First Report of Leaf Spot Disease on Avocado Caused by Bipolaris setariae in China.

Avocado (Persea americana), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as Bipolaris sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with B. setariae (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with B. setariae (MF490833.1, MK144540.1) and B. yamadae (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with B. setariae with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 105 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of B. setariae as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.

The Effects of Sodium Acetate on the Immune Functions of Peripheral Mononuclear Cells and Polymorphonuclear Granulocytes in Postpartum Dairy Cows.

Excessive lipid mobilization will snatch cell membrane lipids in postpartum dairy cows, which may impair the function of immune cells, including peripheral mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). Acetate, as a precursor and the energy source of milk fat synthesis, plays a key role in lipid synthesis and the energy supply of dairy cows. However, there is little information about the effect of sodium acetate (NaAc) on the immune function of PBMC and PMN in postpartum dairy cows. Therefore, this study aimed to evaluate the effects of NaAc on the immune functions of PBMCs and PMNs in postpartum dairy cows. In this experiment, twenty-four postpartum multiparous Holstein cows were randomly selected and divided into a NaAc treatment group and a control group. Our results demonstrated that the dietary addition of NaAc increased (p < 0.05) the number of monocytes and the monocyte ratio, suggesting that these postpartum cows fed with NaAc may have better immunity. These expressions of genes (LAP, XBP1, and TAP) involved in the antimicrobial activity in PBMCs were elevated (p < 0.05), suggesting that postpartum dairy cows supplemented with NaAc had the ability of antimicrobial activity. In addition, the mRNA expression of the monocarboxylate transporters MCT1 and MCT4 in PBMCs was increased (p < 0.05) in diets supplemented with NaAc in comparison to the control. Notably, the expression of the XBP1 gene related to antimicrobial activity in PMN was upregulated with the addition of NaAc. The mRNA expression of genes (TLN1, ITGB2, and SELL) involved in adhesion was profoundly increased (p < 0.05) in the NaAc groups. In conclusion, our study provided a novel resolution strategy in which the use of NaAc can contribute to immunity in postpartum dairy cows by enhancing the ability of antimicrobial and adhesion in PBMCs and PMNs.

Effects of different manganese sources on nutrient digestibility, fecal bacterial community, and mineral excretion of weaning dairy calves.

Introduction: Mn, which is an essential trace mineral for all animals, has functions in skeletal system development, carbohydrate and lipid metabolism. The aim of this study was to clarify the effects of different manganese (Mn) sources in basal diets on nutrient apparent digestibility, fecal microbes, and mineral elements excretion before and after weaning. Methods: A total of 15 Holstein heifer calves (6-week-old, 82.71 ± 1.35, mean ± standard error) were randomly designed into three groups (five each): no extra Mn supplemented (CON), 20 mg Mn/kg (dry matter basis) in the form of chelates of lysine and glutamic acid in a mixture of 1:1 (LGM), and 20 mg Mn/kg (dry matter basis) in the form of MnSO4. All calves were weaned at 8 weeks of age. The experiment lasted for 28 days (14 days before weaning and 14 days after weaning). Dry matter intake (DMI) was recorded daily. The animals were weighed by electronic walk-over, and body size indices were collected using tape on days -14, -1, and 14 of weaning. The feces of calves was collected to measure the apparent digestibility of nutrients (acid insoluble ash was an internal marker) and bacterial community on days -1, 1, 3, 7, and 14 of weaning. Fecal mineral concentration was determined by inductively coupled plasma emission spectroscopy on days -1, 1, 7, and 14 of weaning. Results: The results showed that, compared with the CON group, adding LGM to diets containing 158.82 mg/kg Mn increased the apparent digestibility (P < 0.05). The Chao 1 and Shannon index of fecal bacteria decreased at day 1 in the LGM and MnSO4 groups and increased after weaning. The PCoA results indicated that the LGM group was distinctly separate from the CON and MnSO4 groups during the whole experimental period. Significant differences (P < 0.05) were observed in the relative abundance of two phyla (Proteobacteria and Spirochaetota) and eight genera (Alloprevotella, Prevotellaceae_UCG-001, Clostridia UCG 014, RF39, UCG-010, Pseudomonas, Ralstonia, and Treponema) in three groups. Moreover, the LGM group showed less excretion of Fe, P, and Mn than the MnSO4 group. Discussion: In summary, 20 mg Mn/kg diet supplementation improved nutrient digestibility, changed the fecal microbial community, and reduced mineral excretion. Organic Mn supplementation in the diet had more advantages over the sulfate forms in weaning calves.

GPR41 Regulates the Proliferation of BRECs via the PIK3-AKT-mTOR Pathway.

Short-chain fatty acids (SCFAs) play a pivotal role in regulating the proliferation and development of bovine rumen epithelial cells (BRECs). G protein-coupled receptor 41 (GPR41) is involved in the signal transduction in BRECs as a receptor for SCFAs. Nevertheless, the impact of GPR41 on the proliferation of BRECs has not been reported. The results of this research showed that the knockdown of GPR41 (GRP41KD) decreased BRECs proliferation compared with the wild-type BRECs (WT) (p < 0.001). The RNA sequencing (RNA-seq) analysis showed that the gene expression profiles differed between WT and GPR41KD BRECs, with the major differential genes enriched in phosphatidylinositol 3-kinase (PIK3) signaling, cell cycle, and amino acid transport pathways (p < 0.05). The transcriptome data were further validated by Western blot and qRT-PCR. It was evident that the GPR41KD BRECs downregulated the level of the PIK3-Protein kinase B (AKT)-mammalian target of the rapamycin (mTOR) signaling pathway core genes, such as PIK3, AKT, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and mTOR contrasted with the WT cells (p < 0.01). Furthermore, the GPR41KD BRECs downregulated the level of Cyclin D2 p < 0.001) and Cyclin E2 (p < 0.05) compared with the WT cells. Therefore, it was proposed that GPR41 may affect the proliferation of BRECs by mediating the PIK3-AKT-mTOR signaling pathway.

Effects of supplementation of sodium acetate on rumen fermentation and microbiota in postpartum dairy cows.

The primary product of rumen fermentation is acetic acid, and its sodium salt is an excellent energy source for post-partum cows to manage negative energy balance (NEB). However, it is unknown how adding sodium acetate (NAc) may affect the rumen bacterial population of post-partum cows. Using the identical nutritional total mixed ration (TMR), this research sought to characterize the impact of NAc supplementation on rumen fermentation and the composition of bacterial communities in post-partum cows. After calving, 24 cows were randomly assigned to two groups of 12 cows each: a control group (CON) and a NAc group (ACE). All cows were fed the same basal TMR with 468 g/d NaCl added to the TMR for the CON group and 656 g/d NAc added to the TMR for the ACE group for 21 days after calving. Ruminal fluid was collected before morning feeding on the last day of the feeding period and analyzed for rumen bacterial community composition by 16S rRNA gene sequencing. Under the identical TMR diet conditions, NAc supplementation did not change rumen pH but increased ammonia nitrogen (NH3-N) levels and microbial crude protein (MCP) concentrations. The administration of NAc to the feed upregulated rumen concentrations of total volatile fatty acids (TVFA), acetic, propionic, isovaleric and isobutyric acids without affecting the molar ratio of VFAs. In the two experimental groups, the Bacteroidota, Firmicutes, Patescibacteria and Proteobacteria were the dominant rumen phylum, and Prevotella was the dominant rumen genus. The administration of NAc had no significant influence on the α-diversity of the rumen bacterial community but upregulated the relative abundance of Prevotella and downregulated the relative abundance of RF39 and Clostridia_UCG_014. In conclusion, the NAc supplementation in the post-peripartum period altered rumen flora structure and thus improved rumen fermentation in dairy cows. Our findings provide a reference for the addition of sodium acetate to alleviate NEB in cows during the late perinatal period.

Acetate-Induced Milk Fat Synthesis Is Associated with Activation of the mTOR Signaling Pathway in Bovine Mammary Epithelial Cells.

Acetate is a precursor substance for fatty acid synthesis in bovine mammary epithelial cells (BMECs), and the mTOR signaling pathway plays an important role in milk fat synthesis. However, the mechanism of the regulatory effects of acetate on lipogenic genes via the mTOR signaling pathway in BMEC remains unknown. We hypothesized that acetate can enhance the expression of lipogenic genes and triglyceride (TG) production by activating the mTOR signaling pathway in BMECs. Therefore, the aim of this study was to investigate the network of acetate-regulated lipid metabolism by the mTOR signaling pathway in BMECs. These results showed that TG synthesis was elevated (p < 0.01) in BMECs with acetate treatment. The lipid droplets were increased in the acetate-treated groups compared with those in the control group through the Bodipy staining of the lipids. In addition, the fatty acid profile in BMECs treated with acetate was affected, with an elevation in the proportions of C14:0, C16:0, and C18:0. The mRNA levels of the sterol-response-element-binding protein 1 (SREBP1), stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FAS) genes involved in the lipogenesis and transcriptional factors were upregulated (p < 0.05) in BMECs with acetate treatment. Remarkably, the expression of acetyl-CoA carboxylase α (ACCα) and FAS rate-limiting enzymes involved in lipogenesis was upregulated in BMECs with acetate treatment. Moreover, the addition of acetate enhanced the key protein expression of S6K1, which is related to the mTOR signaling pathway. Taken together, our data suggest that TG accumulation and expression of lipogenic genes induced by acetate are associated with the activation of the mTOR signaling pathway, which provides new insights into the understanding of the molecular mechanism in the expression of mTOR-signaling-pathway-regulated lipogenic genes.

An Extract of Artemisia argyi Leaves Rich in Organic Acids and Flavonoids Promotes Growth in BALB/c Mice by Regulating Intestinal Flora.

In the context of global restrictions on the use of antibiotics, there has been increased research on natural plant-based ingredients as additives. It has been proved that many natural active ingredients contained in plants have positive effects on animal growth regulation. Artemisia argyi (A. argyi) is a traditional Chinese herbal medicine, and its extracts have been reported to have a variety of biological activities. Therefore, in order to explore the potential of the active extract of Artemisia argyi leaves (ALE) as a plant source additive, mice were fed with ALE at different concentrations for 60 days. Finally, the effects of ALE were evaluated by the growth indexes, blood indexes, and intestinal microflora changes of the mice. It was found that a medium concentration of ALE (150 mg/kg) could promote growth, and especially improved the feed efficiency of the mice. However, high concentrations of ALE (300 mg/kg) had some negative effects on the growth of mice, especially liver damage, which significantly increased AST and ALT levels in the blood. Therefore, the 150 mg/kg ALE treatment group was selected for 16S rDNA analysis. It was found that ALE could play a positive role by regulating the proportion of Bacteroidetes and Firmicutes in the intestinal tract. In particular, it can significantly up-regulate the quantities of Akkermansia and Bifidobacterium. These results suggest that ALE at appropriate concentrations can positively regulate animal growth.