In vivo infection of Bursaphelenchus xylophilus by the fungus Esteya vermicola.

BACKGROUND: As the causal agent of pine wilt disease, Bursaphelenchus xylophilus, is a serious pathogen of forest pine trees. Esteya vermicola is a nematophagous fungus of B. xylophilus and exhibits great potential as a biological control agent. However, the in vivo infection mechanism of E. vermicola on B. xylophilus is unclear. Experiments were conducted to study the colonization of host plant and infection of B. xylophilus by E. vermicola inside pine tree xylem. RESULTS: A green fluorescent protein (GFP)-tagged E. vermicola transformant was constructed as a biomarker to study the in vivo colonization and infection of B. xylophilus in pine trees. The in vitro infection of B. xylophilus by E. vermicola was observed through GFP expression. The bacilloid conidia produced by trophic hyphae in the body of the nematode are described. Additionally, the monitoring of in vivo colonization by GFP-tagged E. vermicola showed the germination and hyphal extension of this fungus after inoculation. Moreover, B. xylophilus infected by this biocontrol agent were extracted from healthy seedlings and observed in the xylem of trees that were wilting due to pine wilt disease. CONCLUSION: Evidence of fungal colonization and infection of B. xylophilus by E. vermicola is provided to improve our understanding of the in vivo infection mechanisms used by this nematophagous fungus against B. xylophilus. The infection of B. xylophilus by E. vermicola was inferred to begin with the implantation of propagules, and this inference will require future investigation. The colonization of Esteya vermicola in host pine tree xylem and the in vivo infection of pinewood nematode by E. vermicola were investigated using the green fluorescence protein transformant. © 2020 Society of Chemical Industry.

Effect of Aster tataricus on production of inflammatory mediators in LPS stimulated rat astrocytoma cell line (C6) and THP-1 cells.

Neuroinflammation is the commonest cause of neurodegenerative diseases such as Alzheimer's disease. Present investigation evaluates the inhibitory effect of ethanolic root extract of Aster tataricus (AS) on inflammatory mediators production in lipopolysaccharide (LPS) stimulated C6 cells. C6 cells were treated with AS (20 and 40 mg/kg) and nimesulide (NSL, 1.5 µg/ml) for 1 day. Thereafter various parameters such as production of ROS, release of nitrite, MDA, glutathione level and NF-κB translocation were estimated in C6 cell lines. Effect of AS was estimated on the expressions of tumor necrosis factor α (TNF-α) of human monocytic leukemia cell line (THP-1). It was observed that AS (20 and 40 mg/ml) treated group shows significant (p < 0.01) decrease in production of ROS, Nitrite release and MDA level in LPS activated C6 cell lines compared to negative control group. Moreover, treatment with it decreases glutathione level and inhibits the translocation of NF-κB in LPS activated C6 cell lines compared to negative control group. There were significant (p < 0.01) decreases in expression of TNF-α in AS treated group compared to negative control group in THP-1 cell lines. Present investigation concludes the anti neuroinflammatory effect of ethanolic extract of AS root by decreasing oxidative stress and attenuates the cytokine.

In vivo hair growth-stimulating effect of medicinal plant extract on BALB/c nude mice.

CONTEXT: Chrysanthemum zawadskii var. latilobum (Asteraceae) (CZ) and Polygonum multiflorum Thunb. (Polygonaceae) (PM) have been used traditionally to treat different systemic diseases and acclaimed for various biological activities including hair growth. OBJECTIVE: This study investigates the hair restoration efficacy of selected medicinal plant extracts on nude mice. MATERIALS AND METHODS: Nude mice genetically predisposed to pattern balding were used in this study. Topical methanol extracts of CZ and PM (10 mg/mouse/d) with standardized vehicle formulation, only vehicle (propylene glycol:ethanol:dimethyl sulfoxide, 67:30:3% v/v) and Minoxidil (2%) were applied daily for 40 consecutive days. RESULTS: In our study, the maximum hair score (2.5 ± 0.29) was obtained in the CZ-treated group. Histological observation revealed a significant increase (p < 0.001) in the number of hair follicles (HF) in CZ-treated mice (58.66 ± 3.72) and Minoxidil-treated mice (40 ± 2.71). Subsequently, immunohistochemical analysis also confirmed the follicular keratinocyte proliferation by detection of BrdU-labeling, S-phase cells in Minoxidil and CZ-treated mouse follicular bulb and outer root sheaths. CONCLUSION: Our study revealed the underlying mechanism of stimulating hair growth in athymic nude mice by repair the nu/nu follicular keratin differentiation defect. Thus, the topical application of CZ may represent a novel strategy for the management and therapy of certain forms of alopecia.

The effects of fluctuating culture temperature on stress tolerance and antioxidase expression in Esteya vermicola.

The endoparasitic nematophagous fungus, Esteya vermicola, has shown great potential as a biological control agent against the pine wood nematode, Bursaphelenchus xylophilus. Fluctuating culture temperatures can affect fungal yields and fungal tolerance to desiccation, UV radiation, H2O2, and heat stress, as well as antioxidase expression. To explore these effects, E. vermicola cultured under five temperature ranges, 26°C, 15-26°C, 26-35°C, 20-30°C, and 15-35°C, were compared. The cultures grown at lower temperatures showed better growth, stronger tolerance to desiccation, UV, and H2O2 stresses, and increased catalase expression, However, these cultures also showed weaker heat stress tolerance and lower superoxide dismutase expression than the higher-temperature cultures. In particular, the E. vermicola cultured at 20-30°C, i.e., fluctuating in a narrow range around the optimal temperature, showed the best performance. Therefore, for production in practical applications, this narrowly fluctuating, moderate temperature appears to be optimal for yield and stress tolerance in E. vermicola.

Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode.

The fungus, Esteya vermicola has been proposed as biocontrol agent against pine wilting disease caused by Bursaphelenchus xylophilus. In this study, we reported the effects of temperature and different additives on the viability and biocontrol efficacy of E. vermicola formulated by alginate-clay. The viability of the E. vermicola formulation was determined for six consecutive months at temperature ranged from -70 to 25 °C. The fresh conidia without any treatment were used as control. Under the optimal storage conditions with E. vermicola alginate-clay formulation, the results suggested that E. vermicola alginate-clay formulation with a long shelf life could be a non-vacuum-packed formulation that contains 2 % sodium alginate and 5 % clay at 4 °C. Three conidial formulations prepared with additives of 15 % glycerol, 0.5 % yeast extract and 0.5 % herbal extraction, respectively significantly improved the shelf life. In addition, these tested formulations retained the same biocontrol efficacy as the fresh conidial against pinewood nematode. This study provided a tractable and low-cost method to preserve the shelf life of E. vermicola.

Aqueous extract of red deer antler promotes hair growth by regulating the hair cycle and cell proliferation in hair follicles.

Deer antlers are the only mammalian appendage capable of regeneration. We aimed to investigate the effect of red deer antler extract in regulating hair growth, using a mouse model. The backs of male mice were shaved at eight weeks of age. Crude aqueous extracts of deer antler were prepared at either 4 °C or 100 °C and injected subcutaneously to two separate groups of mice (n = 9) at 1 mL/day for 10 consecutive days, with water as a vehicle control group. The mice skin quantitative hair growth parameters were measured and 5-bromo-2-deoxyuridine was used to identify label-retaining cells. We found that, in both the 4 °C and the 100 °C deer antler aqueous extract-injection groups, the anagen phase was extended, while the number of BrdU-incorporated cells was dramatically increased. These results indicate that deer antler aqueous extract promotes hair growth by extending the anagen phase and regulating cell proliferation in the hair follicle region.

Comparison between conidia and blastospores of Esteya vermicola, an endoparasitic fungus of the pinewood nematode, Bursaphelenchus xylophilus.

Esteya vermicola, an endoparasitic fungus of Bursaphelenchus xylophilus, the pinewood nematode (PWN), exhibits great potential as a biological control agent against this nematode. E. vermicola produces blastospores in liquid media and aerial conidia on solid media. The agent was mass-produced using two kinds of culture media: S (50 % wheat bran and 50 % pine wood powder), L (0.5 g wheat bran and 0.5 g pinewood powder in 200 ml of potato dextrose broth), and two controls: SC (potato dextrose agar), LC (potato dextrose broth). Yields, multiple stress tolerance, storage life, new generation conidial number, and PWN mortality rates of the spores were measured in each of these four media and compared. The spore yields, new generation conidial number, and nematode mortality rates of blastospores were higher than those of conidia. Nevertheless, the conidia had a higher germination rate than the blastospores during the storage process and multiple stress treatments. Considering the number of spores surviving from the process of the storage and multiple stress treatments per unit of mass media, the blastospores from L survived most. Comprehensive analysis indicates that the L culture medium is the most optimal medium for mass production relatively.

Ginsenosides Rb1 and Rd regulate proliferation of mature keratinocytes through induction of p63 expression in hair follicles.

Ginsenosides Rb1 and Rd are the two main types of ginsenosides in Panax ginseng and have been used as an additive to against alopecia. However, the mechanisms involved are largely unknown. To determine how ginsenosides prevent hair loss, we topically applied protopanaxadiol-type ginsenosides Rb1 and Rd over the shaved skin of B57CL/6 mice, and monitored and assessed them for 35 days. We then investigated the effects of ginsenosides on cell genesis in different phases of adult hair follicles (HFs), using 5-bromo-2'-deoxyuridine as a marker for dividing cells. Moreover, p63, a specific marker and a major regulator of keratinocyte progenitor cells of the multi-layered epithelia, was detected in epidermis. Results indicated that treatment with ginsenosides Rb1 and Rd increased cell proliferation in both anagen and telogen of HFs. However, it had no significant effect on the survival of cells in the bulge and upper follicle region. Investigation of p63 demonstrated that up-regulation of p63 expression in the matrix and outer root sheath might be one of the mechanisms by which ginsenosides Rb1 and Rd promote cell proliferation in HFs. Our study reveals a novel mechanism by which ginsenoside promotes hair growth through p63 induction in follicular keratinocytes and indicates that ginsenosides Rb1 and Rd might be developed as a therapeutic agent for the prevention of hair loss.

Growth of Esteya vermicola in media amended with nitrogen sources yields conidia with increased predacity and resistance to environmental stress.

Esteya vermicola , an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study to enhance the sporulation, predacity, and environmental resistance of E. vermicola, various nitrogen sources, such as glycine, L-leucine, and ammonium nitrate, were tested. The supplement of glycine and L-leucine had a significant influence on the growth rate of the colony, enhancing colony dry mass by 5-fold more than did ammonium nitrate or the control. Of the nitrogen sources tested, ammonium nitrate and L-leucine promoted sporulation, yielding more than 6 × 10(6) CFU/g, while glycine enhanced the proportion of lunate spores. Meanwhile, the supplement of nitrogen sources had a significant influence on adhesive rate and mortality rate against Bursaphelenchus xylophilus . Moreover, the supplement of glycine enhanced the survival rate against heat stress by more than 3-fold that of L-leucine, ammonium nitrate, and control. The spores produced in media amended with glycine, L-leucine, and ammonium nitrate had slightly but not significantly higher UV resistance and drought resistance than spores produced without nitrogen sources. These results suggested that the addition of glycine resulted in the production of E. vermicola conidia with increased predacity and resistance to environmental stress that may be more suitable for control of pine wilt disease.