Discovery of 23,24-diols containing ergosterols with anti-neuroinflammatory activity from Penicillium citrinum TJ507.

Citristerones A-E (1-5), five new 23,24-diols containing ergosterols, along with three known analogues, were isolated from the endophytic fungus Penicillium citrinum TJ507 obtained from Hypericum wilsonii N. Robson. Their structures and absolute configurations were determined by NMR, HRESIMS, Snatzke's method, X-ray diffraction analyses and ECD calculation. Subsequently, the anti-neuroinflammatory effects of these isolates were screened using lipopolysaccharide (LPS)-induced BV-2 microglial cells, and citristerone B (2) showed outstanding anti-neuroinflammatory activity, with IC50 value of 0.60 ± 0.04 µM. Moreover, immunofluorescence and western blot analysis suggested that citristerone B not only reduced the release of nitric oxide (NO) and proinflammatory cytokines in LPS-induced BV-2 microglial cells, but also significantly inhibited the expression of TNF-α, iNOS and NF-κB, along with the production of cellular ROS.

Five previously undescribed citrinin derivatives from the endophytic fungus Penicillium citrinum GZWMJZ-836.

Penicillium citrinum GZWMJZ-836 is an endophytic fungus from Drynaria roosii Nakaike. Five previously undescribed citrinin derivatives (1-5) and six intermediates related to their biosynthesis (6-11) were obtained from the extract of this strain's solid fermentation using multiple column chromatography separations, including high-performance liquid chromatography. The structures of these compounds were determined through comprehensive spectroscopic analyses, primarily using NMR and HRESIMS data. The stereochemistry was mainly confirmed by ECD calculations, and the configurations of C-7' in compounds 4 and 5 were determined using 13C NMR calculations. Compounds 4-5 and 8 showed antibacterial activity against five strains, with minimum inhibitory concentration values ranging from 7.8 to 125 µM. Compounds 4 and 7 exhibited inhibitions against three plant pathogenic fungi, with IC50 values ranging from 66.6 to 152.1 µM. Additionally, a putative biosynthetic pathway for compounds 1-5 derived from citrinin was proposed.

Citrisorbicillinol, an undescribed hybrid sorbicillinoid with osteogenic activity from Penicillium citrinum ZY-2.

Citrisorbicillinol (1), along with six other known compounds (2-7), was isolated from an endphyte Penicillium citrinum ZY-2 of Plantago asiatica L. Citrisorbicillinol (1) was characterized as a skeletally unprecedented hybrid sorbicillinoid, and its unique framework is likely formed by intermolecular [4 + 2] cycloaddition between intermediates derived from citrinin and sorbicillinoid biosynthetic gene clusters. Compounds 1 and 2 demonstrated to promote osteoblastic differentiation in MC3T3-E1 cells, and to be osteogenic in the prednisolone induced osteoporotic zebrafish. Compounds 3-7 exhibited moderate cytotoxicity against four human cancer cell lines.

Antimicrobial and Cytotoxic Secondary Metabolites from a Marine-Derived Fungus Penicillium Citrinum VM6.

Investigation of an antimicrobial and cytotoxic ethyl acetate extract prepared from solid fermentation of the marine-derived fungus Penicillium citrinum VM6 led to the isolation of eight metabolites (1-8), including one citrinin dimer dicitrinone F (1). Of these, compound 7 was isolated for the first time from the Penicillium genus and compound 1 with carbon-bridged C-7/C-7' linkage is rarely reported. All compounds (1-8) exhibited selective antimicrobial activity against the tested Gram-positive bacteria and Candida albicans with MICs of 32-256 µg/mL. Compounds 1 and 8 exhibited cytotoxicity against all tested cell lines A549, MCF7, MDA-MB-231, Hela, and AGS with IC50 values of 6.7 ± 0.2 to 29.6 ± 2.2 µg/mL, whereas compound 5 had selective cytotoxicity against the MCF7 cell lines with IC50 of 98.1 ± 7.8 µg/mL.

Penidihydrocitrinins A-C: New Polyketides from the Deep-Sea-Derived Penicillium citrinum W17 and Their Anti-Inflammatory and Anti-Osteoporotic Bioactivities.

Three new polyketides (penidihydrocitrinins A-C, 1-3) and fourteen known compounds (4-17) were isolated from the deep-sea-derived Penicillium citrinum W17. Their structures were elucidated by comprehensive analyses of 1D and 2D NMR, HRESIMS, and ECD calculations. Compounds 1-17 were evaluated for their anti-inflammatory and anti-osteoporotic bioactivities. All isolates exhibited significant inhibitory effects on LPS-stimulated nitric oxide production in murine brain microglial BV-2 cells in a dose-response manner. Notably, compound 14 displayed the strongest effect with the IC50 value of 4.7 µM. Additionally, compounds 6, 7, and 8 significantly enhanced osteoblast mineralization, which was comparable to that of the positive control, purmorphamine. Furthermore, these three compounds also suppressed osteoclastogenesis in a dose-dependent manner under the concentrations of 2.5 µM, 5.0 µM, and 10 µM.

Chemical Constituents of the Deep-sea Gammarid Shrimp-Derived Fungus Penicillium citrinum XIA-16.

One new alkaloid, (S)-2-acetamido-4-(2-(methylamino)phenyl)-4-oxobutanoic acid (1), was isolated from the deep-sea-derived Penicillium citrinum XIA-16, together with 25 known compounds including ten polyketones (2-11), eight alkaloids (12-19), six steroids (20-25), and a fatty acid (26). Their planar and relative structures were determined by an analysis of 1D and 2D nuclear magnetic resonance (NMR) as well as high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) data. The absolute configuration of 1 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Penicitrinol B (6) significantly inhibited RSL3-induced ferroptosis (EC50 =2.0 µM) by reducing lipid peroxidation and heme oxygenase 1 (HMOX1) expression. Under the concentration of 10 µM, penicitrinol A (7) was able to inhibit cuproptosis with the cell viabilities of 68.2 % compared to the negative control (copper and elesclomol) with the cell viabilities of 14.8 %.

Functional analysis of auxin derived from a symbiotic mycobiont.

The biosynthesis of auxin or indole-3-acetic acid by microorganisms has a major impact on plant-microbe interactions. Several beneficial microbiota are known to produce auxin, which largely influences root development and growth in the host plants. Akin to findings in rhizobacteria, recent studies have confirmed the production of auxin by plant growth-promoting fungi too. Here, we show that Penicillium citrinum isolate B9 produces auxin as deduced by liquid chromatography tandem-mass spectrometry analysis. Such fungal auxin is secreted and contributes directly to enhanced root and shoot development and overall plant growth in Arabidopsis thaliana. Furthermore, auxin production by P. citrinum likely involves more than one tryptophan-dependent pathway. Using auxin biosynthesis inhibitor L-Kynurenine, we show that the indole-3-pyruvate pathway might be one of the key biosynthetic routes involved in such auxin production. Confocal microscopy of the DR5rev:GFP Arabidopsis reporter line helped demonstrate that P. citrunum B9-derived auxin is biologically active and is able to significantly enhance auxin signaling in roots during such improved root growth and plant development. Furthermore, the phenotypic growth defects arising from impaired auxin signaling in Arabidopsis taa1 mutant or upon L-Kynurenine treatment of wild-type Arabidopsis seedlings could be significantly alleviated by fungus B9-derived auxin, thus suggesting its positive role in plant growth promotion. Collectively, our results provide clear evidence that the production of auxin is one of the main mechanisms involved in induction of the beneficial plant growth by P. citrinum.

Safety evaluation of the food enzyme ribonuclease P from the non-genetically modified Penicillium citrinum strain AE-RP-4.

The food enzyme ribonuclease P (EC 3.1.26.5) is produced with the non-genetically modified Penicillium citrinum strain AE-RP-4 by Amano Enzyme Inc. It is intended to be used in yeast processing only for the production of yeast extract. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.153 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 134.7 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 880. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Non-monotonic dose-response of di-(2-ethylhexyl) phthalate isolated from Penicillium citrinum XT6 on adipogenesis and expression of PPARγ and GLUT4 in 3T3-L1 adipocytes.

OBJECTIVES: Adipogenesis is the fat cell formation process regulated by peroxisome proliferator-activated receptors (PPARγ). The insulin-responsive glucose transporter 4 (GLUT4) has a major role in glucose uptake and metabolism in insulin target tissues (i.e., adipose and muscle cells). The interplay between PPARγ and GLUT4 is essential for proper glucose homeostasis. This study aimed to isolate, elucidate, and investigate the effect of an isolated compound from Penicillium citrinum XT6 on adipogenesis, PPARγ, and GLUT4 expression in 3T3-L1 adipocytes. METHODS: The isolated compound was determined by analyzing spectroscopic data (LC-MS, FT-IR, Spectrophotometry UV-Vis, and NMR). The adipogenesis activity of the isolated compound in 3T3-L1 cells was determined by the Oil Red O staining method. RT-PCR was used to analyze the gene expression of PPARγ and GLUT4. RESULTS: Di-(2-ethylhexyl)-phthalate (DEHP) was the isolated compound from P.citrinum XT6. The results revealed adipogenesis stimulation and inhibition, as well as PPARγ and GLUT4 expressions. CONCLUSIONS: DEHP showed a non-monotonic dose-response (NMDR) effect on adipogenesis and PPARγ and GLUT4 expression. It is the first study that reveals DEHP's NMDR effects on lipid and glucose metabolism in adipocytes.

Quinolinones Alkaloids with AChE Inhibitory Activity from Mangrove Endophytic Fungus Penicillium citrinum YX-002.

Acetylcholinesterase (AChE) inhibitory activity-guided studies on the mangrove-derived endophytic fungus Penicillium citrinum YX-002 led to the isolation of nine secondary metabolites, including one new quinolinone derivative, quinolactone A (1), a pair of epimers quinolactacin C1 (2) and 3-epi-quinolactacin C1 (3), together with six known analogs (4-9). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses, and compared with data in the literature. The absolute configurations of compounds 1-3 was determined by combination of electronic circular dichroism (ECD) calculations and X-Ray single crystal diffraction technique using CuKα radiation. In bioassays, compounds 1, 4 and 7 showed moderate AChE inhibitory activities with IC50 values of 27.6, 19.4 and 11.2 µmol/L, respectively. The structure-activity relationships (SARs) analysis suggested that the existence of carbonyl group on C-3 and the oxygen atom on the five-membered ring were beneficial to the activity. Molecular docking results showed that compound 7 had a lower affinity interaction energy (-9.3 kcal/mol) with stronger interactions with different sites in AChE activities, which explained its higher activities.

Bioleaching of valuable metals from spent LIBs followed by selective recovery of manganese using the precipitation method: Metabolite maximization and process optimization.

Despite the increased demand for resource recovery from spent lithium-ion batteries (LIBs), low Mn leaching efficiencies have hindered the development of this technology. A novel process was devised to enhance the dissolution of metals by producing citric acid using a molasses medium by Penicillium citrinum. This investigation used response surface methodology to investigate the influence of molasses concentration and media components on citric acid production, which demonstrated that molasses (18.5% w/w), KH2PO4 (3.8 g/L), MgSO4.7H2O (0.11 g/L), and methanol (1.2% (v/v)) were the optimum values leading to the production of 31.50 g/L citric acid. Afterward, optimum inhibitor concentrations (iodoacetic acid: 0.05 mM) were added to accumulate citric acid, resulting in maximum bio-production (40.12 g/L) of citric acid. The pulp density and leaching time effect on metals dissolution was investigated in enriched-citric acid spent medium. The suitable conditions were a pulp density of 70 g/L and a leaching duration of 6 days, which led to the highest dissolution of Mn (79%) and Li (90%). Based on the results of the TCLP tests, the bioleaching residue is non-hazardous, suitable for safe disposal, and does not pose an environmental threat. Moreover, nearly 98% of Mn was extracted from the bioleaching solution with oxalic acid at 1.2 M. XRD, and FE-SEM analyses were utilized for further bioleaching and precipitation mechanism analysis.

Penicillium citrinum Provides Transkingdom Growth Benefits in Choy Sum (Brassica rapa var. parachinensis).

Soil-borne beneficial microbes establish symbioses with plant hosts and play key roles during growth and development therein. In this study, two fungal strains, FLP7 and B9, were isolated from the rhizosphere microbiome associated with Choy Sum (Brassica rapa var. parachinensis) and barley (Hordeum vulgare), respectively. Sequence analyses of the internal transcribed spacer and 18S ribosomal RNA genes combined with colony and conidial morphology identified FLP7 and B9 to be Penicillium citrinum strains/isolates. Plant-fungus interaction assays revealed that isolate B9 showed significant growth promotion effects in Choy Sum plants cultivated in normal soil, as well as under phosphate-limiting conditions. In comparison to the mock control, B9-inoculated plants showed a 34% increase in growth in aerial parts and an 85% upsurge in the fresh weight of roots when cultivated in sterilized soil. The dry biomass of such fungus-inoculated Choy Sum increased by 39% and 74% for the shoots and roots, respectively. Root colonization assays showed that P. citrinum associates directly with the root surface but does not enter or invade the root cortex of the inoculated Choy Sum plants. Preliminary results also indicated that P. citrinum can promote growth in Choy Sum via volatile metabolites too. Interestingly, we detected relatively higher amounts of gibberellins and cytokinins in axenic P. citrinum culture filtrates through liquid chromatography-mass spectrometry analyses. This could plausibly explain the overall growth induction in P. citrinum-inoculated Choy Sum plants. Furthermore, the phenotypic growth defects associated with the Arabidopsis ga1 mutant could be chemically complemented by the exogenous application of P. citrinum culture filtrate, which also showed accumulation of fungus-derived active gibberellins. Our study underscores the importance of transkingdom beneficial effects of such mycobiome-assisted nutrient assimilation and beneficial fungus-derived phytohormone-like metabolites in the induction of robust growth in urban farmed crops.

Anti-IAV indole-diterpenoids from the marine-derived fungus Penicillium citrinum.

A new indole-diterpenoid, penijanthine E (1), and a known analogue (2), were obtained from the PDB culture of the marine-derived fungus Penicillium citrinum ZSS-9. The absolute configuration of 1 was elucidated by calculated TDDFT ECD and DP4plus calculations. The absolute configuration of 2 was confirmed by single-crystal X-ray diffraction analysis and TDDFT ECD calculations. Compounds 1 and 2 showed antiviral activity against influenza A virus (IAV) of A/WSN/33(H1N1) and A/PR/8/34(H1N1) strains with IC50 values ranging from 12.6 to 46.8 µM.

Biodegradation of low density polyethylene (LDPE) by mesophilic fungus 'Penicillium citrinum' isolated from soils of plastic waste dump yard, Bhopal, India.

Low density Polyethylene (LDPE) in various forms has become a part of life. Its accretion due to non degradable nature is concern, endangering life on earth. Amongst various methods of LDPE disposal bioremediation is regarded as ecofriendly & widely accepted. Current investigation was an attempt to isolate potent PE degrading fungus from municipal landfill soils of Bhopal, India loaded with plastic waste.16 fungal isolates were recorded from the site; PE deteriorating fungus was screened using mineral salt agar medium amended with 3% LDPE powder as sole carbon source. The isolate Penicillium citrinum showed fast fungal colony growth in screening medium was selected for biodegradation study. P.citrinum showed 38.82 ± 1.08% weight loss of untreated LDPE pieces; to improve the degradation capacity nitric acid pretreatment was performed; biodegradation was significantly stimulated by 47.22 ± 2.04% after it's pretreatment. Laccase, lipase, esterase & manganese peroxidase activities were assayed by spectrophotometer. LDPE biodegradation was analyzed by weight loss %, change in pH during fungal growth, field emission scanning electron microscopy (FE-SEM), fourier transform infrared spectroscopy (FTIR) & thermogravimetric analysis (TGA). FTIR spectra showed appearance of new functional groups assigned to hydrocarbon biodegradation, confirming enzymatic role in process. Changes in FTIR spectra of LDPE samples (untreated & pretreated) before & after biodegradation & surface changes in the biodegraded LDPE (indicated from FE-SEM) confirmed depolymerization of LDPE. Further changes in thermal decomposition rates of biodegraded samples in comparison to control, validate biodegradation. This is the first report signifying high competence of P.citrinum in LDPE degradation without prior pretreatment.

Chitosan production by Penicillium citrinum using paper mill wastewater and rice straw hydrolysate as low-cost substrates in a continuous stirred tank reactor.

In this study, paper mill wastewater and hemicellulose hydrolysate were evaluated as low-cost substrates for fungal chitosan production using Penicillium citrinum. Submerged fermentation was first studied using a bioreactor operated under batch, fed-batch and continuous modes with paper mill wastewater as the substrate. Very high removal (91%) of organics as chemical oxygen demand (COD) in the wastewater with 160 mg L-1 chitosan production by P. citrinum was obtained using the bioreactor operated under fed-batch mode for 72 h. Moreover, 86% reduction of phenolics in the wastewater with 89% decolourization efficiency was achieved in the fed-batch experiments with the bioreactor. Under the continuous mode of operation with the bioreactor, maximum chitosan production of 170 mg L-1 was observed. The effect of acetic acid addition to the wastewater for enhancing chitosan production by the fungus was further studied in a batch system. Chitosan productivity of 2.33 mg L-1 h-1 was obtained with 50 mg/L acetic acid. Various models, viz. Monod, Haldane, Andrews, Webb and Yano, were fitted to the experimental data for understanding the kinetics involved in the process. Haldane model accurately fitted the experimental data on biomass specific growth rate, acetic acid consumption rate and chitosan production rate by P. citrinum with acetic acid addition to the wastewater. Fungal fermentation of another low-cost substrate, rice straw hydrolysate, was further studied using the batch-operated bioreactor; and a maximum chitosan titre of 911 mg L-1 was achieved using the detoxified rice straw hydrolysate.Highlights Low-cost substrates for chitosan production by Penicillium citrinum are reportedAcetic acid addition to paper mill wastewater enhances chitosan productionBiomass growth and chitosan production follow substrate inhibition kineticsFed-batch -operated bioreactor resulted in 91% wastewater treatment efficiencyMaximum chitosan titre of 911 mg L-1 was achieved with rice straw hydrolysate.

Environmentally sustainable and cost-effective recycling of Mn-rich Li-ion cells waste: Effect of carbon sources on the leaching efficiency of metals using fungal metabolites.

Metals recovery from spent lithium coin cells (SCCs) is enjoying great attention due to environmental problems and metal-rich contents such as Mn and Li. Fungi can generate many organic acids, and metals can be dissolved, but sucrose is not an economical medium. The main objective of this study is to find a suitable carbon substrate in place of sucrose for fungal bioleaching. We have developed an environmentally friendly, cost-effective, and green method for recycling and detoxifying Mn and Li from SCCs using the spent culture medium fromPenicillium citrinumcultivation. Sugar cane molasses and sucrose were selected as carbon sources. Based on the extracted fungal metabolites, the effects of pulp density, temperature, and leaching time were assessed on metal dissolution. The most suitable conditions were 30 g/L of pulp density, a temperature of 40 °C, and 4 days of leaching time in spent molasses medium, which led to a high extraction of 87% Mn and 100% Li. Based on EDX-mapping analyses, it was found that the initial concentration of ∑ (Mn + C) in the SCCs powder was almost 100% while reaching nearly 6.4% after bioleaching. After bioleaching, an analysis of residual powder confirmed that metal dissolution from SCCs was effective owing to fungal metabolites. The economic study showed that the bioleaching method is more valuable for the dissolution of metals than the chemical method; In addition to improving bioleaching efficiency, molasses carbon sources can be used for industrial purposes.

Mass Spectrometry Metabolomics Approach Reveals Anti-Trichomonas vaginalis Scaffolds from Marine Fungi.

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world caused by Trichomonas vaginalis. Failures in the treatment with the 5-nitroimidazole class including parasite resistance to metronidazole elicit new alternatives. Marine natural products are sources of several relevant molecules, presenting a variety of metabolites with numerous biological activities. In this work, we evaluated the anti-T. vaginalis activity of fungi associated with marine invertebrates by mass spectrometry-based metabolomics approaches. After screening of six marine fungi, extract from Penicillium citrinum FMPV 15 has shown to be 100% active against T. vaginalis, and the gel permeation column on Sephadex LH-20® yielded twelve organic fractions which five showed to be active. Metabolomics and statistical analyses were performed with all the samples (extract and fractions), and several compounds were suggested to be related to the activity. These components include citrinin, dicitrinin C, citreoisocoumarin, dihydrocitrinone, decarboxycitrinin, penicitrinone C, and others. The minimum inhibitory concentration (MIC) value of anti-T. vaginalis activity of citrinin was 200 µM. The marine fungi metabolites show potential as new alternatives to overcome drug resistance in T. vaginalis infections.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.

First report of green mold disease caused by Penicillium citrinum on Dictyophora rubrovalvata in China.

Dictyophora rubrovolvata is a saprophytic mushroom widely cultivated in China, including Guizhou Province for its high nutritional, medicinal, and economical values (Chen et al. 2021). In May 2021, green mold disease was observed on the fruiting bodies of D. rubrovolvata, causing its death or preventing it from forming a sporocarp, in an indoor-production facility at Asuo village, Baiyun District Guiyang city, Guizhou Province, China (26°73'51" N, 106°72'88" E). The disease incidence was 60%-70% in the affected 1.33-ha growing area, causing a serious economic loss. To identify the causal agent, a total of 15 samples with symptomatic symptoms were collected. Small pieces (5 mm × 5 mm) were cut from the diseased tissues, surface sterilized in 0.4% NaClO for 5 min, washed three times with sterilized water, placed on potato dextrose agar (PDA) medium, and incubated at 24 °C for 7 days. Twenty-one pure cultures were obtained by single-spore isolation method. The colonies were initially white but after seven days as conidia developed they turned green. Hyphae were hyaline and guttulate. Conidiophores were verrucose stipes, triverticulate, and phialides flask shaped. Conidia were smooth and pale green, with subglobose to globose shape measuring 2.0-2.5 × 1.8-2.5 µm (n=50). Based on these morphological characteristics, the isolates matched the description of the genus Penicillium (Visagie et al. 2014). To confirm the identity, DNA of five representative isolates (QS001, QS005, QS008, QS015, QS017) was extracted according to the manufacturer's instructions (Biomiga Fungal DNA Extraction Kit; CA, USA). Afterwards, PCR was performed to amplify ITS region, calmodulin and ß-tubulin genes using primer pairs ITS1/ITS4 (White et al. 1990), CMD5/CMD6 (Glass et al. 1995), and Bt2a/Bt2b (Hong et al. 2006), respectively. BLASTN analysis of these sequences showed the best matches with Penicillium citrinum CBS 139.45 (ITS region: 98.60% (493/500 bp) identity to accession MH856132.1; CMD: 99.79% (469/470 bp) identity to accession MN969245.1; ß-tubulin:100% (407/407 bp) identity to accession GU944545.1). Representative sequences of the sequenced DNA regions were deposited in GenBank (ITS region: OK446552; CMD: OK492612; ß-tubulin: OK482677). Furthermore, a phylogenetic tree was constructed with MEGA 7 based on the concatenated sequences. Koch's postulates were met to confirm the pathogenicity of the representative isolate (QS001) on D. rubrovolvata. Six discs (5mm×5mm) from actively growing P. citrinum QS001 colonies (5-day-old) were placed on six fruiting bodies of D. rubrovolvata (5-month-old). Mock inoculations were performed using PDA discs only without any fungus. The inoculation sites were wrapped with a sterilized 200-µm nylon mesh. All fruiting bodies were incubated at 23°C ± 2°C under a 0-h/24-h photoperiod and 80% relative humidity (RH) after inoculation. After 14 days, green mold was observed on all P. citrinum QS001 inoculated mushrooms. In contrast, no disease was observed in mock inoculated group. The disease assays were repeated three times. P. citrinum QS001 was isolated from all inoculated D. rubrovolvata and verified via the molecular analysis mentioned above. To the best of our knowledge, this is the first report that P. citrinum causes green mold on D. rubrovalvata in China and further studies should focus on managing this disease to prevent any disease outbreaks.

Bisabolane Sesquiterpenes with Anti-Inflammatory Activities from the Endophytic Fungus Penicillium citrinum DF47.

Seven new bisabolane-type sesquiterpenes (1-7), namely penicibisabolanes A-G, together with eight known analogs (8-15) were obtained from the AcOEt extract of the millet fermentation broth of the endophytic fungus Penicillium citrinum DF47, which was isolated from the fresh root of Codonopsis pilosula (Franch.) Nannf. The gross structures of new metabolites were determined on the basis of the spectroscopic data (HR-ESI-MS, 1D and 2D NMR spectra), while their absolute configurations were resolved by comparison of the experimental and calculated ECD spectra, in association with specific rotation data. Compound 1 is a rare seco-trinor-bisabolane sesquiterpene found in nature, while 3 is the first example of phenolic bisabolanes bearing a methoxy group at C-1. All the isolates were evaluated their inhibitory effects against NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells. Among them, compounds 7 and 13 showed moderately anti-inflammatory effects with the inhibitory rate more than 50 % at the concentration of 20 µM.