of the pilling qualification rate with the change rule of the working parameters is consistent with the discrete coefficient, and can be used as an indirect evaluation index of the quality of pilling. To further determine the optimal combination of working parameters, a quadratic regression orthogonal design test was conducted with the discrete coefficients as the evaluation index, and the second-order regression equations of the red clover seeds were established and analyzed by ANOVA using Design-Expert software. The study shows that, when the rotational speed of the coating pot is 307.204 rpm, the vibration frequency is 2.526 Hz, and the vibration amplitude of the coating pot is 5.843 mm, the predicted coefficient of dispersion at this time is 8.1%. Simulation using the best combination of parameters to obtain the average value of the dispersion coefficient of 8.4%, relative to the predicted value of 3.7%, indicated that the optimization of the experimental regression model is accurate, and the results obtained for the vibration of small seeds under the conditions of the design of the pellet granulation coating machine and the optimization of the pelletization coating process parameters have a certain degree of reference significance.

This study examined the effects of chopping or chopping + blender maceration of red clover or lucerne on pre-fermented juice (PFJ) and determined the effects of PFJs on the quality of red clover silage or lucerne silage. The PFJs from chopping red clover (PFJ-RC) or lucerne (PFJ-LC) had a higher lactic acid bacteria (LAB) count than that from chopping + blender maceration (p < 0.05) and were used as additives. Compared with the Control of both silages, adding PFJ increased LAB, lactic acid (LA), and in vitro digestibility of dry matter (IVDMD) (p < 0.05), while pH, acetic acid (AA), and ammonia nitrogen/total nitrogen (NH3-N/TN) were decreased (p < 0.05). For red clover silages, the PFJ-RC treatment contained the greatest LAB and LA and the lowest pH and NH3-N/TN among treatments (p < 0.05); similar results were observed in PFJ-LC treatment for lucerne silages (p < 0.05). The IVDMD of both silages correlated negatively with pH, AA, and NH3-N/TN and positively with LA (p < 0.05). Overall, chopping alone was a better method for preparing PFJ. Adding PFJ at ensiling increased LA and decreased the pH, AA, and NH3-N/TN of both silages. Ensiling lucerne or red clover with PFJ from the ensiling material had a more positive effect on the fermentation parameters mentioned above. Satisfactory fermentation parameters detected in the present study contributed to improving the IVDMD of both silages.

To locate quantitative trait loci (QTLs) resistant to powdery mildew (Erysiphe), a major disease of red clover, 151 red clover hybrids from crosses between genotypes from a resistant (Ganong PR1) and a susceptible (Minshan) cultivar were used as the mapping population. A genetic linkage map of red clover was constructed, and 30 plants with disease severity levels on a scale of one to nine, were used to locate QTLs correlated with resistance to powdery mildew. A genetic map that included seven linkage groups (LG1–7) was constructed based on 149 amplified fragment length polymorphic markers. QTLs associated with the resistance to powdery mildew were located using the multiple interval mapping method. The linkage map covered 640.5 cM, with an average distance of 4.3 cM between markers. LG1 was the largest (140.6 cM), with the average distance between markers, while LG4 was the smallest (55.2 cM) but had the highest marker density, with a 1.8-cM average distance between markers. Five QTLs (qrp-1–5) correlated with resistance to powdery mildew were located on LG4 and LG5. The phenotypic variance explained by these five QTLs ranged from 29% to 90%, with qrp-1 having the greatest contribution. Therefore, qrp-1 was identified as the main candidate gene controlling resistance to powdery mildew in red clover. This study provides a foundation in red clover for locating genes, transgenesis and marker-assisted breeding.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.

The family Partitiviridae includes plant ( Alphacryptovirus and Betacryptovirus ), fungal ( Partitivirus ) and protozoan ( Cryspovirus ) viruses with bisegmented dsRNA genomes and isometric virions. Cryptic viruses commonly occur in different plant species without causing any symptoms. So far, numerous sequences have been determined for viruses of the genus Alphacryptovirus , but no sequence is available for any assigned member of the genus Betacryptovirus . Following extraction, cloning and sequence analysis of double-stranded RNA in this study, we report the molecular properties of three assigned members of the genus Betacryptovirus, white clover cryptic virus 2, red clover cryptic virus 2 and hop trefoil cryptic virus 2, and two new putative betacryptoviruses found in crimson clover and dill. Betacryptoviruses share sequence motifs with members of the genus Partitivirus . In phylogenetic analyses, members of the genus Betacryptovirus formed a new sub-cluster within the clusters containing members of the genus Partitivirus. Our results provide evidence for a distinct evolutionary lineage of dsRNA viruses of plants and fungi.