Epilobium angustifolium L. Essential Oil-Biological Activity and Enhancement of the Skin Penetration of Drugs-In Vitro Study.

Epilobium angustifolium L. is a popular medicinal plant found in many regions of the world. This plant contains small amounts of essential oil whose composition and properties have not been extensively investigated. There are few reports in the literature on the antioxidant and antifungal properties of this essential oil and the possibility of applying it as a potential promoter of the skin penetration of drugs. The essential oil was obtained by distillation using a Clavenger type apparatus. The chemical composition was analyzed by the GC-MS method. The major active compounds of E. angustifolium L. essential oil (EOEa) were terpenes, including α-caryophyllene oxide, eucalyptol, ß-linalool, camphor, (S)-carvone, and ß-caryophyllene. The analyzed essential oil was also characterized by antioxidant activity amounting to 78% RSA (Radical Scavenging Activity). Antifungal activity against the strains Aspergillus niger, A. ochraceus, A. parasiticum, and Penicillium cyclopium was also determined. The largest inhibition zone was observed for strains from the Aspergillus group. The EOEa enhanced the percutaneous penetration of ibuprofen and lidocaine. After a 24 h test, the content of terpene in the skin and the acceptor fluid was examined. It has been shown that the main compounds contained in the essential oil do not penetrate through the skin, but accumulate in it. Additionally, FTIR-ATR analysis showed a disturbance of the stratum corneum (SC) lipids caused by the essential oil application. Due to its rich composition and high biological activity, EOEa may be a potential candidate to be applied, for example, in the pharmaceutical or cosmetic industries. Moreover, due to the reaction of the essential oil components with SC lipids, the EOEa could be an effective permeation enhancer of topically applied hydrophilic and lipophilic drugs.

Unravelling the Antifungal Effect of Red Thyme Oil (Thymus vulgaris L.) Compounds in Vapor Phase.

The aim of this work was to evaluate the antifungal activity in vapor phase of thymol, p-cymene, and γ-terpinene, the red thyme essential oil compounds (RTOCs). The Minimum Inhibitory Concentration (MIC) of RTOCs was determined against postharvest spoilage fungi of the genera Botrytis, Penicillium, Alternaria, and Monilinia, by measuring the reduction of the fungal biomass after exposure for 72 h at 25 °C. Thymol showed the lowest MIC (7.0 µg/L), followed by γ-terpinene (28.4 µg/L) and p-cymene (40.0 µg/L). In the case of P. digitatum ITEM 9569, resistant to commercial RTO, a better evaluation of interactions among RTOCs was performed using the checkerboard assay and the calculation of the Fractional Inhibitory Concentration Index (FICI). During incubation, changes in the RTOCs concentration were measured by GC-MS analysis. A synergistic effect between thymol (0.013 ± 0.003 L/L) and γ-terpinene (0.990 ± 0.030 L/L) (FICI = 0.50) in binary combinations, and between p-cymene (0.700 ± 0.010 L/L) and γ-terpinene (0.290 ± 0.010 L/L) in presence of thymol (0.008 ± 0.001 L/L) (FICI = 0.19), in ternary combinations was found. The synergistic effect against the strain P. digitatum ITEM 9569 suggests that different combinations among RTOCs could be defined to control fungal strains causing different food spoilage phenomena.

Transcriptome analysis reveals underlying immune response mechanism of fungal (Penicillium oxalicum) disease in Gastrodia elata Bl. f. glauca S. chow (Orchidaceae).

BACKGROUND: Gastrodia elata Bl. f. glauca S. Chow is a medicinal plant. G. elata f. glauca is unavoidably infected by pathogens in their growth process. In previous work, we have successfully isolated and identified Penicillium oxalicum from fungal diseased tubers of G. elata f. glauca. As a widespread epidemic, this fungal disease seriously affected the yield and quality of G. elata f. glauca. We speculate that the healthy G. elata F. glauca might carry resistance genes, which can resist against fungal disease. In this study, healthy and fungal diseased mature tubers of G. elata f. glauca from Changbai Mountain area were used as experimental materials to help us find potential resistance genes against the fungal disease. RESULTS: A total of 7540 differentially expressed Unigenes (DEGs) were identified (FDR < 0.01, log2FC > 2). The current study screened 10 potential resistance genes. They were attached to transcription factors (TFs) in plant hormone signal transduction pathway and plant pathogen interaction pathway, including WRKY22, GH3, TIFY/JAZ, ERF1, WRKY33, TGA. In addition, four of these genes were closely related to jasmonic acid signaling pathway. CONCLUSIONS: The immune response mechanism of fungal disease in G. elata f. glauca is a complex biological process, involving plant hormones such as ethylene, jasmonic acid, salicylic acid and disease-resistant transcription factors such as WRKY, TGA.

Clove Essential Oil as an Alternative Approach to Control Postharvest Blue Mold Caused by Penicillium italicum in Citrus Fruit.

Penicillium italicum causes blue mold disease and leads to huge economic losses in citrus production. As a natural antifungal agent, clove essential oil (CEO), which is a generally recognized as safe (GRAS) substance, shows strong in vitro activity against fungal pathogens. However, few studies on CEO for controlling postharvest blue mold disease caused by P. italicum in citrus fruit have been reported. Our aims were to investigate the control efficacy and possible mechanisms involved of CEO against P. italicum. In the present study, CEO treatment inhibited the disease development of blue mold when applied at 0.05% to 0.8% (v/v), and with the effective concentration being obtained as 0.4% (v/v). Besides its direct antifungal activity, CEO treatment also spurred a rapid accumulation of H2O2 compared with untreated fruits, which might contribute to enhancing an increase in the activities of defense-related enzymes, such as ß-1,3-glucanase (ß-Glu), chitinase (CHI), phenylalanine ammonia-lyase (PAL), peroxidase (POD), polyphenol oxidase (PPO), and lipoxygenase (LOX) in citrus fruit. Results of real time-quantitative polymerase chain reaction (RT-qPCR) showed that the gene expressions of ß-Glu, CHI, PAL, POD and PPO were up-regulated in CEO-treated fruits. At the same time, CEO treatment led to down-regulated expression of the LOX gene in citrus fruit. Clove essential oil effectively control the disease incidence of blue mold decay in citrus fruit by motivating the host-defense responses, suppressing the malondialdehyde (MDA) accumulation while enhancing the activities and gene expressions of defense-related enzymes. Our study provides an alternative preservative applying CEO to reduce postharvest fungal decay in citrus fruit.

Mechanism of Cell Wall Polysaccharides Modification in Harvested 'Shatangju' Mandarin (Citrus reticulate Blanco) Fruit Caused by Penicillium italicum.

Modification of cell wall polysaccharide in the plant plays an important role in response to fungi infection. However, the mechanism of fungi infection on cell wall modification need further clarification. In this study, the effects of Penicillium italicum inoculation on 'shatangju' mandarin disease development and the potential mechanism of cell wall polysaccharides modification caused by P. italicum were investigated. Compared to the control fruit, P. italicum infection modified the cell wall polysaccharides, indicated by water-soluble pectin (WSP), acid-soluble pectin (ASP), hemicellulose and lignin contents change. P. italicum infection enhanced the activities of polygalacturonase (PG), pectin methylesterase (PME), and the expression levels of xyloglucanendotransglucosylase/hydrolase (XTH) and expansin, which might contribute to cell wall disassembly and cellular integrity damage. Additionally, higher accumulation of reactive oxygen species (ROS) via decreasing antioxidant metabolites and the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) also contributed to the cell wall polysaccharides modification. Meanwhile, the gene expression levels of hydroxyproline-rich glycoprotein (HRGP) and germin-like protein (GLP) were inhibited by pathogen infection. Altogether, these findings suggested that cell wall degradation/modification caused by non-enzymatic and enzymatic factors was an important strategy for P. italicum to infect 'shatangju' mandarin.

Population Pharmacodynamics of Amphotericin B Deoxycholate for Disseminated Infection Caused by Talaromyces marneffei.

Amphotericin B deoxycholate (DAmB) is a first-line agent for the initial treatment of talaromycosis. However, little is known about the population pharmacokinetics and pharmacodynamics of DAmB for talaromycosis. Pharmacokinetic data were obtained from 78 patients; among them, 55 patients had serial fungal CFU counts in blood also available for analysis. A population pharmacokinetic-pharmacodynamic model was fitted to the data. The relationships between the area under the concentration-time curve (AUC)/MIC and the time to blood culture sterilization and the time to death were investigated. There was only modest pharmacokinetic variability in the average AUC, with a mean ± standard deviation of 11.51 ± 3.39 mg·h/liter. The maximal rate of drug-induced kill was 0.133 log10 CFU/ml/h, and the plasma concentration of the DAmB that induced the half-maximal rate of kill was 0.02 mg/liter. Fifty percent of patients sterilized their bloodstreams by 83.16 h (range, 13 to 264 h). A higher initial fungal burden was associated with a longer time to sterilization (hazard ratio [HR], 0.51; 95% confidence interval [CI], 0.36 to 0.70; P < 0.001). There was a weak relationship between AUC/MIC and the time to sterilization, although this did not reach statistical significance (HR, 1.03; 95% CI, 1.00 to 1.06, P = 0.091). Furthermore, there was no relationship between the AUC/MIC and time to death (HR, 0.97; 95% CI, 0.88 to 1.08; P = 0.607) or early fungicidal activity {slope = log[(0.500 - 0.003·(AUC/MIC)]; P = 0.319} adjusted for the initial fungal burden. The population pharmacokinetics of DAmB are surprisingly consistent. The time to sterilization of the bloodstream may be a useful pharmacodynamic endpoint for future studies. (This study has been registered at the ISRCTN registry under no. ISRCTN59144167.).

Soil fungal biodiversity and pathogen identification of rotten disease in Aconitum carmichaelii (Fuzi) roots.

Aconitum carmichaelii, commonly known as Fuzi, is a typical traditional Chinese medicine (TCM) herb that has been grown for more than one thousand years in China. Although root rot disease has been seriously threatening this crop in recent years, few studies have investigated root rot disease in Fuzi, and no pathogens have been identified. In this study, fungal libraries from rhizosphere soils were constructed by internal transcribed spacer (ITS) sequencing using the HiSeq 2500 high-throughput platform. A total of 948,843 tags were obtained from 17 soil samples, and these corresponded to 195,583,495 nt. At 97% identity, the libraries yielded 12,266 operational taxonomic units (OTUs), of which 97.5% could be annotated. In sick soils, Athelia, Mucor and Mortierella were the dominant fungi, comprising 10.3%, 10.1% and 7.7% of the fungal community, respectively. These fungi showed 2.6-, 1.53- to 6.31- and 1.38- to 2.65-fold higher enrichment in sick soils compared with healthy soils, and their high densities reduced the fungal richness in the areas surrounding the rotted Fuzi roots. An abundance analysis suggested that A. rolfsii and Mucor racemosus, as the dominant pathogens, might play important roles in the invading Fuzi tissue, and Phoma adonidicola could be another pathogenic fungus of root rot. In contrast, Mortierella chlamydospora, Penicillium simplicissimum, Epicoccum nigrum, Cyberlindnera saturnus and Rhodotorula ingeniosa might antagonize root rot pathogens in sick soils. In addition, A. rolfsii was further verified as a main pathogen of Fuzi root rot disease through hypha purification, morphological observation, molecular identification and an infection test. These results provide theoretical guidance for the prevention and treatment of Fuzi root rot disease.

Incidence, Distribution, and Pathogenicity of Fungi Causing Root Rot in Idaho Long-Term Sugar Beet Storage Piles.

Fungal rots in sugar beet roots held in long-term storage can lead to considerable sucrose loss but the incidence and distribution of fungal rots inside sugar beet piles and pathogenicity for some species is poorly understood. Thus, Idaho sugar beet held in five outdoor and two indoor piles in 2014 and 2015 were investigated. The root surface area covered by fungal growth and discolored and healthy tissue were assessed in nine 1-m2 areas per pile using a stratified random sampling design. Pathogenicity was evaluated indoors via plug inoculation in 2015 and 2016. Botrytis cinerea covered more root surface area inside indoor piles (6 to 22%) than outdoor piles (0 to 3%) (P < 0.0001). No trends were evident for the Athelia-like sp. (0 to 15%) and Penicillium-type spp. (0 to 8%). Penicillium-type isolates comprised the following species: 60% Penicillium expansum, 34% P. cellarum, 3% P. polonicum, and 3% Talaromyces rugulosus. Trace levels (<1% of root surface) of other fungi, including Cladosporium and Fusarium spp., were evident on roots and in isolations. Based on sample location in a pile, there were no trends or differences; however, two outdoor piles (OVP1 and OVP2) had more healthy tissue (90 to 96%) than other piles (28 to 80%) (P < 0.0001). When the pathogenicity tests were analyzed by species, all were significantly different from each other (P < 0.0001), except for P. polonicum and P. expansum: B. cinerea (61 mm of rot), P. polonicum (36 mm), P. expansum (35 mm), P. cellarum (28 mm), Athelia-like sp. (21 mm), T. rugulosus (0 mm; not different from check), and noninoculated check (0 mm). The OVP1 and OVP2 piles had negligible fungal growth on roots after more than 120 days of storage under ambient conditions, which indicates that acceptable storage can be achieved over this time period through covering piles with tarps and cooling with ventilation pipe.

Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance.

Penicillium spp. are the major postharvest pathogens of citrus fruit in Mediterranean climatic regions. The induction of natural resistance constitutes one of the most promising alternatives to avoid the environmental contamination and health problems caused by chemical fungicides. To understand the bases of the induction of resistance in citrus fruit against Penicillium digitatum, we have used a 12k citrus cDNA microarray to study transcriptional changes in the outer and inner parts of the peel (flavedo and albedo, respectively) of elicited fruits. The elicitor treatment led to an over-representation of biological processes associated with secondary metabolism, mainly phenylpropanoids and cellular amino acid biosynthesis and methionine metabolism, and the down-regulation of genes related to biotic and abiotic stresses. Among phenylpropanoids, we detected the over-expression of a large subset of genes important for the synthesis of flavonoids, coumarins and lignin, especially in the internal tissue. Furthermore, these genes and those of ethylene biosynthesis showed the highest induction. The involvement of both phenylpropanoid and ethylene pathways was confirmed by examining changes in gene expression and ethylene production in elicited citrus fruit. Therefore, global results indicate that secondary metabolism, mainly phenylpropanoids, and ethylene play important roles in the induction of resistance in citrus fruit.

Effect of heat shock treatment on stress tolerance and biocontrol efficacy of Metschnikowia fructicola.

The effect of high temperature and oxidative stress on the cell viability of the yeast antagonist, Metschnikowia fructicola was determined. A mild heat shock (HS) pretreatment (30 min at 40 °C) improved the tolerance of M. fructicola to subsequent high temperature (45 °C, 20-30 min) and oxidative stress (0.4 mol L⁻¹ hydrogen peroxide, 20-60 min). HS-treated yeast cells showed less accumulation of reactive oxygen species (ROS) than nontreated cells in response to both stresses. Additionally, HS-treated yeast exhibited significantly greater (P<0.0001) biocontrol activity against Penicillium expansum and a significantly faster (P<0.0001) growth rate in wounds of apple fruits stored at 25 °C compared with the performance of untreated yeast cells. Transcription of a trehalose-6-phosphate synthase gene (TPS1) was upregulated in response to HS and trehalose content also increased. Results indicate that the higher levels of trehalose induced by the HS may contribute to an improvement in ROS scavenging, stress tolerance, population growth in apple wounds and biocontrol activity of M. fructicola.

Response of jujube fruits to exogenous oxalic acid treatment based on proteomic analysis.

In this study, we found that oxalic acid (OA) at the concentration of 5 mM could delay jujube fruit sene-scence by reducing ethylene production, repressing fruit reddening and reducing alcohol content, which consequently increased fruit resistance against blue mold caused by Penicillium expansum. In order to gain a further understanding of the mechanism by which OA delays senescence and increases disease resistance of jujube fruit, we used a proteomics approach to compare soluble proteome of jujube fruits treated with water or 5 mM OA for 10 min. A total of 25 differentially expressed proteins were identified by using electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF-MS/MS). Among these proteins, alcohol dehydrogenase 1, which plays a direct role in ethanol metabolism, was repressed, and the abundances of three photosynthesis-related proteins was enhanced in jujube fruit after OA treatment. The protein identified as a cystathionine beta-synthase domain-containing protein, which can regulate ethylene precursors, was also induced by OA treatment. The activity of 1-aminocyclopropane-1-carboxylic acid synthase was significantly suppressed in OA-treated jujube fruit. In addition, three proteins related to the defense/stress response were up-regulated by OA, and contributed to the establishment of systemic resistance induced by OA in jujube fruits. These results indicated that OA treatment might affect ethanol and ethylene metabolism, resulting in delaying senescence, and increase resistance of jujube fruits against fungal pathogens.

Detección de metabolitos fúngicos con actividad tóxica mediante bioensayo sobre Artemia salina / Detection of fungal metabolites showing toxic activity through Artemia salina bioassay

El objetivo fue aplicar un ensayo de toxicidad sobre Artemia salina para ladetección de metabolitos fúngicos tóxicos, obtenidos a partir de hongoscontaminantes de hierbas medicinales y alimentos.Los extractos fúngicos fueron clasificados, según el porcentaje de mortalidadhallado, como: no tóxico (NT), levemente tóxico (LT), tóxico (T) y muy tóxico(MT). En los tipos T y MT se investigó la presencia de micotoxinas.Seis extractos resultaron de tipo T (8,5%). Penicillium brevicompactumDierckx, aislado de un embutido, fue el único MT, debido principalmente a lapresencia de ocratoxina A y de otros dos metabolitos no identificados(AU)

The aim of this study was to detect toxic metabolites from fungi contaminatingfood and medicinal herbs by applying the toxicity assay to Artemia salina.According to toxicity percentages, the extracts were classified as nontoxic(NT), slightly toxic (ST), toxic (T) and highly toxic (HT). Those classified asT and HT were assayed for mycotoxins.Only 6 out of 71 strains were found to be T (8.5%) for A. salina.Penicillium brevicompactum Dierckx, isolated from sausages, was found tobe HT, mainly due to the presence of ochratoxin A and two other unidentifiedmetabolites(AU)

Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations.

A central component in understanding plant-enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites. Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi. Because moisture and temperature play a key role in the epidemiology of most plant-pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site. We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp. and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography. Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r2 = 0.36 for Saccharomyces, r2 = 0.37 for Penicillium) and number (r2 = 0.34 for Saccharomyces) of antifungal secondary metabolites. This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites. We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r2 = 0.60 for Saccharomyces, r2 = 0.61 for Penicillium). Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds. Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens. The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation. That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses. The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites. The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush.