Secondary Metabolism Rearrangements in Linum usitatissimum L. after Biostimulation of Roots with COS Oligosaccharides from Fungal Cell Wall

In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant–pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2–10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.     

Mass Spectrometric Identification of Antimicrobial Peptides from Medicinal Seeds

Traditional medicinal plants contain a variety of bioactive natural products including cysteine-rich (Cys-rich) antimicrobial peptides (AMPs). Cys-rich AMPs are often crosslinked by multiple disulfide bonds which increase their resistance to chemical and enzymatic degradation. However, this class of molecules is relatively underexplored. Herein, in silico analysis predicted 80–100 Cys-rich AMPs per species from three edible traditional medicinal plants: Linum usitatissimum (flax), Trifolium pratense (red clover), and Sesamum indicum (sesame). Bottom-up proteomic analysis of seed peptide extracts revealed direct evidence for the translation of 3–10 Cys-rich AMPs per species, including lipid transfer proteins, defensins, α-hairpinins, and snakins. Negative activity revealed by antibacterial screening highlights the importance of employing a multi-pronged approach for AMP discovery. Further, this study demonstrates that flax, red clover, and sesame are promising sources for further AMP discovery and characterization.     

Effect of Nitric Oxide on Lachnum YMU50 Extracellular Polyphenol Accumulation and Antioxidant Defense System

The effect of sodium nitroprusside (SNP) of different concentrations (0.05, 0.1, 0.2 mM) on biomass and extracellular polyphenol accumulation and antioxidant defense system of Lachnum YMU50 had been studied in this article. Results showed that, when the NO donor was SNP, both 0.05 and 0.1 mM SNP could promote the biomass accumulation of Lachnum YMU50 and activate the phenylalanine ammonia lyase, thus promoting its extracellular polyphenol accumulation and activate and induce its antioxidant defense system by activating the activities of catalase, superoxide dismutase, and ascorbate peroxidase, and 0.1 mM SNP has a stronger effect than 0.05 mM SNP. SNP 0.2 mM was adverse to the accumulation of the biomass and extracellular polyphenol of Lachnum YMU50, decreasing the activities of catalase, superoxide dismutase, and ascorbate peroxidase and increasing the contents of hydrogen peroxide and malondialdehyde, which indicated that 0.2 mM SNP damaged the antioxidant defense system of Lachnum YMU50.     

Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-α. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.     

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.     

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC₅₀ values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.     

A nanosized Ag-silica hybrid complex prepared by γ-irradiation activates the defense response in Arabidopsis

Silver nanoparticles have antimicrobial activity against many pathogenic microbes. Here, the preparation of a nanosized Ag-silica hybrid complex (NSS) prepared by γ-irradiation is described. The effects of both NSS and reduced Ag nanoparticles (Ag⁰) on the growth of the model plant Arabidopsis thaliana were tested. The application of 1-10 ppm NSS complex improved Arabidopsis growth in soil, whereas 100 ppm NSS resulted in weakly curled leaves. In addition, supplementation of Murashige and Skoog (MS) growth medium with 1 ppm NSS promoted the root growth of Arabidopsis seedlings, but root growth was inhibited by supplementation with 10 ppm NSS. To investigate whether the NSS complex could induce plant defense responses, the expression of pathogenesis-related (PR) genes that are implicated in systemic acquired resistance (SAR) in Arabidopsis plants was examined. PR1, PR2 and PR5 were significantly up-regulated by each application of 10 ppm NSS complex or Ag⁰ to the rosette leaves. Furthermore, pretreatment with the NSS complex induced more pathogen resistance to the virulent pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) compared to water treatment in Arabidopsis plants.     

Intramolecular charge transfer effects on 3-aminobenzoic acid

Effect of solvents, buffer solutions of different pH and β-cyclodextrin on the absorption and fluorescence spectra of 3-aminobenzoic acid (3ABA) have been investigated. The solid inclusion complex of 3ABA with β-CD is discussed by UV–Vis, fluorimetry, semiempirical quantum calculations (AM1), FT-IR, ¹H NMR and Scanning Electron Microscope (SEM). The thermodynamic parameters (ΔH, ΔG and ΔS) of the inclusion process are also determined. The experimental results indicated that the inclusion processes is an exothermic and spontaneous. The large Stokes shift emission in solvents with 3ABA are correlated with different solvent polarity scales suggest that, 3ABA molecule is more polar in the S₁ state. Solvent, β-CD studies and excited state dipole moment values confirms that the presence of intramolecular charge transfer (ICT) in 3ABA. Acidity constants for different prototropic equilibria of 3ABA in the S₀ and S₁ states are calculated. β-Cyclodextrin studies shows that 3ABA forms a 1:1 inclusion complex with β-CD. β-CD studies suggest COOH group present in non-polar part and amino group present in hydrophilic part of the β-CD cavity. A mechanism is proposed to explain the inclusion process.     

AtTrxh3, a Thioredoxin,Is Identified as an Abscisic AcidBinding Protein in Arabidopsis thaliana

Abscisic acid (ABA, 1) is a plant hormone that regulates various plant physiological processes such as seed developing and stress responses. The ABA signaling system has been elucidated; binding of ABA with PYL proteins triggers ABA signaling. We have previously reported a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, a protein cross-linker, and a bioactive small molecule with an azido group (azido probe). This method was used to identify the unknown ABA binding protein of Arabidopsis thaliana. As a result, AtTrxh3, a thioredoxin, was isolated as an ABA binding protein. Our developed method can be applied to the identification of binding proteins of bioactive compounds.     

Microbiological hydroxylation of (+)- and (−)-bornyl acetate with Helminthosporium sativum and Fusarium culmorum.

The regiospecificity of hydroxylation of (+)- and (?)-bornyl acetateby cultures of Helminthosporium sativum and Fusarium culmorum has been determined.     

Effectiveness of the Influence of Selected Essential Oils on the Growth of Parasitic Fusarium Isolated from Wheat Kernels from Central Europe

The aim of the study was to determine the effectiveness of selected seven commercial essential oils (EsO) (grapefruit, lemongrass, tea tree (TTO), thyme, verbena, cajeput, and Litsea cubeba) on isolates of common Central European parasitic fungal species of Fusarium obtained from infected wheat kernels, and to evaluate the oils as potential natural fungicides. The study was conducted in 2 stages. At each stage, the fungicidal activity of EsO (with concentrations of 0.025; 0.05; 0.125; 0.25; 0.50; 1.0, and 2.0%) against Fusarium spp. was evaluated using the disc plate method and zones of growth inhibition were measured. At the first stage, the fungistatic activity of EsO was evaluated against four species of Fusarium from the Polish population (F. avenaceum FAPL, F. culmorum FCPL, F. graminearum FGPL and F. oxysporum FOPL). The correlation coefficient between the mycelial growth rate index (T) and the fungistatic activity (FA) was calculated. At the second stage, on the basis of the mycelium growth rate index, the effectiveness of the EsO in limiting the development of Fusarium isolates from the German population (F. culmorum FC1D, F. culmorum FC2D, F. graminearum FG1D, F. graminearum FG2D and F. poae FP0D) was assessed. The first and second stage results presented as a growth rate index were then used to indicate essential oils (as potential natural fungicides) effectively limiting the development of various common Central European parasitic species Fusarium spp. Finally, the sensitivity of four Fusarium isolates from the Polish population and five Fusarium isolates from the German population was compared. The data were compiled in STATISTICA 13.0 (StatSoft, Inc, CA, USA) at the significance level of 0.05. Fusarium isolates from the German population were generally more sensitive than those from the Polish population. The sensitivity of individual Fusarium species varied. Their vulnerability, regardless of the isolate origin, in order from the most to the least sensitive, is as follows: F. culmorum, F. graminearum, F. poae, F. avenaceum and F. oxysporum. The strongest fungicidal activity, similar to Funaben T, showed thyme oil (regardless of the concentration). Performance of citral oils (lemongrass and Litsea cubeba) was similar but at a concentration above 0.025%.     

Effects of Coumarinyl Schiff Bases against Phytopathogenic Fungi,the Soil-Beneficial Bacteria and Entomopathogenic Nematodes: Deeper Insight into the Mechanism of Action

Coumarin derivatives have been reported as strong antifungal agents against various phytopathogenic fungi. In this study, inhibitory effects of nine coumarinyl Schiff bases were evaluated against the plant pathogenic fungi (Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Macrophomina phaseolina and Sclerotinia sclerotiourum). The compounds were demonstrated to be efficient antifungal agents against Macrophomina phaseolina. The results of molecular docking on the six enzymes related to the antifungal activity suggested that the tested compounds act against plant pathogenic fungi, inhibiting plant cell-wall-degrading enzymes such as endoglucanase I and pectinase. Neither compound exhibited inhibitory effects against two beneficial bacteria (Bacillus mycoides and Bradyrhizobium japonicum) and two entomopathogenic nematodes. However, compound 9 was lethal (46.25%) for nematode Heterorhabditis bacteriophora and showed an inhibitory effect against acetylcholinesterase (AChE) (31.45%), confirming the relationship between these two activities. Calculated toxicity and the pesticide-likeness study showed that compound 9 was the least lipophilic compound with the highest aquatic toxicity. A molecular docking study showed that compounds 9 and 8 bind directly to the active site of AChE. Coumarinyl Schiff bases are promising active components of plant protection products, safe for the environment, human health, and nontarget organisms.     

ROS Dependent Antifungal and Anticancer Modulations of Piper colubrinum Osmotin

Osmotin, a plant defense protein, has functional similarity to adiponectin, an insulin sensitizingsensitising hormone secreted by adipocytes. We speculated that Piper colubrinum Osmotin (PcOSM) could have functional roles in obesity-related cancers, especially breast cancer. Immunofluorescence assays, flow cytometry, cell cycle analysis and a senescence assay were employed to delineate the activity in MDAMB231 breast cancer cell line. PcOSM pre-treated P. nigrum leaves showed significant reduction in disease symptoms correlated with high ROS production. In silico analysis predicted that PcOSM has higher binding efficiency with adiponectin receptor compared to adiponectin. PcOSM was effectively taken up by MDAMB231 cancer cells which resulted in marked increase in intracellular ROS levels leading to senescence and cell cycle arrest in G2/M stage. This study provides evidence on the ROS mediated direct inhibitory activity of the plant derived osmotin protein on the phytopathogen Phytophthora capsici, and the additional functional roles of this plant defense protein on cancer cells through inducing ROS associated senescence. The strong leads produced from this study could be pursued further to obtain more insights into the therapeutic potential of osmotin in human cancers.     

Toward Understanding the Molecular Recognition of Fungal Chitin and Activation of the Plant Defense Mechanism in Horticultural Crops

Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.     

Identification of the roselle root rot pathogen and its sensitivity to different fungicides

Root rot is the main disease affecting roselle plantings and production and can seriously affect the yield and quality of calyces. Thus, it is urgent to identify the pathogen causing roselle root rot and screen effective pesticides to control the disease. In the present study, morphological observation, pathogenicity assays and molecular biology methods were used to identify the pathogen causing roselle root rot in Nanning, Guangxi Province, and four biological and four chemical fungicides were evaluated for their effects on the mycelial growth rate of the pathogen. The results showed that the pathogen causing roselle root rot in Nanning, Guangxi, was Fusarium solani, marking the first report of this fungus causing root rot of roselle in China. The fungicidal activity screening revealed differences in the inhibitory effects of the eight fungicides on the colony growth of F. solani. For the biofungicides, Bacillus amyloliquefaciens exhibited the best fungistatic effect, with an IC₅₀ of 1.10 mg/mL. When the mass concentration was 2.5 mg/mL, the mycelial growth of the pathogen was 100% inhibited, while Bacillus subtilis had the worst inhibitory effect, with an IC₅₀ of 46.78 mg/mL. When its mass concentration was 80 mg/mL, mycelial growth was only inhibited by 74.67%. For the chemical fungicides, carbendazim and thiophanate-methyl presented the strongest effects on F. solani, with IC₅₀ values of 0.0082 mg/mL and 0.0243 mg/mL, respectively. When the mass concentrations were 0.03 mg/mL and 0.075 mg/mL, mycelial growth was inhibited by 100%. These results provide a scientific basis for rationally selecting fungicides to control roselle root rot in field production.     

13C NMR spectra and biological activity of N-(1H-benzimidazol-2-yl)benzamides

Derivatives of 1H-benzimidazol-2-amine and halo-, nitro-, methoxy-, and hydroxy-substituted benzoic acids were synthesized, and their fungicide activity against pure Fusarium culmorum and Helminthosporium sativum cultures and pathogenic microflora of wheat and barley seeds in laboratory and field tests was studied. Some compounds were found to exhibit a strong fungicide activity.     

Computational Strategy for Minimizing Mycotoxins in Cereal Crops: Assessment of the Biological Activity of Compounds Resulting from Virtual Screening

Cereal crops are frequently affected by toxigenic Fusarium species, among which the most common and worrying in Europe are Fusarium graminearum and Fusarium culmorum. These species are the causal agents of grain contamination with type B trichothecene (TCTB) mycotoxins. To help reduce the use of synthetic fungicides while guaranteeing low mycotoxin levels, there is an urgent need to develop new, efficient and environmentally-friendly plant protection solutions. Previously, F. graminearum proteins that could serve as putative targets to block the fungal spread and toxin production were identified and a virtual screening undertaken. Here, two selected compounds, M1 and M2, predicted, respectively, as the top compounds acting on the trichodiene synthase, a key enzyme of TCTB biosynthesis, and the 24-sterol-C-methyltransferase, a protein involved in ergosterol biosynthesis, were submitted for biological tests. Corroborating in silico predictions, M1 was shown to significantly inhibit TCTB yield by a panel of strains. Results were less obvious with M2 that induced only a slight reduction in fungal biomass. To go further, seven M1 analogs were assessed, which allowed evidencing of the physicochemical properties crucial for the anti-mycotoxin activity. Altogether, our results provide the first evidence of the promising potential of computational approaches to discover new anti-mycotoxin solutions     

Elicitation of Andrographolide in the Suspension Cultures of Andrographis paniculata

Andrographis paniculata belonging to the family Acanthaceae produces a group of diterpene lactones, one of which is the pharmaceutically important??andrographolide. It is known to possess various important biological properties like anticancer, anti-HIV, anti-inflammatory, etc. This is the first report on the production of andrographolide in the cell suspension cultures of Andrographis paniculata by ??elicitation??. Elicitation was attempted to enhance the andrographolide content in the suspension cultures of Andrographis paniculata and also to ascertain its stimulation under stress conditions or in response to pathogen attack. The maximum andrographolide production was found to be 1.53?mg/g dry cell weight (DCW) at the end of stationary phase during the growth curve. The biotic elicitors (yeast, Escherichia coli, Bacillus subtilis, Agrobacterium rhizogenes 532 and Agrobacterium tumefaciens C 58) were more effective in eliciting the response when compared to the abiotic elicitors (CdCl₂, AgNO₃, CuCl₂ and HgCl₂). Yeast has shown to stimulate maximum accumulation of 13.5?mg/g DCW andrographolide, which was found to be 8.82-fold higher than the untreated cultures.