Assessment of Antimicrobial Properties of Phenolic Acid Extracts from Grain Infected with Fungi from the Genus Fusarium

Problems related with biological contamination of plant origin raw materials have a considerable effect on prevention systems at each stage of food production. Concerning the antimicrobial action of phenolic acids, studies were undertaken to investigate antibacterial properties against bacterial strains of Escherichia coli (EC), Pseudomonas fluorescence (PF), Micrococcus luteus (ML) and Proteus mirabilis (PM), as well as antifungal properties targeting microscopic fungi Fusarium spp., extracts of phenolic compounds coming from inoculated grain from various genotypes of cereals. This study evaluated the antimicrobial action of phenolic acids extracts obtained from both naturally infested and inoculated with microorganisms. For this purpose a total of 24 cereal cultivars were selected, including 9 winter and 15 spring cultivars. The analyses showed a bactericidal effect in the case of 4 extracts against Micrococcus luteus (ML), 14 extracts against Pseudomonas fluorescence (PF), 17 extracts against Escherichia coli (EC) as well as 16 extracts against Proteus mirabilis (PM). It was found that 3 out of the 24 extracts showed no antibacterial activity. In turn, fungicidal action was observed in the case of 17 extracts against Fusarium culmorum (FC) (NIV), 16 extracts against FC (3AcDON), 12 extracts against Fusarium graminearum (FG) (3AcDON), while 12 other extracts showed antifungal action against FG (NIV) and 19 extracts against Fusarium langsethiae (FL). Based on the conducted analyses it was found that grain of small-grained cereals exposed to fungal infection is a source of bioactive compounds exhibiting antimicrobial properties. It was observed that the qualitative and quantitative profiles of polyphenols vary depending on the cereal cultivar. This extracts may be used to develop an antimicrobial preparation applicable in organic farming.     

Chemical Composition,Antifungal and Anti-Biofilm Activities of Volatile Fractions of Convolvulus althaeoides L. Roots from Tunisia

The antifungal drugs currently available and mostly used for the treatment of candidiasis exhibit the phenomena of toxicity and increasing resistance. In this context, plant materials might represent promising sources of antifungal agents. The aim of this study is to evaluate for the first time the chemical content of the volatile fractions (VFs) along with the antifungal and anti-biofilm of Convolvulus althaeoides L. roots. The chemical composition was determined by gas chromatography coupled to a flame ionization detector and mass spectrometry. In total, 73 and 86 chemical compounds were detected in the n-hexane (VF1) and chloroform (VF2) fractions, respectively. Analysis revealed the presence of four main compounds: n-hexadecenoic acid (29.77%), 4-vinyl guaiacol (12.2%), bis(2-ethylhexyl)-adipate (9.69%) and eicosane (3.98%) in the VF extracted by hexane (VF1). n-hexadecenoic acid (34.04%), benzyl alcohol (7.86%) and linoleic acid (7.30%) were the main compounds found in the VF extracted with chloroform (VF2). The antifungal minimum inhibitory concentrations (MICs) of the obtained fractions against Candida albicans, Candida glabrata and Candida tropicalis were determined by the micro-dilution technique and values against Candida spp. ranged from 0.87 to 3.5 mg/mL. The biofilm inhibitory concentrations (IBF) and sustained inhibition (BSI) assays on C. albicans, C. glabrata and C. tropicalis were also investigated. The VFs inhibited biofilm formation up to 0.87 mg/mL for C. albicans, up to 1.75 mg/mL against C. glabrata and up to 0.87 mg/mL against C. tropicalis. The obtained results highlighted the synergistic mechanism of the detected molecules in the prevention of candidosic biofilm formation.     

Simultaneous densitometric determination of shikonin,acetylshikonin, and β‐acetoxyisovaleryl‐shikonin in ultrasonic‐assisted extracts of four Arnebia species using reversed‐phase thin layer chromatography

A simple, precise, and rapid high‐performance thin‐layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin ( 1 ) together with its derivatives acetylshikonin ( 2 ), and β‐acetoxyisovalerylshikonin ( 3 ) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP‐18 F_254S TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100–600 ng for shikonin and acetylshikonin, and 100–1800 ng for β‐acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1 – 3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.     

Overexpression of an Endochitinase Gene (<Emphasis Type="Italic">ThEn-42</Emphasis>) in <Emphasis Type="Italic">Trichoderma atroviride</Emphasis> for Increased Production of Antifungal Enzymes and Enhanced Antagonist Action Against Pathogenic Fungi

Trichoderma is one of the most promising biocontrol agents against plant fungal diseases. In this study, a transgenic strain of Trichoderma atroviride was characterized. The transgenic strain contains an endochitinase gene (ThEn-42) driven by the cellulase promoter cbh1 of T. reesei for overexpression of ThEn-42. The culture filtrates of the transformant and the parental strain grown in eight different media were evaluated for chitinase and antifungal enzyme production based on activity gels, protein profiles, and antifungal activities. Results demonstrated that chitinases are important components and synergistic interactions play a key role in the antagonistic action of T. atroviride. Moreover, altering medium nutrient concentration and composition led to enhanced production of antifungal enzymes, a potential strategy for mass production. Two of the culture filtrates contained almost pure endochitinase, and could be excellent commercial sources for this enzyme. Several culture filtrates were highly antifungal. Two filtrates were so effective in biocontrol of a fungal pathogen, Penicillium digitatum, that they not only inhibited spore germination but destroyed the spores completely when 20 μl of culture filtrate (corresponding to approximately 104 μg of total protein) was applied in a total volume of 150 μl (approximately 0.7 mg protein ml⁻¹).     

Isolation,characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in Acinetobacter strains from rhizosphere of wheat

Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobacter genospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography. Purified IAA was confirmed by ¹H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 × 10⁻⁵, and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead. This is the first report of plasmid-encoded IAA production in the genus Acinetobacter.     

Synthesis,Biological Evaluation,and Structure–Activity Relationships of 4-Aminopiperidines as Novel Antifungal Agents Targeting Ergosterol Biosynthesis

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.     

Microbial inactivation in cloudy apple juice by multi-frequency Dynashock power ultrasound

The study determined the efficacy of Dynashock wave power ultrasound as an alternative processing technique for apple juice against a number of pathogenic and spoilage microorganisms. The effects of several implicit, intrinsic and extrinsic properties on the Dynashock wave inactivation of the microorganisms were also investigated. Results showed that acid adaptation increased the resistance of Escherichia coli O157:H7 and Salmonella spp. but decreased that of Listeria monocytogenes. Spoilage yeast mixed inoculum composed of Debaryomyces hansenii, Torulaspora delbrueckii, Clavispora lusitaniae, Pichia fermentans and Saccharomyces cerevisiae was found to be more resistant than any of the adapted or non-adapted pathogens. Among the individual, acid-adapted E. coli O157:H7, the MN-28 isolate was found most resistant; while three other individual isolates had greater resistance than the composited E. coli inoculum. Increased in pulp content decreased the efficacy of Dynashock waves, but co-treatment with ultraviolet-C rays significantly enhanced inactivation in the cloudy apple juice. The results demonstrated the potential of Dynashock wave technology, together with other antimicrobial hurdles as alternative juice processing technique/s.     

Clove Essential Oil and Its Main Constituent,Eugenol, as Potential Natural Antifungals against Candida spp. Alone or in Combination with Other Antimycotics Due to Synergistic Interactions

The occurrence of candidiasis, including superficial infections, has recently increased dramatically, especially in immunocompromised patients. Their treatment is often ineffective due to the resistance of yeasts to antimycotics. Therefore, there is a need to search for new antifungals. The aim of this study was to determine the antifungal effect of clove essential oil (CEO) and eugenol (EUG) towards both reference and clinical Candida spp. strains isolated from the oral cavity of patients with hematological malignancies, and to investigate their mode of action and the interactions in combination with the selected antimycotics. These studies were performed using the broth microdilution method, tests with sorbitol and ergosterol, and a checkerboard technique, respectively. The CEO and EUG showed activity against all Candida strains with a minimal inhibitory concentration (MIC) in the range of 0.25–2 mg/mL. It was also found that both natural products bind to ergosterol in the yeast cell membrane. Moreover, the interactions between CEO and EUG with several antimycotics—cetylpyridinium chloride, chlorhexidine, silver nitrate and triclosan—showed synergistic or additive effects in combination, except nystatin. This study confirms that the studied compounds appear to be a very promising group of phytopharmaceuticals used topically in the treatment of superficial candidiasis. However, this requires further studies in vivo.     

Application Potential of Bacterial Volatile Organic Compounds in the Control of Root-Knot Nematodes

Plant-parasitic nematodes (PPNs) constitute the most damaging group of plant pathogens. Plant infections by root-knot nematodes (RKNs) alone could cause approximately 5% of global crop loss. Conventionally, chemical-based methods are used to control PPNs at the expense of the environment and human health. Accordingly, the development of eco-friendly and safer methods has been urged to supplement or replace chemical-based methods for the control of RKNs. Using microorganisms or their metabolites as biological control agents (BCAs) is a promising approach to controlling RKNs. Among the metabolites, volatile organic compounds (VOCs) have gained increasing attention because of their potential in the control of not only RKNs but also other plant pathogens, such as insects, fungi, and bacteria. This review discusses the biology of RKNs as well as the status of various control strategies. The discovery of VOCs emitted by bacteria from various environmental sources and their application potential as BCAs in controlling RKNs are specifically addressed.     

Capillary and gel electromigration techniques and MALDI-TOF MS – Suitable tools for identification of filamentous fungi

Microbial strains are now spreading out of their original geographical areas of incidence and previously adequate morphological identification methods often must be accompanied by a phenotypic characterization for the successful microbial identification. The fungal genus Monilinia represents a suitable example. Monilinia species represent important fruit pathogens responsible for major losses in fruit production. Four closely related spp. of Monilinia: Monilinia laxa, Monilinia fructigena, Monilinia fructicola and Monilia polystroma have been yet identified. However, the classical characterization methods are not sufficient for current requirements, especially for phytosanitary purposes.