Anoectochilus Roxburghii Research Articles

Improved 1D convolutional neural network adapted to near-infrared spectroscopy for rapid discrimination of Anoectochilus roxburghii and its counterfeits

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is a rare traditional Chinese medicine. For seeking high profit, some traditional Chinese medicine sellers usually adulterated A. roxburghii with Goodyera Schlechtendaliana and Ludisia discolor or directly fake A. roxburghii using Anoectochilus formosanus. These counterfeits with similar appearance greatly influence the prescription efficacy. Therefore, there is an urgent need for an effective and fast authentication method to identify A. roxburghii and its counterfeits. In this paper, the near-infrared spectroscopy (NIRS) data of A. roxburghii and its counterfeits are mearsured. Then, an improved inception architecture based 1-dimensional convolutional neural network (Improved 1D-Inception-CNN) is designed for processing the NIRS data and identifying A. roxburghii and its counterfeits. The Improved 1D-Inception-CNN has less parameters and high calculation efficiency which makes the identification model more practical. The experimental results show that compared with traditional structured CNN models, the complexity of the Improved 1D-Inception-CNN is reduced by 40 %, the parameters are reduced by 50 % and the performances are improved by 1.01 %. Therefore, the Improved 1D-Inception-CNN model based on NIRS technology can effectively and quickly identify A. roxburghii and its counterfeits.

Characterization of Anoectochilus roxburghii polysaccharide and its therapeutic effect on type 2 diabetic mice

Anoectochilus roxburghii is a traditional herb in China that can be potentially used to treat diabetes. A novel polysaccharide ARLP-W was isolated from Anoectochilus roxburghii by chromatography on DEAE-52 cellulose. Chemical analysis indicated that ARLP-W (8.1 × 104 Da) was mainly composed of mannose and glucose. The main linkages of glycosidic bonds of ARLP-W were β-1, 4-Manp and α-1, 4-Glcp. The terminal Glcp was connected to Manp-via O-3. RT-qPCR and western blotting analysis showed that ARLP-W caused a significant reduction in the levels of the key gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) in the liver. The results of the insulin resistance tests indicated that ARLP-W increased glucose absorption. These results indicate that ARLP-W has a good therapeutic effect on type 2 diabetes and can assist with further development and application treatment of diabetes.

Purification, structure and conformation characterization of a novel glucogalactan from Anoectochilus roxburghii

Anoectochilus roxburghii (AR) has been used in food, medicine and ornamental industries for a long time. Anion exchange resin was proposed to purify the sub-fraction of water-extracted AR polysaccharide (ARPP-70), and a homogeneous polysaccharide ARPP-70a was obtained. The structural features of ARPP-70a were characterized using gas chromatography–mass spectrometry (GC–MS), nuclear magnetic resonance (NMR) spectroscopy, and high performance size exclusion chromatograph coupled with multi-angle laser light scattering (HPSEC-MALLS). The relative weight average molecular weight for ARPP-70a was determined to be 14.8 kDa, and the molar ratio of glucose to galactose was 1.0:3.2. The structure of ARPP-70a was elucidated to be glucogalactan, with backbone comprising β-1,4-linked Galp and some α-1,4-linked Glcp. The conformation characteristics of ARPP-70a were supposed to exist as a random coil chain in 0.1 M NaNO3 solution. Moreover, in vitro antioxidant activity assays revealed ARPP-70a exhibited appreciable antioxidant potential. To the best of our knowledge, this is the first study to obtain this type of glucogalactan, and provide systematic information on its structural and conformational properties. This study improved the understanding of the physicochemical characteristics of AR polysaccharide, which is beneficial for its further application in food and medicinal industry.

Synergic cloud-point extraction using [C4mim][PF6] and Triton X-114 as extractant combined with HPLC for the determination of rutin and narcissoside in Anoectochilus roxburghii (Wall.) Lindl. and its compound oral liquid

A green, novel and efficacious method for the simultaneous extraction and enrichment of rutin and narcissoside from the compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid (CAROL) and Anoectochilus roxburghii (Wall.) Lindl. was developed. Ionic liquid-surfactant synergic cloud-point extraction (IL-CPE) was used to enrich two analytes, which were determined by high-performance liquid chromatography (HPLC). Some parameters affecting IL-CPE were optimized, such as ratio and volume of 1-butyl-3-methyl-imidazolium hexafluorophosphate and Triton X-114, pH of the sample, NaCl concentration, total extraction volume, incubation temperature and time, centrifuge rate and time. The corresponding linearity range for two analytes exhibited good linearity (r2＞0.9997), with the average added recoveries ranging from 92.1% to 98.9%. The limits of detection of rutin and narcissoside were 0.26 and 0.30 ng/mL, respectively. The method was successfully applied for the determination of two flavonoids in the complex-matrix sample, i.e. CAROL and the water extract of A. roxburghii. The mass spectrum data showed that the sample contained rutin and narcissoside. Compared with conventional extraction methods, IL-CPE exhibited higher extraction efficiency and better extraction selectivity. This method may provide a novel platform for the determination of active ingredients in compound Chinese medicine oral liquid and herb.

Development of a HPTLC method with iterative calibration approach for quantitative evaluation of kinsenoside content in different types of Anoectochilus roxburghii

Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) or more commonly known as Jin Xian Lian is a widely adapted medicinal herb in the Asian continent. However, till date there are limited rapid analytical method such as thin layer chromatography method that acts as standard for quantifying the active ingredients of A. roxburghii. In this research, a simple and rapid method high performance thin layer chromatography (HPTLC)-anisaldehyde-sulfuric acid derivative with iterative calibration method was developed and validated to quantify the non UV sensitive bioactive compound kinsenoside in wild, cultivated and tissue cultured A. roxburghii plants. The optimum mobile phase identified for kinsenoside in A. roxburghii was chloroform/methanol/water (45:30:4,%v/v/v). It was also proved that HPTLC method for determination of kinsenoside showed good accuracy, precision, stability and repeatability, with wide working concentration range of 0.099–6.365 mg/mL. Intra-day and inter-day precision (% RSD) of the developed HPTLC method were 5.08–7.57% and 6.72–8.72%, respectively. The % RSD of iterative calibration method was 3.139%. Through this study, it can be concluded that the proposed HPTLC method with iterative calibration approach was successfully applied for quantitative analysis of kinsenoside in three types of A. roxburghii plants.

Network Pharmacology Study of the Hepatoprotective Effects of Quercetin-Containing Traditional Chinese Medicine, Anoectochilus roxburghii, and Validation of Quercetin as an Anti-Liver Injury Agent in a Mouse Model of Liver Injury.

BackgroundAnoectochilus roxburghii (Orchidaceae) (AR) has been widely used to treat liver injury in China, but its underlying mechanisms remain elusive. Network pharmacology was utilized to assess the hepatoprotective effects of quercetin (Que)-containing AR, and to validate the anti-liver injury effects of Que in a mouse model of liver injury.Material/MethodsNetwork pharmacology analysis was performed to determine bio-active compounds in AR. The core therapeutic targets of AR against liver injury were identified using a protein–protein interaction network. Biological function and pathway enrichment were analyzed based on the identified core therapeutic targets. The hepatoprotective effects of Que in a mouse model of liver injury induced by CCl4 were assessed to verify the reliability of network pharmacology analysis.ResultsSeven bio-active compounds of AR met drug screening criteria and 17 core therapeutic targets of AR against liver injury were identified. Biological function analysis demonstrated that the therapeutic effects of AR against liver injury were chiefly associated with the suppression of inflammation and immunity; and pathway enrichment analysis showed that nuclear factor-kappa B (NF-κB) and tumor necrosis factor (TNF) signaling pathways were associated with the inflammatory responses. Experimental validation in a mouse model showed that AR exerted anti-inflammatory effects by regulating the NF-κB signaling pathway, a finding that also confirmed the reliability of network pharmacology analysis.ConclusionsThe bio-active compounds identified in AR and the elucidation of their mechanisms of action against liver injury provide a theoretical basis for designing agents that can prevent or suppress liver injury.

Simultaneous determination of five pesticides residues in Anoectochilus roxburghii by HPLC-MS/MS method

Objective： To establish a HPLC-MS/MS method for simultaneous determination of five pesticides residues including tebuconazole，metalaxyl-M，thiophanate-methyl，oxytetracycline and abamectin in Anoectochilus roxburghii.Methods： The samples were extracted by acetonitrile solution and purified by QuEChERS method.Separation was performed on Agilent Eclipse Plus C18 column（50 mm×2.1 mm，1.8 μm） with the mobile phase consisting of 0.1% formic acid（A） and acetonitrile（B） by gradient elution（0-1.0 min，90% A；1.0-3.0 min，90%-70% A;3.0-5.0 min，70%-30% A;5.0-8.0 min，30%-10% A;8.0-14.0 min，10% A;14.0-15.0 min，10%-90% A;15.0-17.0 min，90% A）.Detection was performed on a triple quadrupole tandem mass spectrometry with electrospray ionization source operating in positive ion mode under multiple reaction monitoring（MRM） mode.Results： The calibration curves of five pesticides showed good linearity coefficients（r>0.99）.The average recoveries ranged from 72.7% to 94.9%（n=6），and the relative standard derivations（RSDs） were within 1.8%-10.3%（n=6）.The detection limits of tebuconazole，metalaxyl-M，thiophanate-methyl，oxytetracycline and abamectin were 0.6，0.2，0.3，0.2，5.0 μg/kg，and the quantitation limits of 5 pesticides were 2.0，0.5，1.0，0.6 and 10.0 μg/kg respectively.Conclusion： The established method was proved to be sensitive，simple and reliable，and could be applied for the determination of these five pesticides in Anoectochilus roxburghii and quality control in the planting process.

LED Lights Promote Growth and Flavonoid Accumulation of Anoectochilus roxburghii and Are Linked to the Enhanced Expression of Several Related Genes.

Anoectochilus roxburghii is a wild edible species and has been traditionally used for a wide range of diseases in many countries. Our research aims to find the optimal light-emitting diode (LED) lighting conditions to improve the growth and development of A. roxburghii seedling at the acclimation stage. Two-month-old explants were cultured under the various lighting conditions including red (R), blue (B), BR (one blue: four red), BRW151 (one blue: five red: one white), BRW142 (one blue: four red: two white), and fluorescent lamp (FL). The results showed that the lighting conditions not only affect the growth and morphology of plants but also the accumulation of total flavonoids. Single wavelengths (B or R LED) inhibited the growth and secondary biosynthesis of A. roxburghii, while the BR LED showed an enhancement in both growth and biomass accumulation. A. roxburghii plants were grown under BR LED light has average plant height (7.18 cm), stem diameter (17.6mm), number of leaves (5.78 leaves/tree), leaf area (4.67 cm2), fresh weight (0.459 g/tree), dry matter percentages (11.69%), and total flavonoid (1.811 mg/g FW) is considered to be superior to FL lamps and other LEDs in the experiment. This indicates that both blue and red wavelengths are required for the normal growth of A. roxburghii. To learn more about how light affects flavonoid biosynthesis, we evaluated the expression of genes involved in this process (pal, chs, chi, and fls) and found that BR LED light enhances the expression level of chi and fls genes compared to fluorescent lamps (1.18 and 1.21 times, respectively), leading to an increase in the flavonoid content of plant. Therefore, applying BR LED during in vitro propagation of A. roxburghii could be a feasible way to improve the medicinal value of this plant.

Improving the yield of Anoectochilus roxburghii by Bacillus licheniformis cultured in Agaricus bisporus industrial wastewater

There is a large amount of industrial wastewater produced by the mushroom industry during the canning processing each year, which could provide abundant carbon, nitrogen and inorganic salts for microbial growth. The aim of this study was to optimize the culture conditions for Bacillus licheniformis cultured in the Agaricus bisporus industrial wastewater to produce the agricultural microbial fertilizer. In this work, the maximal biomass of B. licheniformis could be obtained under the following culture conditions: 33.7°C, pH 7.0, 221 rpm shaking speed, 0.5% wastewater, 2 (v:v, %) inoculum dose, loading liquid of 60 mL/250 mL and a culture time of 24 h, and the average experimental value obtained was 1.35 ± 0.04 × 10 9 Obj/mL, which was within the 95% confidence interval of the predicted model (1.29–1.38 × 10 9 Obj/mL), and met the national microbial fertilizers' standard in China. Furthermore, the field experiment results showed that the fermentation broth of B. licheniformis could significantly improve the yield of Anoectochilus roxburghii . Agaricus bisporus industrial wastewater can be used to produce agricultural microbial fertilizer. How to cite: Huang J, Huang A, Lu L, et al. Improving the yield of Anoectochilus roxburghii by Bacillus licheniformis cultured in Agaricus bisporus industrial wastewater. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.08.002.

Chemical Constituents from Endophytic Fungus Annulohypoxylon cf. stygium in Leaves of Anoectochilus roxburghii.

The chemical investigation on endophytic fungus Annulohypoxylon cf. stygium in leaves of Anoectochilus roxburghii (Wall.) Lindl. has been performed. Sixteen compounds were isolated and their structures were identified as (-)-notoamide A, (-)-notoamide B, (+)-versicolamide B, notoamide C, notoamide D, stephacidin A, sterigmatocystin, dihydrosterigmatocystin, secosterigmatocystin, versiconol, averufanin, kipukasin D, kipukasin E, diorcinal, palmarumycin CP2 and (-)-(3R)-mellein methyl ether, respectively, by spectroscopic analysis and comparison with literature data. All the compounds were isolated from Annulohypoxylon genus for the first time. Sterigmatocystin and palmarumycin CP2 showed selective cytotoxic activities against HepG2, HeLa, MCF-7 and HT-29.

An integrated analysis of metabolomic and transcriptomic profiles reveals flavonoid metabolic differences between Anoectochilus roxburghii and Anoectochilus formosanus

Anoectochilus roxburghii (Wall.) Lindl (Ar) and Anoectochilus formosanus Hayata (Af) are two species of valuable Chinese herbal medicines that belong to the genus Anoectochilus in Orchidaceae and have often been applied as the Chinese herbal medicine “Jin Xian Lian”. However, these species exhibit obvious differences in leaf color, and the difference in their medicinal value remains unclear. In this study, the metabolomes of leaf samples of Ar and Af were analyzed by high-performance liquid chromatography-mass spectrometry (LC–MS) in the positive and negative ion modes. The analysis of the leaves of both species in the positive ion mode identified 4013 metabolites enriched in 106 metabolic pathways, and the analysis in the negative ion mode found 1530 metabolites enriched in 98 metabolic pathways. A differential metabolic profiling analysis of the results obtained in positive ion mode found that 816 differentially accumulated metabolites were enriched in 80 metabolic pathways, whereas 297 differentially accumulated metabolites enriched in 55 metabolic pathways were obtained from the analysis in the negative ion mode. The number of flavonoids was higher than those of alkaloids and terpenoids. A quantitative analysis of the differentially accumulated metabolites showed that the most highly upregulated metabolites (Af/Ar) among the top 20 differentially accumulated metabolites in Af were mainly related to amino acid-related metabolic pathways, whereas the most highly downregulated metabolites (Af/Ar) in Af were mainly associated with flavonoid-related metabolic pathways. In addition, the transcriptome sequencing of Ar and Af leaf samples yielded 40.12 Gb of transcriptomic data. The analysis of differentially expressed genes (DEGs) classified 555 metabolism-related DEGs into 23 metabolism-related KEGG pathways, and the top DEGs were involved in the phenylpropanoid biosynthetic pathways, which included up to 138 DEGs associated with the flavonoid metabolism pathway. In addition, significant differences in the flavonoid-related KEGG pathways were found. A comprehensive analysis of the metabolomes and transcriptomes showed that the genes annotated as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), caffeoyl-CoA O-methyltransferase (CCOAOMT) and anthocyanidin synthase (ANS) were downregulated in Af leaves compared with Ar leaves, which might account for the downregulation of flavonoid-related metabolites in Af. In summary, both the metabolomic and transcriptomic analyses showed significant differences in flavonoid metabolism between the Ar and Af leaves, and the mechanisms underlying the molecular regulation of these differences were preliminarily analyzed.

A new abietane-type diterpenoid and a new long-chain alkenone from fungus Daldinia sp. TJ403-LS1

A new abietane-type diterpenoid, dalterpenoid A (1), a new long-chain alkenone derivative, (3E,5E,10E)-8-hydroxytrideca-3,5,10,12-tetraen-2-one (2), together with six known compounds (3–8), namely epi-guaidiol A (3), xylaranol A (4), daldinone C (5), trans-3,4-dihydroxy-3,4-dihydro-anofinic acid (6), (R)-6-hydroxymellein (7), helicascolide A (8), were obtained from fungus Daldinia sp. TJ403-LS1, which was originally isolated from roots of the medicinally valuable plant Anoectochilus roxburghii. The structures of compounds 1 and 2 were established based on widespread spectroscopic methods, mainly including 1D & 2D NMR and HRESIMS analyses, and the absolute configuration of 1 was further confirmed by electronic circular dichroism (ECD) calculation. All new compounds were tested for the in vitro cytotoxicity against five human cancer cell lines.

Scavenging of hydroxyl radical by kinsenoside originated from anoectochilus roxburghii: A DFT approach

- A density functional theory (DFT) study on the hydroxyl radical scavenging properties of Kinsenoside originated from Anoectochilus roxburghii is presented. Two mechanisms, single electron transfer (SET) and hydrogen atom transfer (HAT) are considered. The thermochemical results demonstrate that the SET mechanism is not plausible to occur. With respect to the HAT, the interaction of OH• radical with Kinsenoside is also studied in detail by establishing potential energy surface (PES). This result strongly confirms that the C8H bond decides the radical scavenging activity of Kinsenoside with activation Gibbs free energy (∆G≠) and rate constants (k) of 1.1 kcal/mol and 5.9×10-8 cm3/molecules, respectively

Coculture with two Bacillus velezensis strains enhances the growth of Anoectochilus plants via promoting nutrient assimilation and regulating rhizosphere microbial community

The potentiality of PGPR and biocontrol bacteria in agriculture is steadily increased as it offers an attractive way to replace the use of chemical fertilizers, pesticides and other supplements. This study aimed to investigate the effects and potential mechanism of inoculation with two Bacillus velezensis strains on the growth and quality improvement of Anoectochilus roxburghii (Wall.) Lindl. (MRH) and Anoectochilus formosanus (Wall.) Lindl. (YYB). The control treatment was non-inoculated (CK) and the coculture treatments were inoculated with Bacillus velezensis D2WM (D2), Bacillus velezensis ZJ-11 (ZJ-11), Bacillus velezensis D2WM and Bacillus velezensis ZJ-11 (D2+ZJ-11). The fresh weight, plant length, amount of active compounds, as well as chlorophyll content were significantly increased under D2, ZJ-11, and D2+ZJ-11 treatments compared with MRH and YYB plants in CK. In particular, D2+ZJ-11 treatment resulted in the greatest growth promotion. In MRH and YYB plants, the fresh weight increased by 82.6 % and 106.6 %, the kinsenoside content increased by 9.33 % and 21.65 % per gram, and the flavonoid content increased by 44.70 % and 21.07 % per gram, respectively. D2WM and ZJ-11 were capable of secreting siderophore, phytase, indole-3-acetic acid (IAA), and zeatin to promote plant nutrient assimilation and growth. Moreover, when D2WM and ZJ-11 were both inoculated with Anoectochilus roxburghii, the rhizosphere soil enzyme activities of urease, phosphatase, and invertase were significantly higher than control, thus helped to provide more nutrients to Anoectochilus. Further, the analysis of microbial community diversity indicated the increase of abundance of beneficial microorganisms, such as nitrogen-fixing bacteria, Basidiomycota and Ascomycota both of which are known to fuel their plant partners with mineral nutrients from the soil. Additionally, the abundance of pathogens belonging to Xanthobacteraceae as well as Cladophialophora and Penicillium were reduced. Hence, coculture with beneficial microorganisms can improve the growth and quality of Anoectochilus plants via promoting nutrient assimilation and regulating rhizosphere microbial community. This technique is of great application value in promoting the efficient cultivation and production of high-quality plant materials.

Anoectochilus roxburghii polysaccharide prevents carbon tetrachloride-induced liver injury in mice by metabolomic analysis

Anoectochilus roxburghii (Wall.) Lindl, a traditional Chinese medicine, is used for the effective treatment of liver disease in China. Anoectochilus roxburghii polysaccharide (ARPT) is an important constituent of Anoectochilus roxburghii. ARPT exerts a hepatoprotective effect and contributes directly to the therapeutic benefit of Anoectochilus roxburghii. However, the hepatoprotective mechanism of ARPT requires further elucidation. The present study was designed to assess the effects and underlying mechanism of ARPT when used to pretreat carbon tetrachloride (CCl4)-induced liver injury in mice. Mice were randomly divided into three groups: control group (no ARPT treatment or liver injury), model group (liver injury induced with CCl4), and the ARPT group (ARPT pretreatment followed by liver injury). A metabolomic method, based on liquid chromatography combined with mass spectrometry (LC-MS) and pattern recognition analysis, was applied. The data were analyzed with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA), to determine differentiating metabolites in the serum and liver tissue between the experimental groups. The PCA and OPLS-DA scores plots of the serum and liver tissue samples based on the LC-MS data showed a clear separation between the control and liver injury model group, while the ARPT-treated group showed a trend of close with the control. There were eleven metabolites [PS(O-18:0/0:0), phosphocholine, phenylalanine, hippuric acid, α-ketoisovaleric acid, metyrosine, leucinic acid, ketoleucine, Cer(d18:1/19:0), α-kamlolenic acid, and 4-formyl indole] were identified as candidate biomarkers in the serum samples, eight such metabolites (valine, phosphohydroxypyruvic acid, phosphocholine, ornithine, indole, xanthine, uridine, and glucose 6-phosphate) were found in the liver tissue samples, and one metabolite (phosphocholine) was observed in both the serum and liver tissue samples. These endogenous metabolites are considered to be in response to the hepatoprotective effects of ARPT and are involved in amino acid metabolism, lipid metabolism, gut bacteria metabolism, energy metabolism, and the methylation pathway. These findings suggest that ARPT mitigates the metabolic effect of CCl4-induced hepatotoxicity in mice by affecting at least five different pathways. LC-MS-based metabolomics provides a powerful approach for identifying potential biomarkers and for elucidating the protective mechanisms of traditional Chinese medicines against disease.

Three-phase hollow fiber liquid-phase microextraction combined with HPLC for determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl.

The analysis of plant growth regulators presents a challenge due to their trace quantities and complex matrices. A novel, simple, and effective analytical method for the determination of three trace acidic plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl was developed to address this issue. Three-phase hollow fiber liquid-phase microextraction combined with high-performance liquid chromatography was applied for the enrichment, purification, and determination of three acidic plant growth regulators, namely, indole-3-acetic-acid, indole-3-butyric-acid, and (+)-abscisic acid. The factors affecting extraction performance, including extractant species, pH of donor and acceptor phases, salt addition dosage, extraction time, temperature, and stirring rate, were investigated and optimized. Under optimum conditions, the proposed method provided good linearity (R2 , 0.9994-0.9999), low limit of detection (0.038-0.12ng/mL), and acceptable relative recoveries (56.7-117.6%). The enrichment factors were between 153 and 328. The developed method was successfully applied to the enrichment and determination of plant growth regulators in Anoectochilus roxburghii (Wall.) Lindl and exhibited increased purification capacity, higher sensitivity, and decreased organic solvent consumption compared with conventional sample preparation methods. This method may provide atesting platform for the monitoring of plant growth regulator residues, ensuring the safe and effective use of traditional Chinese medicine.

First Report of Gray Mold on Jinxianlian (Anoectochilus roxburghii) Caused by Botrytis cinerea in China

Anoectochilus roxburghii is one of the important herbal medicines in China. As a national rare and endangered plant, A. roxburghii is presently cultivated in bottles through greenhouse productions. The cultivation of A. roxburghii in Zhejiang Province accounts for a large proportion in China. Gray mold was found to severely affect the production of A. roxburghii. In 2018, the incidence of gray mold indicated by water-soaked gray mold symptoms with white to gray mycelia and conidiogenous apparatus on infected stems and leaves was observed for 6% of seedling bottles. When the conditions are suitable, gray mold spreads to the whole leaf and then gradually turns into grayish black and produces brown dead plaque, which causes the plant to wrinkle in severe cases. Thirty-one diseased leaves collected from different greenhouses in Jinhua City were picked with a needle on 2% potato dextrose agar (PDA) and cultured in the dark at 23°C. The colonies on the PDA were initially grayish white, followed by brownish black, and produced one-celled, round conidia (8.78 ± 0.4 × 12.60 ± 0.3 μm). These morphological features are consistent with Botrytis cinerea. In order to confirm the identification based on morphological features, DNA-dependent RNA polymerase subunit II (RPB2), heat-shock protein 60 (HSP60), and glyceraldehyde-3-phosphate (G3PDH) were amplified from the DNA extracted from the mycelium produced on PDA (Berbee et al. 1999; Liu et al. 1999; Smith 1989). The sequences were deposited in NCBI under accession numbers RPB2 (MN464111, MN464112, MN464113), HSP60 (MN464108, MN464109, MN464110), and G3PDH (MN464105, MN464106, MN464107). BLAST analysis of the RPB2 (1,106 bp) segment showed 100% identity to the RPB2 sequence of B. cinerea isolate S4GBR40 (GenBank accession no. MK3880531). The HSP60 (956 bp) segment showed 100% identity to the HSP60 sequence of B. cinerea isolate F2-17 (GenBank accession no. MK256903). And the G3PDH (994 bp) gene segment showed 100% identity to B. cinerea isolate GZFQ-1 (GenBank accession no. MH479930). Phylogenetic analysis based on RPB2, HSP60, and G3PDH showed that the pathogen was B. cinerea. Uninfected A. roxburghii healthy plants were sprayed with 2 × 10⁶ conidia/ml in distilled water as inoculum, and plants inoculated with only distilled water were adopted as the blank control. For each isolate, 16 intact plants were inoculated. Plants were then kept at 25°C and 95% relative humidity in a 12-h light/dark incubation. After 10 days, all three tested isolates caused gray mold symptoms on A. roxburghii, whereas no symptoms appeared in the controls. Cultures reisolated from lesions and cultured on PDA exhibited the same morphological characteristics as B. cinerea (Carmichael et al. 2019), confirming Koch’s postulates. The inoculations were performed twice. To the best of our knowledge, this is the first report of the gray mold in A. roxburghii, which is the main factor limiting its productions. Measures must be taken to manage the disease in a timely manner.

New secondary metabolites with immunosuppressive activity from the phytopathogenic fungus Bipolaris maydis.

Three previously undescribed compounds, including a meroterpenoid, guignardone T (1), and two ophiobolin-type sesterterpenoids, maydispenoids A and B (2 and 3), along with four known compounds (4-7), were isolated from the phytopathogenic fungus Bipolaris maydis collected from Anoectochilus roxburghii (Wall.) Lindl leaves. The structures of all undescribed compounds were elucidated by spectroscopic analysis, electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction. Structurally, maydispenoids A was characterized by a fascinating decahydro-3-oxacycloocta[cd]pentalene fragment. It is notable that the compounds 2 and 3 exhibited potential inhibitory activity in anti-CD3/anti-CD28 monoclonal antibodies (mAbs) stimulated murine splenocytes proliferation, with IC50 values of 5.28 and 9.38μM, respectively, and also suppress the murine splenocytes proliferation activated by lipopolysaccharide (LPS), with IC50 values of 7.25 and 16.82μM, respectively. This is the first report of ophiobolin-type sesterterpenoids as immunosuppressor, and may provide new chemical templates for the development of new immunosuppressive drugs for autoimmune disease treatment.

New bioactive secondary metabolites from the Anoectochilus roxburghii endophytic fungus Aspergillus versicolor

Six new compounds, including two new isochromane lactones, versicoisochromanes A and B (1 and 2), two new benzolactones, versicobenzos A and B (3 and 4), one furancarboxylic derivate, asperfuran A (6) and one ergosterol-type steroid, asperergoster A (7), along with five known steroids (8–12), were isolated from the Anoectochilus roxburghii endophytic fungus Aspergillus versicolor. The structures of these new compounds were determined by extensive spectroscopic techniques and electronic circular dichroism (ECD) calculations. It is notable that the new compound 7 exhibited obvious IL-1β, NO and TNF-α inhibitory activity in LPS-stimulated RAW264.7 macrophages, with IC50 values of 35.5, 33.9 and 31.3 μM, respectively. Furthermore, compounds 7 and 8 displayed potential inhibitory effects on murine splenocytes proliferation stimulated by anti-CD3/anti-CD28 monoclonal antibodies (mAbs), meanwhile suppress the lipopolysaccharide (LPS) irritated murine splenocytes proliferation.

Beneficial Effects of Endophytic Fungi from the Anoectochilus and Ludisia Species on the Growth and Secondary Metabolism of Anoectochilus roxburghii.

Endophytic fungi possess favorable effects on their host plants, including disease-resistance improvement, secondary metabolite induction, and growth promotion. It is therefore a promising and sustainable strategy to utilize endophytic fungi for the quality improvement of medicinal herbs or important crops. In our study, a collection of 277 strains of endophytic fungi were isolated from Anoectochilus and Ludisia orchids. Two strains J162 and J211 can be symbiotically cocultured with the tissue culture seedlings of Anoectochilus roxburghii, a popular medicinal and edible plant in southern China. Both strains can significantly enhance the biomass of A. roxburghii and induce the biosynthesis and accumulation of its active ingredients, including flavonoids, kinsenoside, and polysaccharides. J162 and J211 were further identified as Chaetomium globosum and Colletotrichum gloeosporioides based on multilocus phylogenetic analysis. Immunocytochemical staining indicated that J162 and J211 mainly colonized the intercellular gap of xylem parenchyma cells of A. roxburghii roots without obvious harm. In addition, quantitative real-time polymerase chain reaction showed that the expression of three growth-related genes, namely, uracil phosphoribosyl transferase, amino acid transmembrane transporter, and maturase K, were significantly altered in A. roxburghii plants when treated with J162 and J211. In conclusion, the two strains are highly beneficial microbial resources for the growth and accumulation of active ingredients of A. roxburghii in agricultural cultivation.