Analysis of Wheat Grain Infection by Fusarium Mycotoxin-Producing Fungi Using an Electronic Nose, GC-MS, and qPCR.

Fusarium graminearum and F. culmorum are considered some of the most dangerous pathogens of plant diseases. They are also considerably dangerous to humans as they contaminate stored grain, causing a reduction in yield and deterioration in grain quality by producing mycotoxins. Detecting Fusarium fungi is possible using various diagnostic methods. In the manuscript, qPCR tests were used to determine the level of wheat grain spoilage by estimating the amount of DNA present. High-performance liquid chromatography was performed to determine the concentration of DON and ZEA mycotoxins produced by the fungi. GC-MS analysis was used to identify volatile organic components produced by two studied species of Fusarium. A custom-made, low-cost, electronic nose was used for measurements of three categories of samples, and Random Forests machine learning models were trained for classification between healthy and infected samples. A detection performance with recall in the range of 88-94%, precision in the range of 90-96%, and accuracy in the range of 85-93% was achieved for various models. Two methods of data collection during electronic nose measurements were tested and compared: sensor response to immersion in the odor and response to sensor temperature modulation. An improvement in the detection performance was achieved when the temperature modulation profile with short rectangular steps of heater voltage change was applied.

The Floral Signals of the Inconspicuous Orchid Malaxis monophyllos: How to Lure Small Pollinators in an Abundant Environment.

Many orchid species have evolved complex floral signals to ensure pollination efficiency. Here, the authors combined analyses of anatomical flower structure with analyses of the volatile composition and flower-visiting insects' behaviour, as well as characterised features that can attract pollinators of the inconspicuous orchid Malaxis monophyllos. During field observations, the authors found that only small Diptera (e.g., mosquitos, drosophilids, fungus gnats) visit and are interested in the flowers of M. monophyllos, which was reflected in the characterised flower features that combine well with the pollination system, which engages dipterans. Analyses of the M. monophyllos floral scent revealed substantial concentrations of aliphatic compounds, e.g., 1-octen-3-ol and 1-octanol, which condition the mushroom-like scent and a substantial fraction of alkanes, some of which have been previously described as sex mimicry and aggregation pheromones in orchids' deceptive systems. The labellum anatomical structure exhibits a highly diverse cell cuticle surface and pronounced metabolic and secretory activity of the epidermal and subepidermal cells from all parts of the labellum. Moreover, our study provides evidence for the subsequent decoys of M. monophyllos flowers, including visual signals, such as raphides located on the labellum margin and the rewarding ones connected with lipid secretion limited to the area behind the column. Taking an integrative approach to studying M. monophyllos pollination biology, the authors provide new insight into its previously vague pollination strategies and provide evidence for complex floral signal operation in luring potential pollinators. The synergistic effect of M. monophyllos flowers' volatile and visual signals, together with additional rewarding for nectar/fungus/microbe-feeding pollinators, requires further detailed investigation that will be invaluable in explaining the evolution of Diptera-specific pollination systems in orchids.

Secondary metabolites of downy birch buds (Betula pubescens Erch.).

The subject of this study is the composition of low-molecular-weight metabolites in downy birch (Betula pubescens) buds and their participation in protection from various kinds of stress. Using the GC-MS, 640 compounds were detected, of which 314 were identified in downy birch buds for the first time. The volatile components detected using the SPME technique mainly consisted (about 70% of the total ionic current of the chromatogram, TIC) of mixtures of sesquiterpenoids. The exudate covering the buds, along with sesquiterpenoids (approximately 60% of TIC), included flavonoids (25% of TIC). The main part of the material extracted by supercritical carbon dioxide from buds comprised sesquiterpenoids and triterpenoids (47 and 28% of TIC, respectively). Via column chromatography, 25 known compounds (mainly flavonoids and triterpenoids) were isolated, most of which were first discovered in the buds of downy birch. Many compounds of these classes have strong biological activity and probably either directly or indirectly perform a protective function in birch buds. An assumption is made about the biological role of a number of secondary metabolites (such as volatile isomeric megastigmatriens and triterpene seco-acids) as well as about these compounds' possible means of biosynthesis, which were first discovered in the buds of downy birch.

Santin and cirsimaritin from Betula pubescens and Betula pendula buds induce apoptosis in human digestive system cancer cells.

Flavonoids are bioactive secondary metabolites of plants, which exert anti-cancer effects. However, metabolism in enterocytes and the liver can influence the biological activity of flavonoids contained in the diet. Therefore, results from in vitro studies on cancer cells from the digestive tract and liver may reflect the real effects in the human body. Previously, we have found that the extract from birch buds exerts antiproliferative activity in a panel of cancer cells. In the present study, the anti-cancer activity of ten flavonoids isolated from the buds of Betula pubescens and Betula pendula was characterized. Among them, santin and cirsimaritin significantly reduced viability, proliferation and clonogenicity of gastric (AGS), colon (DLD-1) and liver (HepG2) cancer cells. Both flavonoids induced apoptosis, accompanied by activation of caspase-3, caspase-7, caspase-8 and caspase-9. Moreover, upregulation of p53 was detected only in wild-type p53 harbouring cells. Together, our results suggest that santin and cirsimaritin exhibit promising anti-cancer activity in cultures of digestive system cancer cells.

Electronic Nose Differentiation between Quercus robur Acorns Infected by Pathogenic Oomycetes Phytophthora plurivora and Pythium intermedium.

Identification of the presence of pathogenic oomycetes in infected plant material proved possible using an electronic nose, giving hope for a tool to assist nurseries and quarantine services. Previously, species of Phytophthora plurivora and Pythium intermedium have been successfully distinguished in germinated acorns of English oak Quercus robur L. Chemical compound analyses performed by HS-SPME/GC-MS (Headspace Solid-Phase Microextraction/Gas Chromatography-Mass Spectrometry) revealed the presence of volatile antifungal molecules produced by oak seedlings belonging to terpenes and alkanes. Compounds characteristic only of Phytophthora plurivora or Pythium intermedium were also found. Methylcarveol occurred when germinated acorns were infected with Pythium, while neophytadiene (isomer 2 and 3) occurred only when infected with Phytophthora. Moreover, isopentanol was found in acorns infected with Phytophthora, while in control, isopentyl vinyl ether was not observed anywhere else. Among the numerous volatile compounds, isopentanol only occurred in acorns infected with Phytophthora and methylcarveol in acorns infected with Pythium.

Detection of Fungi and Oomycetes by Volatiles Using E-Nose and SPME-GC/MS Platforms.

Fungi and oomycetes release volatiles into their environment which could be used for olfactory detection and identification of these organisms by electronic-nose (e-nose). The aim of this study was to survey volatile compound emission using an e-nose device and to identify released molecules through solid phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) analysis to ultimately develop a detection system for fungi and fungi-like organisms. To this end, cultures of eight fungi (Armillaria gallica, Armillaria ostoyae, Fusarium avenaceum, Fusarium culmorum, Fusarium oxysporum, Fusarium poae, Rhizoctonia solani, Trichoderma asperellum) and four oomycetes (Phytophthora cactorum, P. cinnamomi, P. plurivora, P. ramorum) were tested with the e-nose system and investigated by means of SPME-GC/MS. Strains of F. poae, R. solani and T. asperellum appeared to be the most odoriferous. All investigated fungal species (except R. solani) produced sesquiterpenes in variable amounts, in contrast to the tested oomycetes strains. Other molecules such as aliphatic hydrocarbons, alcohols, aldehydes, esters and benzene derivatives were found in all samples. The results suggested that the major differences between respective VOC emission ranges of the tested species lie in sesquiterpene production, with fungi emitting some while oomycetes released none or smaller amounts of such molecules. Our e-nose system could discriminate between the odors emitted by P. ramorum, F. poae, T. asperellum and R. solani, which accounted for over 88% of the PCA variance. These preliminary results of fungal and oomycete detection make the e-nose device suitable for further sensor design as a potential tool for forest managers, other plant managers, as well as regulatory agencies such as quarantine services.

Heterobasidion annosum Induces Apoptosis in DLD-1 Cells and Decreases Colon Cancer Growth in In Vivo Model.

Application of substances from medicinal mushrooms is one of the interesting approaches to improve cancer therapy. In this study, we commenced a new attempt in the field of Heterobasidion annosum (Fr.) Bref. sensu lato to further extend our knowledge on this basidiomycete fungus. For this purpose, analysis of the active substances of Heterobasidion annosum methanolic extract and also its influence on colorectal cancer in terms of in vitro and in vivo experiments were performed. In vivo studies on mice were conducted to verify its acute toxicity and to further affirm its anticancer potential. Results indicated that all the most common substances of best known medicinal mushrooms that are also responsible for their biological activity are present in tested extracts. In vitro tests showed a high hemocompatibility and a significant decrease in viability and proliferation of DLD-1 cells in a concentration-dependent manner of Heterobasidion annosum extract. The studies performed on xenograft model of mice showed lower tendency of tumor growth in the group of mice receiving Heterobasidion annosum extract as well as mild or moderate toxicity. Obtained results suggest beneficial potential of Heterobasidion annosum against colon cancer as cytotoxic agent or as adjuvant anticancer therapy.

Geissospermiculatine, a New Alkaloid from Geissospermum reticulatum Bark.

A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, 1H-15N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30 µg/mL) was comparable with staurosporine (10 µM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0-72 h); toxicity was not evident at 30 µg/mL.

Cytotoxicity of Triterpene Seco-Acids from Betula pubescens Buds.

The present study investigated the magnitude and mechanism of the cytotoxic effect on selected cancer cell lines of 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (1), 3,4-seco-olean-4(24)-en-19-oxo-3-oic acid (2), and 3,4-seco-urs-4(23),20(30)-dien-19-ol-3-oic acid (3) isolated from downy birch (Betula pubescens) buds by carbon dioxide supercritical fluid extraction and gradient column chromatography. Cell viability in six human cancer lines exposed to these compounds was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was quantified by annexin V/propidium iodide staining of gastric cancer AGS and colorectal cancer DLD-1 cells. To evaluate the mechanism of apoptosis, the expression of apoptosis-related proteins was analyzed by Western blot. Compound 1 exhibited non-specific toxicity, while compounds 2 and 3 were specifically toxic to colon and stomach cancer cells. The toxicity of compounds 2 and 3 against these two cell lines was greater than for compound 1. Cleavage of caspase-8, -9, and -3 was found in AGS and DLD-1 cells treated with all three seco-acids, indicating the induction of apoptosis via extrinsic and intrinsic pathways. Therefore, triterpene seco-acids (1-3) decreased cell viability by apoptosis induction. AGS and DLD-1 cells were more susceptible to seco-acids with an oxidized C19 than normal fibroblasts. Hence, it made them a new group of triterpenes with potential anticancer activity.