The Physiological Basis of Alfalfa Plant Height Establishment.

Plant height plays an important role in crop yield, product quality, and cultivation management. However, the physiological mechanisms that regulate the establishment of plant height in alfalfa plants remain unclear. Herein, we measured plant height traits, leaf characteristics, photosynthetic physiology, cell wall composition, and endogenous hormone contents of tall- and short-stalked alfalfa materials at different reproductive periods. We analyzed the physiology responsible for differences in plant height. The results demonstrated that the number of internodes in tall- and short-stalked alfalfa materials tended to converge with the advancement of the fertility period. Meanwhile, the average internode length (IL) of tall-stalked materials was significantly higher than that of short-stalked materials at different fertility periods, with internode length identified as the main trait determining the differences in alfalfa plant height. Leaf characteristics, which are closely related to photosynthetic capacity, are crucial energy sources supporting the expression of plant height traits, and we found that an increase in the number of leaves contributed to a proportional increase in plant height. Additionally, a significant positive correlation was observed between plant height and leaf dry weight per plant during the branching and early flowering stages of alfalfa. The leaves of alfalfa affect plant height through photosynthesis, with the budding stage identified as the key period for efficient light energy utilization. Plant height at the budding stage showed a significant positive correlation with soluble sugar (SS) content and a significant negative correlation with intercellular CO2 concentration. Moreover, we found that alfalfa plant height was significantly correlated with the contents of indole-3-acetic acid in stem tips (SIAA), gibberellin A3 in leaves (LGA3), zeatin in stem tips (SZT), and abscisic acid in leaves (LABA). Further investigation revealed that SS, SIAA, and LGA3 contents were important physiological indicators affecting alfalfa plant height. This study provides a theoretical basis for understanding the formation of alfalfa plant height traits and for genetic improvement studies.

Emerging role of the crosstalk between gut microbiota and liver metabolome of subterranean herbivores in response to toxic plants.

Plant secondary metabolites (PSMs) are a defense mechanism against herbivores, which in turn use detoxification metabolism to process ingested and absorbed PSMs. The feeding environment can cause changes in liver metabolism patterns and the gut microbiota. Here, we compared gut microbiota and liver metabolome to investigate the response mechanism of plateau zokors (Eospalax baileyi) to toxic plant Stellera chamaejasme (SC) in non-SC and SC grassland (-SCG and +SCG). Our results indicated that exposure to SC in the -SCG population increased liver inflammatory markers including prostaglandin (PG) in the Arachidonic acid pathway, while exposure to SC in the +SCG population exhibited a significant downregulation of PGs. Secondary bile acids were significantly downregulated in +SCG plateau zokors after SC treatment. Of note, the microbial taxa Veillonella in the -SCG group was significantly correlated with liver inflammation markers, while Clostridium innocum in the +SCG group had a significant positive correlation with secondary bile acids. The increase in bile acids and PGs can lead to liver inflammatory reactions, suggesting that +SCG plateau zokors may mitigate the toxicity of SC plants by reducing liver inflammatory markers including PGs and secondary bile acids, thereby avoiding liver damage. This provides new insight into mechanisms of toxicity by PSMs and counter-mechanisms for toxin tolerance by herbivores.

Anabolic metabolism of autotoxic substance coumarins in plants.

Background: Autotoxicity is an intraspecific manifestation of allelopathy in plant species. The specialized metabolites and their derivatives that cause intraspecific allelopathic inhibition in the plant are known as autotoxic substances. Consequently, autotoxic substances production seriously affects the renewal and stability of ecological communities. Methods: This article systematically summarizes the types of autotoxic substances present in different plants. They mainly include phenolic compounds, terpenoids, and nitrogenous organic compounds. Phenolic coumarins are the main autotoxic substances in many plants. Therefore, we also discuss differences in coumarin types and content among plant varieties, developmental stages, and tissue parts, as well as their mechanisms of autotoxicity. In addition, we review the metabolic pathways involved in coumarin biosynthesis, the key enzymes, genes, and transcription factors, as well as factors affecting coumarin biosynthesis. Results: Coumarin biosynthesis involves three stages: (1) the formation of the coumarin nucleus; (2) acylation, hydroxylation, and cyclization; (3) structural modification. The key enzymes involved in the coumarin nuclear formation stage include PAL, C4H, 4CL, HCT, CAOMT, COSY, F6'H, and CCoAOMT1, and the key genes involved include BGA, CYP450 and MDR, among others. Ortho-hydroxylation is a key step in coumarin biosynthesis and PS, COSY and S8H are the key enzymes involved in this process. Finally, UGTs are responsible for the glycosylation modification of coumarins, and the MaUGT gene may therefore be involved in coumarin biosynthesis. Conclusion: It is important to elucidate the autotoxicity and anabolic mechanisms of coumarins to create new germplasms that produce fewer autotoxic substances.

The antennal transcriptome analysis and characterizations of odorant-binding proteins in Megachile saussurei (Hymenoptera, Megachilidae).

BACKGROUND: Odorant-binding proteins (OBPs) are essential in insect's daily behaviors mediated by olfactory perception. Megachile saussurei Radoszkowski (Hymenoptera, Megachilidae) is a principal insect pollinating alfalfa (Medicago sativa) in Northwestern China. The olfactory function have been less conducted, which provides a lot of possibilities for our research. RESULTS: Our results showed that 20 OBPs were identified in total. Multiple sequence alignment analysis indicated MsauOBPs were highly conserved with a 6-cysteine motif pattern and all belonged to the classic subfamily, coding 113-196 amino acids and sharing 41.32%-99.12% amino acid identity with known OBPs of other bees. Phylogenetic analysis indicated there were certain homologies existed among MsauOBPs and most sequences were clustered with that of Osmia cornuta (Hymenoptera, Megachilidae). Expression analysis showed the identified OBPs were mostly enriched in antennae instead of other four body parts, especially the MsauOBP2, MsauOBP3, MsauOBP4, MsauOBP8, MsauOBP11 and MsauOBP17, in which the MsauOBP2, MsauOBP4 and MsauOBP8 presented obvious tissue-biased expression pattern. Molecular docking results indicated MsauOBP4 might be the most significant protein in recognizing alfalfa flower volatile 3-Octanone, while MsauOBP13 might be the most crucial protein identifying (Z)-3-hexenyl acetate. It was also found the lysine was a momentous hydrophilic amino acid in docking simulations. CONCLUSION: In this study, we identified and analyzed 20 OBPs of M. saussurei. The certain homology existed among these OBPs, while some degree of divergence could also be noticed, indicating the complex functions that different MsauOBPs performed. Besides, the M. saussurei and Osmia cornuta were very likely to share similar physiological functions as most of their OBPs were clustered together. MsauOBP4 might be the key protein in recognizing 3-Octanone, while MsauOBP13 might be the key protein in binding (Z)-3-hexenyl acetate. These two proteins might contribute to the alfalfa-locating during the pollination process. The relevant results may help determine the highly specific and effective attractants for M. saussurei in alfalfa pollination and reveal the molecular mechanism of odor-evoked pollinating behavior between these two species.

Enriched endogenous free Spd and Spm in alfalfa (Medicago sativa L.) under drought stress enhance drought tolerance by inhibiting H2O2 production to increase antioxidant enzyme activity.

Drought stress is a major factor limiting agricultural development, and exogenous polyamines (PAs) can increase plant drought resistance by enhancing antioxidant activity, but few studies have examined whether endogenous PAs enhance the plant antioxidant system. Here, to investigate the effects of endogenous PAs on the antioxidant system of alfalfa under drought stress and the underlying mechanisms, two alfalfa cultivars, Longzhong (drought resistant) and Gannong No. 3 (drought sensitive), were used as test materials, and their seedlings were treated with polyethylene glycol (PEG-6000) for 8 days at -1.2 MPa to simulate drought stress. The levels of free PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)], hydrogen peroxide (H2O2), malondialdehyde (MDA), key PA metabolism enzyme [arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), polyamine oxidase (PAO), and diamine oxidase (DAO)] activities, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD)] activities were measured. These physiological indicators were used for correlation analysis to investigate the relationship between PA metabolism and the antioxidant enzyme system. The results showed that PA synthesis in alfalfa under drought stress was dominated by the ADC pathway. Spd and Spm played an important role in improving drought tolerance. The high levels of ADC and SAMDC activities were facilitated by the conversion of Put to Spd and Spm. H2O2 generation by oxidative decomposition of PAs was mainly dependent on the oxidative decomposition of DAO but not PAO. Low DAO activity favored low H2O2 production. Spd, Spm, ADC, ODC and SAMDC were positively correlated with the antioxidant enzymes SOD, CAT and POD in both cultivars under drought. Therefore, we concluded that high ADC and SAMDC activities in alfalfa promoted the conversion of Put to Spd and Spm, leading to high accumulation of Spd and Spm and low Put accumulation. Low Put levels led to low H2O2 production through low DAO activity, and low H2O2 levels induced the expression of antioxidant enzyme-encoding genes to improve antioxidant enzyme activity and reduce MDA accumulation and thereby enhanced drought resistance in alfalfa.

The impact of fertilization on ammonia-oxidizing bacteria and comammox Nitrospira communities and the subsequent effect on N2O emission and maize yield in a semi-arid region.

The control of nitrous oxide (N2O) emissions through nitrification and the optimization of maize yield are important in agricultural systems. However, within the semi-arid region, the impact of fertilization on the function of nitrification communities and its connection with N2O emissions in the rhizosphere soil is still unclear. Our study investigates the influence of fertilization treatments on the communities of ammonia-oxidizing bacteria (AOB) and the complete ammonia oxidizers of the Nitrospira known as comammox (CAOB) in a maize agroecosystem. Nitrous oxide production, potential nitrification activity (PNA), maize yield, and nitrogen use efficiency (NUE) were determined for the same samples. The fertilizer treatments included a control group without fertilization (NA), inorganic fertilizer (CF), organic fertilizer (SM), combined inorganic and organic fertilizer (SC), and maize straw (MS). The SC treatment indicated a lower cumulative N2O emission than the CF treatment in the 2020 and 2021 cropping seasons. The AOB community under the CF, MS, and SM treatments was predominantly composed of Nitrosospira cluster 3b, while the SC treatment was associated with the comammox Nitrospira clade A.1 lineage, related to key species such as Ca. Nitrospira inopinata and Ca. Nitrospira nitrificans. Network analysis demonstrated a positive potential for competitive interaction between hub taxonomy and distinct keystone taxa among AOB and comammox Nitrospira nitrifiers. The structural equation model further revealed a significant positive association between AOB nitrifiers and N2O emission, PNA, soil pH, SOC, NO3--N, and DON under organic fertilization. The keystone taxa in the comammox Nitrospira nitrifier and network Module II exhibited a positive correlation with maize productivity and NUE, likely due to their functional activities stimulated by the SC treatment. It is noteworthy that the AOB community played a more significant role in driving nitrification compared to the composition of comammox Nitrospira. Collectively, combined inorganic and organic fertilizer (SC) treatment exhibits high potential for reducing N2O emissions, enhancing maize productivity, increasing NUE, and increasing the sustainability of the nitrogen dynamics of maize agroecosystems in the semi-arid Loess Plateau.

Study on the Key Autotoxic Substances of Alfalfa and Their Effects.

Alfalfa is a leguminous plant with strong autotoxicity, which seriously affects regeneration and stability. In order to clarify the relationship between the key autotoxic substances and autotoxicity of alfalfa, this experiment determined the content of phenolic autotoxic substances in different varieties of alfalfa and the effect of different concentrations of alfalfa extracts on seed germination, seedling growth and physiology. The results showed that the content of single autotoxic substances in the eight alfalfa varieties was highest for total coumarin. The variety with the highest total coumarin content was "LZ", and the lowest content was "656". Principal component analysis of the autotoxicity of eight alfalfa varieties revealed that the variety with the strongest autotoxicity was "LZ" and the weakest was "656". After treatment with extracts, the germination potential, germination rate, germination index and vigor index of 656 were higher than those of LZ, and the seeds of LZ and 656 did not germinate when the concentration was higher than C0.025 and C0.05, respectively. Compared with LZ, 656 had stronger osmotic regulation and antioxidant capacity, while the degree of membrane lipid peroxidation and ROS accumulation were lower. Further correlation analysis between the autotoxic substance content and autotoxicity observed that the content of total coumarin and autotoxic substances showed a significant positive association with autotoxicity (p < 0.01), and the total coumarin content showed a significant positive correlation with the content of autotoxic substances (p < 0.05). The total coumarin content is the major contributor to autotoxicity, and the higher the coumarin content, the higher the autotoxic substance content and the stronger the autotoxicity. Eight alfalfa varieties were systematically clustered on the basis of total coumarin content and autotoxicity, and the high-autotoxic alfalfa variety "LZ" and low-autotoxic alfalfa variety "656" were screened.

Nitrogen fertilization promoted microbial growth and N2O emissions by increasing the abundance of nirS and nosZ denitrifiers in semiarid maize field.

Nitrous oxide (N2O) emissions are a major source of gaseous nitrogen loss, causing environmental pollution. The low organic content in the Loess Plateau region, coupled with the high fertilizer demand of maize, further exacerbates these N losses. N fertilizers play a primary role in N2O emissions by influencing soil denitrifying bacteria, however, the underlying microbial mechanisms that contribute to N2O emissions have not been fully explored. Therefore, the research aimed to gain insights into the intricate relationships between N fertilization, soil denitrification, N2O emissions, potential denitrification activity (PDA), and maize nitrogen use efficiency (NUE) in semi-arid regions. Four nitrogen (N) fertilizer rates, namely N0, N1, N2, and N3 (representing 0, 100, 200, and 300 kg ha-1 yr.-1, respectively) were applied to maize field. The cumulative N2O emissions were 32 and 33% higher under N2 and 37 and 39% higher under N3 in the 2020 and 2021, respectively, than the N0 treatment. N fertilization rates impacted the abundance, composition, and network of soil denitrifying communities (nirS and nosZ) in the bulk and rhizosphere soil. Additionally, within the nirS community, the genera Cupriavidus and Rhodanobacter were associated with N2O emissions. Conversely, in the nosZ denitrifier, the genera Azospirillum, Mesorhizobium, and Microvirga in the bulk and rhizosphere soil reduced N2O emissions. Further analysis using both random forest and structural equation model (SEM) revealed that specific soil properties (pH, NO3--N, SOC, SWC, and DON), and the presence of nirS-harboring denitrification, were positively associated with PDA activities, respectively, and exhibited a significant association to N2O emissions and PDA activities but expressed a negative effect on maize NUE. However, nosZ-harboring denitrification showed an opposite trend, suggesting different effects on these variables. Our findings suggest that N fertilization promoted microbial growth and N2O emissions by increasing the abundance of nirS and nosZ denitrifiers and altering the composition of their communities. This study provides new insights into the relationships among soil microbiome, maize productivity, NUE, and soil N2O emissions in semi-arid regions.

Comparative transcriptomes reveal geographic differences in the ability of the liver of plateau zokors (Eospalax baileyi) to respond and adapt to toxic plants.

BACKGROUND: Environmental changes are expected to intensify in the future. The invasion of toxic plants under environmental changes may change herbivore feeding environments. Herbivores living long-term in toxic plant-feeding environments will inevitably ingest plant secondary metabolites (PSMs), and under different feeding environments are likely to have unique protection mechanisms that support improved adaptation to PSMs in their habitat. We aimed to compare different subterranean herbivore population responses and adaptations to toxic plants to unveil their feeding challenges. RESULTS: Here, we investigated the adaptive capacity of the liver in two geographically separated populations of plateau zokors (Eospalax baileyi) before and after exposure to the toxic plant Stellera chamaejasme (SC), at the organ, biochemical, and transcriptomic levels. The results showed no significant liver granules or inflammatory reactions in the Tianzhu (TZ) population after the SC treatment. The transaminase level in the TZ population was significantly lower than that in the Luqu population. Transcriptome analysis revealed that the TZ population exhibited interactions with other detoxification metabolic pathways by oxytocin pathway-associated genes, including diacylglycerol lipase alpha (Dagla), calcium/calmodulin dependent protein kinase II Alpha (Camk2a), and CD38 molecule (Cd38). The phase II process of liver drug metabolism increased to promote the rate of metabolism. We found that alternative splicing (AS) and the expression of the cyclin D (Ccnd1) gene interact-a TZ population hallmark-reduced liver inflammatory responses. CONCLUSION: Our study supports the detoxification limitation hypothesis that differences in liver detoxification metabolism gene expression and AS are potential factors in herbivore adaptation to PSMs and may be a strategy of different herbivore populations to improve toxic plant adaptability.

Diverse Virulence Attributes of Pantoea alfalfae sp. nov. CQ10 Responsible for Bacterial Leaf Blight in Alfalfa Revealed by Genomic Analysis.

Alfalfa is widely grown worldwide for its excellent nutritional value. Pantoea species living in alfalfa seeds can easily spread over great distances with frequent trade. However, the pathogenic properties of this dangerous hitchhiker on alfalfa have not been evaluated. Here, we identified the taxonomic status of Pantoea strain CQ10 isolated from the interior of alfalfa seeds based on the whole genome sequence. The diverse virulence attributes of strain CQ10 during host infection were characterized through pathogenicity assays and functional and genomic analyses. We report that strain CQ10 belongs to a novel species in the genus Pantoea, which was phylogenetically close to Pantoea vagans and Pantoea agglomerans. Strain CQ10 caused bacterial leaf blight of alfalfa after inoculation from the roots. We found that strain CQ10 possesses a large number of pathogenic genes involved in shaping the virulence properties during bacteria-host interactions, including motility, biofilm, type VI secretion system, and nutrient acquisition. Compared with P. vagans and P. agglomerans, the unique virulence factors of strain CQ10 were mainly involved in motility and biofilm, which were confirmed by in vitro experiments. Taken together, our results suggest that strain CQ10 is the first Pantoea species to infect alfalfa, and it possesses diverse virulence attributes among which motility and biofilm may be the best weapons.

Exogenous application of salicylic acid improves freezing stress tolerance in alfalfa.

Freezing stress is one of the most detrimental environmental factors that can seriously impact the growth, development, and distribution of alfalfa (Medicago sativa L.). Exogenous salicylic acid (SA) has been revealed as a cost-effective method of improving defense against freezing stress due to its predominant role in biotic and abiotic stress resistance. However, how the molecular mechanisms of SA improve freezing stress resistance in alfalfa is still unclear. Therefore, in this study, we used leaf samples of alfalfa seedlings pretreatment with 200 µM and 0 µM SA, which were exposed to freezing stress (-10°C) for 0, 0.5, 1, and 2h and allowed to recover at normal temperature in a growth chamber for 2 days, after which we detect the changes in the phenotypical, physiological, hormone content, and performed a transcriptome analysis to explain SA influence alfalfa in freezing stress. The results demonstrated that exogenous SA could improve the accumulation of free SA in alfalfa leaves primarily through the phenylalanine ammonia-lyase pathway. Moreover, the results of transcriptome analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway-plant play a critical role in SA alleviating freezing stress. In addition, the weighted gene co-expression network analysis (WGCNA) found that MPK3, MPK9, WRKY22 (downstream target gene of MPK3), and TGACG-binding factor 1 (TGA1) are candidate hub genes involved in freezing stress defense, all of which are involved in the SA signaling pathway. Therefore, we conclude that SA could possibly induce MPK3 to regulate WRKY22 to participate in freezing stress to induced gene expression related to SA signaling pathway (NPR1-dependent pathway and NPR1-independent pathway), including the genes of non-expresser of pathogenesis-related gene 1 (NPR1), TGA1, pathogenesis-related 1 (PR1), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and heat shock protein (HSP). This enhanced the production of antioxidant enzymes such as SOD, POD, and APX, which increases the freezing stress tolerance of alfalfa plants.

Potential Effect of DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on Alleviating the Autotoxic Coumarin Stress in Alfalfa (Medicago sativa) Seedlings.

The allelopathic theory has garnered considerable attention in the field of agricultural production for its efficient plant protection, rapid crop yield increase, and scientific establishment of the crop rotation system. To study the effects of the main maize allelochemical DIMBOA (2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one) on the growth and development of alfalfa under autotoxic coumarin stress, we treated alfalfa seedlings with DIMBOA under coumarin stress and non-stress conditions in this study. Results show that 0.0342 mM coumarin significantly inhibited alfalfa seed germination percentage(Gp), germination potential(GP), radicle length, germ length, seeding height, and simple viability index (SVI), with decreases of 37.29%, 59.91%, 7.60%, 30.90%, 13.27%, and 45.70%, respectively. An amount of 0.6 mM DIMBOA could promote alfalfa seed Gp, GP, radicle length, germ length, seeding height, dry fresh ratio, and SVI, with increases of 12.38%, 23.91%, 48.69%, 48.65%, 48.68%, 295.12%, and 67.17%, respectively. However, the addition of DIMBOA under conditions of coumarin stress could effectively alleviate coumarin effects on alfalfa seedlings. Coumarin + DIMBOA treatment for 24 h mainly decreased reactive oxygen species (ROSs) and malondialdehyde (MDA) as well as soluble protein and soluble sugar, increasing some antioxidant enzyme activities and antioxidant content to alleviate the oxidative damage of alfalfa caused by coumarin stress. Administration of treatment for 72 h significantly promoted the morphological development of alfalfa seeding roots. Administration of treatment for 96 h significantly enhanced the photosynthetic capacity of alfalfa seedlings. The results of principal component analysis demonstrated that chlorophyll b(Chl b)and net photosynthetic rate(Pn) were the key indicators for coumarin + DIMBOA treatment to promote photosynthesis in alfalfa seedlings. Additionally, root length, mean root diameter, and root volume were the key indicators of root growth and development. Coumarin + DIMBOA treatment primarily increased catalase(CAT), peroxidase (POD), and ascorbate peroxidase (APX) activity and antioxidants(ASA) while reducing MDA and superoxide anion radical(O2•-). This study strongly suggested that DIMBOA can effectively improve the tolerance of alfalfa seedlings to coumarin stress through a combination of effects on root morphology, photosynthesis, and physiological indicators.

Cross-Breeding Improvement and Performance Analysis of Dominant Production Traits in Grazing-Type Alfalfa (Medicago sativa L.).

Rhizome-rooted Medicago sativa L. "Qingshui" is an excellent germplasm for establishing grazing and ecological grasslands but inferior in yield, in which both high production and ecological values can be achieved by cross-breeding. We have obtained valuable rhizome-rooted hybrid strains (RSA-01, RSA-02, and RSA-03) by crossing of Qingshui and the high-yielding Medicago sativa L. "WL168." In this study, the Qingshui plants with low production performance were crossed for improvement, and progenies with better production and higher quality than those of Qingshui were selected. The results reveal that the branch number, crude protein (CP) content, and relative feed value (RFV) of RSA-01; the stem thickness, CP content, and ether extract (EE) content of RSA-02; and the plant height, stem thickness, branch number, and dry hay yield of RSA-03 were higher than those of Qingshui. Except for the leaf/stem ratio and plant height of RSA-01, leaf/stem ratio of RSA-02, and plant height of RSA-03, the coefficient of variation (CV) of yield traits of the hybrid strains was lower than those of Qingshui, ranging from 0.1% to 4.28%. In addition to the lignin and acid detergent fiber content of RSA-01 as well as EE content of RSA-02 and RSA-03, the CV of the nutritional traits of the hybrid strains was low, ranging from 0.60% to 3.43%. The tested samples were ranked as follows based on yield performance and nutritional values: WL168 > RSA - 03 > RSA - 01 > RSA - 02 > Qingshui and RSA - 01 > WL168 > RSA - 03 > Qingshui > RSA - 02, respectively. Compared with parental Qingshui, RSA-01, RSA-02, and RSA-03 show better yield performance; meanwhile, RSA-01 and RSA-03 had higher nutritional traits. RSA-01 shows heterosis in branch number, CP content, and RFV; RSA-02 shows heterosis in stem thickness and RSA-03 in plant height, stem thickness, branch number, fresh yield, dry hay yield, and CP content. Notably, the low production performance of Qingshui was improved after crossing it with WL168, substantially resulting in an abundant rhizome-rooted germplasm resource for the establishment of grazing grasslands.

Ratio of carbon and nitrogen in fertilizer treatment drives distinct rhizosphere microbial community composition and co-occurrence networks.

Fertilization is the main strategy to accelerate vegetation restoration and improve the rhizosphere microbial community in the northeast China. However, the responses of rhizosphere microbial community structure, specific microbial community and symbiotic pattern to manure fertilization in grassland (alfalfa only) are not well clear. In this study, the variation of bacterial community structures in R_Manure (extracted liquid of fermented cow manure), E_Manure (extracted residue of fermented cow manure), F_Manure (full fermented cow manure), and Control (without fermented cow manure) collected from the rhizosphere microbial community of alfalfa were analyzed by the application of an Illumina HiSeq high-throughput sequencing technique. A total of 62,862 microbial operational taxonomic units (OTUs) were detected and derived from 21 phyla of known bacteria. The dominant bacteria in the rhizosphere include Proteobacteria (70.20%), Acidobacteria (1.24%), Actinobacteria (2.11%), Bacteroidetes (6.15%), Firmicutes (4.21%), and Chlorofexi (2.13%) accounting for 86% of the dominant phyla in all treatments. At the genus level, the dominant genus include NB1-j, Lysobacter, Alphaproteobacteria, Subgroup_6, Actinomarinales, Saccharimonadales, Aneurinibacillus, MO-CFX2, SBR1031, Caldilineaceae, and so on with the average relative abundance (RA) of 1.76%, 1.52%, 1.30%, 1.24%, 1.61%, 2.39%, 1.36%, 1.42%, 1.27%, and 1.03%, respectively. Bacterial diversities and community structures were significantly differentiated by different treatments of fertilization. The results of community structure composition showed that R_Manure treatment significantly increased the population abundance of Firmicutes, Chlorofexi, and Patescibacteria by 34.32%, 6.85%, and 2.70%, and decreased the population abundance of Proteobacteria and Actinobacteria by 16.83% and 1.04%, respectively. In addition, it showed that all treatments significantly resulted in an increase or decrease at the genus level. R_Manure had the higher richness and diversity of the bacterial community, with the greatest topology attributes of the co-occurrence networks. Through the analysis of the molecular ecological network (MENA), the co-occurrence networks had a shorter average path distance and diameter in R_Manure than in others, implying more stability to environmental changes. Redundancy analysis (RDA) showed that the ratio of carbon and nitrogen (C/N) was the main factor affecting rhizosphere microbial community composition while driving distinct rhizosphere bacterial community and its co-occurrence networks. The R_Manure associated with more C/N had relatively complex microbial co-occurrence network with a large number of nodes and edges, while the microbial network of others associated with less C/N had fewer taxa with loose mutual interactions. These results suggested that organic fertilizer with high C/N can regulate the rhizosphere microorganism, while high C/N can determine bacterial community structures, specific bacterial taxa, and their relationships with the nodule size of alfalfa. These significant changes can be used to evaluate soil fertility and fertilizer management in the artificial grassland system, while the potential biological indicators of the rhizosphere microbial community will play an important role in future eco-agriculture.

Seed-Borne Erwinia persicina Affects the Growth and Physiology of Alfalfa (Medicago sativa L.).

Seed-borne Erwinia persicina can be transmitted globally via alfalfa (Medicago sativa L.) seed trade, but there is limited information about the impact of this plant-pathogenic bacterium on alfalfa plants. In this study, strain Cp2, isolated from alfalfa seeds, was confirmed by whole-genome sequencing to belong to E. persicina. Subsequently, the effects of Cp2 on alfalfa growth and physiology were evaluated by constructing a rhizosphere infection model. Strain Cp2 had a strong inhibitory effect on the elongation and growth of alfalfa roots, which was very unfavorable to these perennial plants. Furthermore, an increased number of leaf spots and yellowing symptoms were observed in plants of the Cp2 group from day 10 to day 21 and the strain Cp2 was re-isolated from these leaves. Correlation between growth and photosynthetic parameters was analyzed and the significant decreases in fresh weight and root and plant lengths in the Cp2 group were related to the marked reduction of chlorophyll b, carotenoid, transpiration rate, and stomatal conductance of leaves (r > 0.75). In addition, nine physiological indicators of root, stem, and leaf were measured in the plants 21 days after treatment with Cp2. The physiological response of root and leaf to Cp2 treatment was stronger than that of stem. The physiological indicators with the greatest response to Cp2 infection were further explored through principal component analysis, and superoxide dismutase, peroxidase, ascorbate peroxidase, and soluble protein showed the greatest changes in roots, stems, and leaves (P < 0.001). Among tissues, the commonality was the change of soluble protein. Therefore, soluble protein is speculated to be a physiological marker during alfalfa-E. persicina interactions. These findings indicate that once E. persicina spreads from alfalfa seeds to the rhizosphere, it can invade alfalfa roots and cause disease. This study demonstrates that this plant pathogenic bacterium may be a potential threat to new environment when they spread via seed trade and these "dangerous hitchhikers" warrant further attention, especially in the study of bacterial diseases in pasture-based production systems.

Plateau zokors (Eospalax baileyi) respond to secondary metabolites from the roots of Stellera chamaejasme by enhancing hepatic inflammatory factors and metabolic pathway genes.

Herbivores rarely consume toxic plants. An increase in the proportion of toxic plant secondary metabolites (PSMs) in poisonous plants can promote detoxification and related metabolic capacity of animals. Poisonous plants with thick taproots like Stellera chamaejasme (SC) are important stored food for the plateau zokor (Eospalax baileyi) during the winter and promote the development of detoxification mechanisms in this animal. In this study, plateau zokors were administered gavages of 0.2, 1.05, and 2.10 ml/kg SC water extracts. Serum samples were collected from plateau zokors to measure the levels of transaminases and oxidative stress. Transcriptome analysis was conducted to evaluate the differential genes of multiple metabolic pathways to investigate the relationship between the physiological processes and metabolic adaptation capacity of these animals in response to SC. After SC administration, plateau zokors showed significant hepatic granular degeneration and inflammatory reactions in the liver and aspartate aminotransferase, alanine aminotransferase, and malondialdehyde levels increased in a dose-dependent manner. Further, differential expression was also found in the plateau zokor livers, with most enrichment in inflammation and detoxification metabolism pathways. The metabolic adaptation responses in P450 xenobiotic clearance, bile secretion, and pancreatic secretion (Gusb, Hmgcr, Gstm1, Gstp1, and Eobag004630005095) were verified by mRNA network analysis as key factors related to the mechanism. Plateau zokors respond to SC PSMs through changes in liver physiology, biochemistry, and genes in multiple metabolic pathways, validating our hypothesis that plateau zokors can metabolize PSMs when they ingest toxic plants.

Comparative Transcriptome Analysis Reveals New Insight of Alfalfa (Medicago sativa L.) Cultivars in Response to Abrupt Freezing Stress.

Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa (Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance "Gannong NO.3" and freezing-sensitive "WL326GZ" exposure to -10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in "Gannong NO.3" were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in "WL326GZ" were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca2+ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.

Optimization of the Extraction of Total Phenols from Medicago sativa and Its Antioxidant Capacity.

The phenolic compounds from alfalfa (Medicago sativa L.) are used as antioxidants and in native medicine. They play an indispensable role in defense and signal transduction of the plant under stress conditions. This exploration of the optimal extraction parameters of the total phenols from alfalfa by using ultrasonic-assisted extraction (UAE) was aimed at providing a theoretical basis for better utilization of the total phenols in alfalfa. In this study, the effects of solvent volume fraction (A), extraction time (B), solid-liquid ratio (C) and extraction temperature (D) on the total phenols content and the total antioxidant capacity of Medicago sativa L. WL363HQ after thrips infestation were determined through single-factor experiments. Additionally, the extraction conditions of total phenols were optimized by using Box-Behnken design (BBD) of response surface methodology (RSM). The results showed that the proposed model had a good fitting degree for total phenols extraction (R2 =0.9564). The total phenols extraction from WL363HQ had significant relationship with solid-liquid ratio (C) and extraction temperature (D) (P<0.05). The influence levels of the four factors on total phenols extraction were as follows: extraction temperature (D) > solid-liquid ratio (C)>acetone volume fraction (A)>extraction time (B). The optimum extraction conditions of total phenols from WL363HQ were 50 % acetone, solid-liquid ratio of 1 : 20 (g/mL), extraction time of 45 min and extraction temperature of 60 °C. The corresponding content and total antioxidant capacity under the optimized conditions were 15.76 mg g-1 and 28.79 µmol Trolox g-1 . These results provided a new extraction method of total phenols from alfalfa.

Characterization and expression analysis of tandemly duplicated nicotinic acetylcholine receptors in pearl oysters after stimulation of pathogen-related molecular patterns.

Nicotinic acetylcholine receptors (nAChRs) are a class of ligand-gated ion channels that participate in signal transduction and are reported to play an important role in the immunomodulation of vertebrates and invertebrates. Previous studies have shown that the nAChRs in mollusks have undergone large-scale expansion after tandem repeats and retrotransposition, with the most expansion observed in bivalves. This study characterized the sequence of a tandem repeat nAChR unique to several bivalve mollusks and investigated its functions in Pinctada fucata martensii. Firstly, phylogenetic analysis revealed that the tandem arrays of nAChRs existed before bivalve differentiation and m ost tandem-replicated nAChR genes have a conserved genomic structure and domain combination. In present study, five tandemly duplicated nAChR genes were cloned from P. f. martensii and designated as PmnAChR-1 to PmnAChR-5. qRT-PCR analysis revealed that five PmnAChRs were specifically expressed in adult gills. In addition, after PAMP stimulation, the expression of PmnAChRs in hemocytes of P. f. martensii were strongly induced but exhibited different responses to different stimuli. PmnAChR-1, PmnAChR-4, and PmnAChR-5 exhibited strong and wide responsiveness to lipopolysaccharide (LPS) stimulation but had no response to peptidoglycan (PGN) stimulation. PmnAChR-2 expression was notably upregulated at 6 h after PGN challenge but had no response to LPS stimulation. Polyinosinic-polycytidylic acid challenge upregulated nearly all PmnAChRs, except for PmnAChR-5. Furthermore, Pm-miR-873-3p, Pm-miR-4577, Pm-miR-103a-3p, and Pm-miR-6753-3p were identified as the regulatory miRNA of PmnAChR-1, PmnAChR-3, PmnAChR-4, and PmnAChR-5, respectively. These findings suggested that these tandem arrays of nAChRs are unique to bivalves, and the tandem duplication of nAChR genes may be involved in the immune regulation process after pathogen stimulation.

Physiological and biochemical response of different resistant alfalfa cultivars against thrips damage.

To investigate physiological and biochemical changes of thrips-resistant alfalfa (Medicago sativa L. cv. Gan-nong No. 9), we aimed at clarifying the response mechanisms of alfalfa against thrips. Medicago sativa L. cv. including thrips-resistant Gan-nong No.9 (G9), thrips-susceptible Gan-nong No.3 (G3) and highly thrips-susceptible WL363HQ (363) were infested with different thrips densities (3, 5, 7 and 9-thrips per branch). The quantitative change in specific nutrients, secondary metabolites, defensive and antioxidant enzymes were measured at seedling stage of the three alfalfa cultivars. The results showed that with the increase of thrips densities, the damage indices, SS, Pro, flavonoids, tannin and H2O2 in G9, G3 and 363 were significantly increased, but PPO and SOD significantly reduced, compared with CK. Furthermore, the tannin and lignin contents of G9 were significantly higher compared to 363, but SP content was significantly lower than G3 and H2O2 content which was further significantly less compared to 363. Correlation analysis observed that the damage index of the three alfalfa cultivars showed a significant positive association with SS, Pro, flavone, tannin, and H2O2 (P < 0.01), while damage index and DW, Chl (a, b, a + b), PPO and SOD showed a significant negative correlation (P < 0.01). Based on principal component comprehensive evaluation, the 5-thrips adults per branch were the critical inoculation threshold for G9 against thrips injury because the score was - 0.048. These results revealed that thrips damage significantly increased the contents of SS, Pro, flavonoids, tannins and H2O2, as well as significantly declined the activities of PPO and SOD in the three cultivars (P < 0.05), moreover, thrips-resistant G9 markedly accumulated lignin content, POD and CAT activity, inhibited Chl (a + b, b) and SP biosynthesis to resist thrips damage.