Purification and Characterization of a CkTLP Protein from Cynanchum komarovii Seeds that Confers Antifungal Activity

BACKGROUND: Cynanchum komarovii Al Iljinski is a desert plant that has been used as analgesic, anthelminthic and antidiarrheal, but also as a herbal medicine to treat cholecystitis in people. We have found that the protein extractions from C. komarovii seeds have strong antifungal activity. There is strong interest to develop protein medication and antifungal pesticides from C. komarovii for pharmacological or other uses. METHODOLOGY/PRINCIPAL FINDINGS: An antifungal protein with sequence homology to thaumatin-like proteins (TLPs) was isolated from C. komarovii seeds and named CkTLP. The three-dimensional structure prediction of CkTLP indicated the protein has an acid cleft and a hydrophobic patch. The protein showed antifungal activity against fungal growth of Verticillium dahliae, Fusarium oxysporum, Rhizoctonia solani, Botrytis cinerea and Valsa mali. The full-length cDNA was cloned by RT-PCR and RACE-PCR according to the partial protein sequences obtained by nanoESI-MS/MS. The real-time PCR showed the transcription level of CkTLP had a significant increase under the stress of abscisic acid (ABA), salicylic acid (SA), methyl jasmonate (MeJA), NaCl and drought, which indicates that CkTLP may play an important role in response to abiotic stresses. Histochemical staining showed GUS activity in almost the whole plant, especially in cotyledons, trichomes and vascular tissues of primary root and inflorescences. The CkTLP protein was located in the extracellular space/cell wall by CkTLP::GFP fusion protein in transgenic Arabidopsis. Furthermore, over-expression of CkTLP significantly enhanced the resistance of Arabidopsis against V. dahliae. CONCLUSIONS/SIGNIFICANCE: The results suggest that the CkTLP is a good candidate protein or gene for contributing to the development of disease-resistant crops

Glucogenic and lipogenic diets affect in vitro ruminal microbiota and metabolites differently

This study was conducted to evaluate the effects of two glucogenic diets (C: ground corn and corn silage; S: steam-flaked corn and corn silage) and a lipogenic diet (L: sugar beet pulp and alfalfa silage) on the ruminal bacterial and archaeal structures, the metabolomic products, and gas production after 48 h in vitro fermentation with rumen fluid of dairy cows. Compared to the C and S diets, the L dietary treatment leaded to a lower dry matter digestibility (DMD), lower propionate production and ammonia-nitrogen concentration. The two glucogenic diets performed worse in controlling methane and lactic acid production compared to the L diet. The S diet produced the greatest cumulative gas volume at any time points during incubation compared to the C and L diet. The metabolomics analysis revealed that the lipid digestion especially the fatty acid metabolism was improved, but the amino acid digestion was weakened in the L treatment than in other treatments. Differences in rumen fermentation characteristics were associated with (or resulting from) changes in the relative abundance of bacterial and archaeal genera. The rumen fluid fermented with L diet had a significantly higher number of cellulolytic bacteria, including the genera of Ruminococcus, Butyrivibrio, Eubacterium, Lachnospira, unclassified Lachnospiraceae, and unclassified Ruminococcaceae. The relative abundances of amylolytic bacteria genera including Selenomonas_1, Ruminobacter, and Succinivibrionaceae_UCG-002 were higher in samples for diets C and S. The results indicated that the two glucogenic diets leaded to a higher relative abundance of bacteria which functions in succinate pathway resulting in a higher propionate production. The steam-flaked corn diet had a higher gas production and lower level of metabolites in fatty acids and amino acids. Most highly abundant bacteria were observed to be not sensitive to dietary alterations of starch and fiber, except for several amylolytic bacteria and cellulolytic bacteria. These finding offered new insights on the digesting preference of ruminal bacteria, which can assist to improve the rumen functioning

Metagenomic insights into the relationship between gut microbiota and residual feed intake of small-sized meat ducks

IntroductionThe objective of this study was to determine the regulatory effects of gut microbiota on the feed efficiency (FE) of small-sized meat ducks by evaluating correlations between gut microbiota and residual feed intake (RFI).MethodsA total of 500 21-day-old healthy male ducks with similar initial body weights (645 ± 15.0 g) were raised contemporaneously in the same experimental facility until slaughter at 56 days of age. In total, nine low-RFI (LR) and nine high-RFI (HR) birds were selected for further gut microbiota composition and functional analyses based on the production performance, and the RFI was calculated from 22 to 56 days of age.ResultsGrowth performance results indicated a significantly lower RFI, feed conversion ratio, feed intake, and average daily feed intake in the LR ducks (P < 0.05). Taxonomy results of gut microbiota showed the identification of 19 kinds of phyla and more than 250 kinds of genera in all samples. No significant discrepancies in cecal bacterial α-diversity were discovered between the LR and HR groups, which indicated that the microbial modulatory effects on RFI may be attributed to the bacterial composition, rather than the species diversity. Differential analysis of bacterial communities between the LR and HR groups showed a significant increment of Firmicutes and a significant decline of Bacteroidetes in the LR group (P < 0.05). Specifically, genera of Erysipelatoclostridium, Parasutterella, Fournierella, and Lactococcus significantly proliferated, while Bacteroides significantly decreased in the LR group (P < 0.05). Furthermore, correlation analysis showed that the RFI was significantly correlated with carbohydrate metabolism-related bacteria including Bacteroides, Alistipes, Bifidobacterium, Ruminiclostridium_9, Sellimonas, Oscillibacter, Escherichia-Shigella, Lactococcus, and Streptococcus.ConclusionIn conclusion, the communities related to carbohydrate metabolism had positive regulatory effects on the FE of small-sized meat ducks, promoting it by improving the relative abundance and utilization of these communities. The present study provides valuable insight into the dynamics of gut microbiota underlying the variations in the FE of small-sized meat ducks

Effects of Partial Replacment of Dietary Forage Using Kelp Powder (Thallus laminariae) on Ruminal Fermentation and Lactation Performances of Dairy Cows

Background: Kelp powder, which was rich in novel oligosaccharides and iodine might be utilized by the rumen microbiome, promoted the ruminal fermentation and finally enhanced the lactation performance of dairy cows. Therefore, the purpose of this study was to investigate the effects of kelp powder partially replacing dietary forage on rumen fermentation and lactation performance of dairy cows. (2) Methods: In the present study, 20 Chinese Holstein dairy cows were randomly divided into two treatments, a control diet (CON) and a kelp powder replacing diet (Kelp) for a 35-d long trial. Dry matter intake (DMI), milk production, milk quality, ruminal fermentable parameters, and rumen microbiota were measured to investigate the effects of kelp powder feeding on dairy cows. (3) Results: On the lactation performance, kelp significantly increased milk iodine content and effectively enhanced milk production and milk fat content. On the fermentable aspects, kelp significantly raised TVFA while reducing the ammonia-N content. On the rumen microbial aspect, kelp feeding significantly promoted the proliferation of Firmicutes and Proteobacteria while suppressing Bacteroidetes. (4) Conclusion: kelp powder as an ingredient of feedstuff might promote the rumen fermentation ability and effectively increase milk fat and iodine content, and consequently improve the milk nutritional value

Effects of monochromatic lights on the growth performance, carcass characteristics, eyeball development, oxidation resistance, and cecal bacteria of Pekin ducks

Objective: Light is a significant component of housing environment in commercial poultry industry. This study was conducted to investigate whether Pekin ducks perform better under monochromatic lights than under white light with respect to their growth performance, carcass quality, eyeball development, oxidation resistance, and cecal bacterial communities. Methods: A total of 320 one-day-old male Pekin ducklings were randomly distributed into five rooms with different light treatments, white, red, yellow, green, and blue light. Each room consisted of 4 replicated pens with 16 ducklings per pen. Results: Blue light significantly decreased fat deposition by decreasing abdominal fat. Long wavelength light, such as red, green, and yellow light, considerably increased the back-to-front eyeball diameter and the red light potentially enlarged the side-to-side eyeball diameter. Besides, the blue light had adverse effects on the oxidation resistance status in terms of increasing the product malonaldehyde of lipid oxidation and decreasing the plasma concentration of total superoxide dismutase. The phyla of Firmicutes had the greatest abundance in the green and blue treatments, while Bacteroidetes in blue treatment was the least. The genus of Faecalibacterium was significantly lower under the red light. Conclusion: The high risk of cecal health status and decreased anti-oxidation activity were observed under blue light. Red, yellow, and green light might increase the risk of oversized eyeball and cecal illness. Therefore, monochromatic lights compared to white light did not show advantages on the performance of housing ducks, it turns out that the white light is the best light condition for grow-out ducks.</p

Metagenome sequencing to analyze the impacts of thiamine supplementation on ruminal fungi in dairy cows fed high-concentrate diets

Abstract Ruminal thiamine deficiencies occur when dairy cows are overfed with high-concentrate diet, and thiamine supplementation has been proved to attenuate high-concentrate diet induced SARA. However, there is limited knowledge of the relationship between thiamine supplementation in high-concentrate diets and ruminal fungi. In order to investigate the impacts of thiamine supplementation on ruminal fungi, twelve Chinese Holstein dairy cows were randomly assigned into three treatments: control diet (CON; 20% starch, dry matter basis), high-concentrate diet (HC; 33.2% starch, dry matter basis) and high-concentrate diet supplemented with 180 mg thiamine/kg dry matter intake. Dry matter intake and milk production were recorded during the experimental periods. On day 21, rumen fluid samples were collected at 3 h postfeeding and ruminal pH, thiamine concentration and volatile fatty acids were measured. Metagenome sequencing method was conducted to detect ruminal fungi composition. Feeding HC significantly decreased dry matter intake, milk production, ruminal pH, ruminal acetate and thiamine concentration, however, significantly increased propionate and isovalerate (P < 0.05). These changes were inversed by thiamine supplementation (P < 0.05). Totally, seven phyla and almost 1050 species of rumen fungi were identified across all samples in which especially, 3 genera and 10 species of strictly anaerobic fungi phylum Neocallimastigomycota was found. Principal coordinate analysis indicated that feeding HC and thiamine supplementation caused a significant inverse in ruminal fungi composition. Feeding HC significantly decreased the abundance of fungi compared with CON (P < 0.05) while thiamine supplementation significantly increased the abundance of ruminal fungi (P < 0.05). These results indicated that thiamine supplementation may effectively attenuate rumen metabolic disorder caused by HC diet through buffering the ruminal pH, shifting the rumen fermentation pattern and increasing the abundance of ruminal fungi. The findings in this study could therefore contribute to the further understanding of the mechanism of thiamine’s function in dairy cows

Metagenomic insights into effects of thiamine supplementation on ruminal non-methanogen archaea in high-concentrate diets feeding dairy cows

Abstract Background Overfeeding of high-concentrate diet (HC) frequently leads to subacute ruminal acidosis (SARA) in modern dairy cows’ production. Thiamine supplementation has been confirmed to attenuate HC induced SARA by increasing ruminal pH and ratio of acetate to propionate, and decreasing rumen lactate, biogenic amines and lipopolysaccharide (LPS). The effects of thiamine supplementation in HC on rumen bacteria and fungi profile had been detected in our previous studies, however, effects of thiamine supplementation in HC on rumen non-methanogen archaea is still unclear. The objective of the present study was therefore to investigate the effects of thiamine supplementation on ruminal archaea, especially non-methanogens in HC induced SARA cows. Results HC feeding significantly decreased dry matter intake, milk production, milk fat content, ruminal pH and the concentrations of thiamine and acetate in rumen fluid compared with control diet (CON) (P < 0.05), while the concentrations of propionate and ammonia-nitrogen (NH3-N) were significantly increased compared with CON (P < 0.05). These changes caused by HC were inversed by thiamine supplementation (P < 0.05). The taxonomy results showed that ruminal archaea ranged from 0.37 to 0.47% of the whole microbiota. Four characterized phyla, a number of Candidatus archaea and almost 660 species were identified in the present study. In which Euryarchaeota occupied the largest proportion of the whole archaea. Furthermore, thiamine supplementation treatment significantly increased the relative abundance of non-methanogens compared with CON and HC treatments. Thaumarchaeota was increased in HC compared with CON. Thiamine supplementation significantly increased Crenarchaeota, Nanoarchaeota and the Candidatus phyla, however decreased Thaumarchaeota compared with HC treatment. Conclusions HC feeding significantly decreased ruminal pH and increased the content of NH3-N which led to N loss and the increase of the relative abundance of Thaumarchaeota. Thiamine supplementation increased ruminal pH, improved the activity of ammonia utilizing bacteria, and decreased Thaumarchaeota abundance to reduce the ruminal NH3 content and finally reduced N loss. Overall, these findings contributed to the understanding of thiamine’s function in dairy cows and provided new strategies to improve dairy cows’ health under high-concentrate feeding regime