Extract Broth Research Articles

The selective breeding and mutagenesis mechanism of high‐yielding surfactin Bacillus subtilis strains with atmospheric and room temperature plasma

Surfactin, a good biological surfactant, is derived from the metabolites of microorganisms. However, the ability of natural strains to produce surfactin is low, and so the presented study aimed to use a novel mutagenesis technology to increase their yields. Atmospheric and room temperature plasma (ARTP) was used to conduct mutation breeding of Bacillus subtilis CICC 10721, and a mutant strain M45 with a higher surfactin yield of 34.2% and a stable subculture was screened out. From the fermentation kinetics study, it was found that the maximum cell dry weight, maximum growth rate and surfactin synthesis parameters of the mutant strain M45 were all greater than that of the original strain. Scanning electron microscope and laser scanning confocal microscope observations showed that the spore morphology changed after ARTP treating, and the intracellular Ca2+ concentration of the mutant increased. Genome resequencing analysis showed that 66 single nucleotide poymorphism non-synonymous mutation sites occurred in M45, and the identification results of the fermentation broth extract from M45 showed that it is composed of C12 -C16 surfactin. ARTP mutagenesis was found to change the morphology of bacteria, membrane permeability and genes related to the synthesis and secretion of surfactin. The present study provides a basis for industrial production of surfactin and an understanding of the mutagenesis mechanism. © 2021 Society of Chemical Industry.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

CULTURE MEDIA EVALUATION ON THE Leucoagaricus gongylophorus AND Escovopsis sp. FUNGI DEVELOPMENT

This study aimed to evaluate the in vitro interference of different culture media, Czapek Dox Agar, Nutrient Agar, Starch Casein agar, Malt Extract Broth Base, Nutrient Broth, and the medium containing Peptone, Malt, Agar and crushed peach palm pulp (Bactris gasipaes) and tucumã (Astrocaryum aculeatum) on the fungus Leucoagaricus gongylophorus and the parasitic fungus Escovopsis sp. growth. The fungi were inoculated in different culture media and then kept in acclimatized chambers at 28 ºC in the dark for 42 days, being analyzed at weekly intervals, totaling six evaluations. Fungal colonies were evaluated considering the factor "f" (cm), obtained from the product between the width (W) and length (L) of the diameter of the colonies (f = W x L). The results showed that the symbiotic fungus and the leafcutter ant parasitic fungus showed a growth increase compared to the media provided in the plates, and the media added with tucumã peel, tucumã peel+pulp, fresh peach palm, and cooked peach palm had a significant effect in the L. gongylophorus fungus development. However, these treatments were not promising for the Escovopsis sp. fungus compared to other culture media.

Molecular intervention of colon cancer and inflammation manifestation by tannin capped biocompatible controlled sized gold nanoparticles from Terminalia bellirica: A green strategy for pharmacological drug formulation based on nanotechnology principles.

Among the diverse nanomaterials, gold nanoparticles (AuNps) are utilised for various therapeutic application due to the distinct physical, chemical properties and biocompatibility. Synthesis of gold nanoparticles using plants is the promising route. This method is low cost, eco-friendly and higher biological activities. In this present study, Gold nanoparticles were synthesised from fruit extract of Terminalia bellirica fruit extract. Their anticancer and anti-inflammatory activity was evaluated against colorectal cancer cell line (HT29) and TNBS-induced zebrafish model. Highly stable tannin capped gold nanoparticles were synthesised from fruit extract broth of Terminalia bellirica rapidly. Structural and functional properties of the synthesised nanoparticles were studied by Fourier transform infrared spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM) equipped with energy-dispersive atomic X-ray spectroscopy (EDAX) and X-ray diffraction (XRD). All the characterisation studies reveal highly stable, crystalline, phytochemicals, mainly tannin doped, spherical, 28nm controlled sized gold nanoparticles. The molecular mechanism of anticancer activity was studied by determining cancer markers' expression, which was studied using quantitative real-time polymerase chain reaction (qPCR). Antioxidative enzymes' status and apoptosis changes were also investigated. Synthesised nanoparticles brought a drastic reduction of all the tested cancer markers' expression. Notable changes in antioxidative enzymes' status and a good sign of apoptosis were observed in nanoparticles' treatment. The anti-inflammatory activity was studied against TNBS-induced zebrafish model, which was confirmed by determining inflammatory markers' expression TNF-α, iNOS (induced Nitric Oxide Synthase) and histopathological examination. Nanoparticles' treatment recorded a drastic reduction of inflammatory markers' expression. No marked sign of inflammation was also observed in histopathological analysis of the nanoparticles' treatment group. The present study suggests the possible utilisation of T. bellirica-mediated gold nanoparticles as an effective therapeutic agent against a prolonged inflammatory disease that progressively develops into cancer.

Antibacterial and antioxidant potential of ethyl acetate extract from Streptomyces AIA12 and AIA17 isolated from gut of Chanos chanos

Abstract. Kurnianto MA, Kusumaningrum HD, Lioe HN, Chasanah E. 2021. Antibacterial and antioxidant potential of ethyl acetate extract from Streptomyces AIA12 and AIA17 isolated from gut of Chanos chanos. Biodiversitas 22: 3196-3206. Streptomyces has been recognized as a promising and productive source of antibacterial and antioxidant compounds. The phenotypic and genomic characterizations indicated AIA12 and AIA17 are closely related to the S. globisporus ARGB01 and S. misionensis S1-SC15, respectively. Incubation on yeast-malt extract broth for nine days (AIA12) and 11 days (AIA17) proved to be the best growth medium and optimum antibacterial production time for these two strains. Crude-extract of extracellular secondary metabolites, obtained by ethyl acetate extraction, demonstrated broad-spectrum inhibitory activity against Pseudomonas aeruginosa InaCC B52, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 14028, and Listeria monocytogenes with minimum inhibitory and minimum bactericidal concentrations from 2.5 to 0.31 and 5.0 to 0.31 mg mL-1, respectively. Evaluation of antioxidant showed AIA17 crude-extract had moderate DPPH scavenging and antioxidant activities of 65.122% ± 0.56 and 28.178 ± 0.24 mg ascorbic acid equivalent antioxidant capacity (AEAC g-1), respectively. The identification of compounds through profiling with RP-HPLC showed optimum absorbance at 210 and 214 nm, which showed the presence of peptide groups in the constituent compounds' molecular structure. These findings indicate that Chanos chanos-derived Streptomyces produces valuable bioactive compounds with various promising biological activities.

Optimization of Growth Medium and Physicochemical Parameters for High-Yield Biomass Production and Active Metabolites from Aspergillus terreus

The selection of a nutrient medium and optimization of physicochemical parameters play a crucial role in the growth enhancement and production of bioactive secondary metabolites of fungi. The current study was to evaluate the influence of cultural conditions and physicochemical parameters affecting the growth and biomass production of Aspergillus terreus. Nutrient medium ((Potato dextrose broth, Czapek’s Dox broth, Sabouraud dextrose broth, Potato Carrot broth and malt extract broth) in conjunction with physicochemical parameters such as temperature, pH, nitrogen sources, carbon sources, and salinity were assessed for optimal biomass production. The growth rate of the fungus was measured by determining the mycelial dry weight, and enhancement of antioxidant activity through DPPH scavenging potency in the culture broth. The highest growth rate of the fungus was observed in potato dextrose broth (PDB) compared to the other nutrient media at a significant level (p< 0.05). The present study demonstrated that PDB supplemented with cellulose and urea as carbon and nitrogen sources with a pH of 5.5 and 4% salinity at 28°C are the suitable conditions for maximal growth and biomass production. However, the antioxidants activity was at its peak level in PDA enriched with sucrose and ammonium sulphate at pH 5.5, and 7% salinity at 28°C. Thus, culture conditions, including nutrient components and physicochemical parameters play an important role in the growth rate A. terreus, to exploit the maximum yield for various biomedical applications.

Ecological Aspect of Antibiotic Batumin Synthesis by Pseudomonas batumici

The species Pseudomonas batumici, isolated from the rhizosphere of eucalyptus in the humid subtropical zone, is a producer of the polyketide antibiotic batumin with highly selective activity against staphylococci. Batumin biosynthesis operon includes 28 genes or 74 151 bp. According to modern notions, the biosynthesis of energy-intensive metabolites, which probably includes batumin, is justified in the case of its multifunctionality for producers. The species P. batumici, as a representative of rhizosphere bacteria, must interact with plants and compete with the surrounding microbiota. Aim. To determine the role of batumin in the ecology of the rhizosphere producer strain P. batumici UCM B-321. Methods. The batumin producing strain P. batumici UCM B-321T was obtained from the Ukrainian Collection of Microorganisms. Antibiotic batumin was obtained by fermentation of P. batumici UCM B-321. Extraction was carried out from acidified P. batumici fermentation broth by chloroform (1:2). Chromatographic analysis of fermentation broth obtained after centrifugation was carried out by HPLC using liquid chromatograph Agilent 1200 with mass spectrometric detector Agilent G1956B. Batumin derivatives were obtained after the extraction of the fermentation broth of P. batumici using thin layer chromatography (TLC) on silica gel plates (Merck, USA) in the benzene-isopropanol system (5:1). Disc-diffusion method on phytopathogenic test-strains was used for bioautography. Biofilm formation by P. batumici strain was studied according to O’Toole by growing strain B-321 at 25 0C for 48 hours in 96-well plates on LB medium. Batumin effect upon bacterial mobility was studied using Volf and Berg method in Petri dishes with 0.5% semisolid bacterial agar. To research chemotaxis the soil strain Bacillus subtilis IMV B-7023 and the following concentrations of batumin were used: 20, 50, and 150 μg/mL. The studies were performed using Tso and Adler method. Results. Growth inhibition zones for phytopathogenic bacteria strains were the following (in mm): Pseudomonas syringae pv. syringae UCM B-1027T – 19±3, P. fluorescens IMV 8573 – 22±3, Pectobacterium carotovorum UCM B-1075T – 17±2. Activity against Xantomonas campestris pv. campestris UCM B-1049, Clavibacter michiganensis subsp. michiganensis IMV 102, Agrobacterium tumefaciens UCM B-1000 was not detected. Minimum inhibitory concentrations (MIC) in the range from 8 to 64 μg/mL for P. carotovorum UCM B-1075T, Erwinia aroidea IMV 1058, Proteus vulgaris UCM B-905 and P. fluorescens IMV 8573 are hardly comparable with the discovered against staphylococci. TLC analysis of its broth extract revealed five separate compounds with different values of retention factors: Rf1=0.42; Rf2=0.38; Rf3=0.31; Rf4=0.28; Rf5=0.25. The main component of extract was batumin, other four substances were present in minor quantities. All found substances had similar absorption maxima with the minimum differences between isomeric forms: descarbamoyl batumin-enol (Mr=505, λ=226 nm), descarbamoyl batumin-keto (Mr=505, λ=231 nm), batumin (Mr=548, λ=231 nm), batumin-enol (Mr=548, λ=228 nm) and 17-hydroxy-batumin (Mr=550, λ=229 nm). The largest inhibition zone (P. carotovorum UCM B-1075T) was on the third compound placement which represents of batumin, tiny inhibition zones were found around keto and enol form of descarbamoyl batumin. Observation of live bacterial cells in light microscope confirmed a serious disruption of motility in all these bacteria by batumin in the concentration far lower than the MIC for these organisms. Proteus actively moved in the control, but in presence of 10 μg/mL of batumin was almost no growth. The biofilm formation by P. batumici UCM B-321 was stimulated by supplementing batumin into the medium. The stimulation effect by batumin on the biofilm formation was equally strong when the compound was applied in the concentrations of 1 and 10 μg/mL. Batumin was not an attractant of the producer strain. However, in one of our experiments batumin has shown the properties of positive effector (attractant) for B. subtilis UCM B-7023 strain. Conclusion. The discovered features allow to consider the antibiotic batumin synthesized by P. batumici UCM B-321 as the essential tool for survival and competition of the producer strain in a natural habitat.

Diversity of Marine Macro-Algicolous Endophytic Fungi and Cytotoxic Potential of Biscogniauxia petrensis Metabolites Against Cancer Cell Lines.

Hypersaline environments are known to support diverse fungal species from various orders. The production of secondary metabolites is one of the strategies that fungi adopt to thrive under such extreme environments, bringing up the stress tolerance response. Some such unique secondary metabolites also exhibit clinical significance. The increasing prevalence of drug resistance in cancer therapy demands further exploration of these novel bioactive compounds as cancer therapeutics. In the present study, a total of 31 endophytic fungi harboring inside red, green, and brown marine algae have been isolated and identified. The maximum likelihood analysis and diversity indices of fungal endophytes revealed the phylogenetic relationship and species richness. The genus Aspergillus was found to be the dominating fungus, followed by Cladosporium spp. All the isolated endophytic fungal extracts were tested for their cytotoxicity against HeLa and A431 cancer cell lines. Nine isolates were further analyzed for their cytotoxic activity from the culture filtrate and mycelia extract. Among these isolates, Biscogniauxia petrensis showed potential cytotoxicity with CC50 values of 18.04 and 24.85 μg/ml against HeLa and A431 cells, respectively. Furthermore, the media and solvent extraction optimization revealed the highest cytotoxic active compounds in ethyl acetate extract from the potato dextrose yeast extract broth medium. The compound-induced cell death via apoptosis was 50–60 and 45% when assayed using propidium iodide-live/dead and loss of mitochondrial membrane potential assay, respectively, in HeLa cells. Four bioactive fractions (bioassay-based) were obtained and analyzed using chromatography and spectroscopy. This study reports, for the first time, the cytotoxic activity of an endophytic fungal community that was isolated from marine macro-algae in the Rameswaram coastal region of Tamil Nadu, India. In addition, B. petrensis is a prominent apoptotic agent, which can be used in pharmaceutical applications as a therapeutic.

Beef-Based Medium Influences Biofilm Formation of Escherichia coli O157:H7 Isolated from Beef Processing Plants

Beef-based medium beef extract (BE) and standard medium tryptic soy broth (TSB) are used as minimally processed food models to study the effects on Escherichia coli O157:H7 biofilm formation. The effects of temperatures (4, 10, 25, 37, and 42°C), pH values (4.5, 5.0, 5.5, 6.0, 7.0, and 8.0), strain characteristics, and the expression of functional genes on the biofilm formation ability of the bacteria were determined. The three tested E. coli O157:H7 strains produced biofilm in both media. Biofilm formation was greater in BE than in TSB (P < 0.05). The strongest biofilm formation capacity of E. coli O157:H7 was achieved at 37°C and pH 7.0. Biofilm formation was significantly inhibited for three tested strains incubated at 4°C. Biofilm formation ability was correlated with swarming in TSB. Biofilm formation was significantly and positively correlated with autoaggregation or hydrophobicity in BE (P < 0.05). At the initial stage of biofilm formation, the expressions of luxS, sdiA, csgD, csgA, flhC, adrA, and rpoS were significantly higher in BE than in TSB (P < 0.05). At the maturity stage, the expressions of luxS, sdiA, csgD, csgA, flhC, csrA, adrB, adrA, iraM, and rpoS were significantly higher in TSB than in BE (P < 0.05). Such information could help in the development of effective biofilm removal technologies to deal with risks of E. coli O157:H7 biofilms in the beef industry.

Diversity of Endophytic Actinomycetes Producing Indole-3- Acetic Acid and In vitro Evaluation of Plant Growth-promoting Activity on Brassica oleracea L.

The evaluation of endophytic actinomycetes diversity, growth-promoting strain effects on cauliflower seeds germination, and in vitro organogenesis are the objectives in this study. Moreover, 15 strains from 125 isolates were determined to produce indole-3- acetic acid (IAA), where majority was obtained from roots (66.67%), followed by from branches (26.67%) and leaves (6.67%). Specifically, Jatropha sp. is a plant species with the most endophytic actinomycetes content compared to others. In addition, all endophytic Streptomyces strains were screened based on IAA production ability in vitro on yeast extract–malt extract broth (YMG) broth medium. The results showed the tendency for one strain with code Streptomyces sp. KMR-1E to generate a maximum IAA isolate from Cinnamomun sp. plant. Furthermore, the molecular taxonomy and phylogenetic analysis were recreated from 16S rRNA gene sequences, which attributed the KMR- 1E to genus Streptomyces. Meanwhile, plant growth promotion was evaluated under in vitro condition. This exposed the individual ability to enhance the shoot and root length of cauliflowers. The untreated cultures with a strain free agar block was used as control.

Effects of Anaerobic Fermentation on Black Garlic Extract by Lactobacillus: Changes in Flavor and Functional Components.

The purpose of this study is to investigate the potential application of probiotics in the development of novel functional foods based on black garlic. The single-factor analysis (extraction temperatures, solid-to-liquid ratios, and extraction times) and the response surface methodology were firstly used to optimize hot water extraction of soluble solids from black garlic. The optimal extraction conditions were temperature 99.96°C, solid-to-liquid ratio 1:4.38 g/ml, and extracting 2.72 h. The effects of Lactobacillus (Lactobacillus plantarum, Lactobacillus rhamnosus, and co-culture of them) fermentation on the physicochemical properties of black garlic extract broth were studied for the first time. Artificial and electronic sensory evaluations demonstrated that fermentation significantly influenced the sensory characteristics. The variations of metabolites in different broth samples (S1, unfermented; S2, 1-day fermentation by L. plantarum; S3, 2-day fermentation by L. rhamnosus; and S4, 1-day fermentation by co-cultured Lactobacillus) were further investigated by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry/mass spectrometry analysis. As a result, Lactobacillus fermentation significantly reduced the pH; increased the contents of the total acid, amino nitrogen, total polyphenol, and total flavonoid; and reduced the content of 5-hydroxymethylfurfural (a carcinogenic component) by 25.10–40.81% in the black garlic extract. The contents of several components with unpleasant baking flavors (e.g., furfural, 2-acetylfuran, and 5-methyl furfural) were reduced, whereas the contents of components with green grass, floral, and fruit aromas were increased. More importantly, the contents of several functional components including lactic acid, Gly-Pro-Glu, sorbose, and α-CEHC (3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-propanoic acid) were increased after Lactobacillus fermentation. The results demonstrated the potential of probiotic fermentation to improve the quality of black garlic. This work will provide an insight into the strategic design of novel black garlic products and facilitate the application of black garlic in functional foods.

Antimicrobial properties of marine fungi from sponges and brown algae of Mauritius

ABSTRACT Purpose of the study: Marine fungi of Mauritius have been poorly studied. There are numerous reports on the bioactive secondary metabolites that are produced by fungi around the world. Yet, research on the molecular characterisation and the pharmaceutical potential of marine fungi in Mauritius is rather scanty. Method: The samples, which consisted of three sponges Haliclona sp., Iotrochota sp. and Biemna sp. and two brown algae Turbinaria conoides and Sargassum portierianum, were collected in the North of Mauritius during winter. No sporulating structures were observed from the fungal cultures making morphological analysis impossible. The molecular characterisation of the selected isolates was carried out by the amplification of the ITS regions and phylogenetic analysis. The antimicrobial properties were then determined using the disc diffusion and the minimum inhibitory concentration (MIC) assay. Results: Genus level identification was made from molecular data and for some isolates, species-level identification was even possible. Twelve fungi that showed the best antimicrobial properties were identified as Peniophora sp., Aspergillus cristatus, Acremonium sp., Cordyceps memorabilis, Aspergillus ochraceus, Biscogniauxia sp., Aspergillus keratitidis, Exserohilum rostratum, Chromocleista sp., Nigrospora oryzae, Aspergillus flavipes and Mycosphaerella. The lowest MIC result of 0.0098 mg/mL was obtained with Chromocleista sp. mycelium extract against Staphylococcus aureus. The MIC of the mycelium extracts was lower than the broth extracts for most isolates indicating that the antimicrobial compounds are not secreted. Conclusion: Marine fungi from the Mauritian waters have immense potential in the search for natural products against antibiotic-resistant bacteria.

Hydrazine-Containing Heterocycle Cytochalasan Derivatives From Hydrazinolysis of Extracts of a Desert Soil-Derived Fungus Chaetomium madrasense 375.

“Diversity-enhanced extracts” is an effective method of producing chemical libraries for the purpose of drug discovery. Three rare new cytochalasan derivative chaetoglobosins B1-B3 (1–3) were obtained from chemically engineered crude broth extracts of Chaetomium madrasense 375 prepared by reacting with hydrazine monohydrate and four known metabolite chaetoglobosins (4–7) were also identified from the fungus. The structures were identified by NMR and MS analysis and electronic circular dichroism simulation. In addition, the antiproliferative activities of these compounds were also evaluated, and the drug-resistant activities of cytochalasans were evaluated for the first time. Compound 6 possessed potent activity against four human cancer cells (A549, HCC827, SW620, and MDA-MB-231), and two drug-resistant HCC827 cells (Gefitinib-resistant, Osimertinib-resistant) compared with the positive controls.

Marinoterpins A-C: Rare Linear Merosesterterpenoids from Marine-Derived Actinomycete Bacteria of the Family Streptomycetaceae.

The chemical examination of two undescribed marine actinobacteria has yielded three rare merosesterterpenoids, marinoterpins A–C (1–3, respectively). These compounds were isolated from the culture broth extracts of two marine-derived actinomycetes associated with the family Streptomycetaceae, (our strains were CNQ-253 and AJS-327). The structures of the new compounds were determined by extensive interpretation of 1D and 2D NMR, MS, and combined spectroscopic data. These compounds represent new chemical motifs, combining quinoline-N-oxides with a linear sesterterpenoid side chain. Additionally, consistent in all three metabolites is the rare occurrence of two five-ring ethers, which were derived from an apparent cyclization of methyl group carbons to adjacent hydroxy-bearing methylene groups in the sesterterpenoid side chain. Genome scanning of AJS-327 allowed for the identification of the marinoterpin (mrt) biosynthetic cluster, which consists of 16 open-reading frames that code for a sesterterpene pyrophosphate synthase, prenyltransferase, type II polyketide synthase, anthranilate:CoA-ligase, and several tailoring enzymes apparently responsible for installing the N-oxide and bis-tetrahydrofuran ring motifs.

COMPARATIVE EFFICACY OF FOUR ORGANIC SOLVENT FRACTIONS OF LEAF EXTRACT OF HEMP AGAINST Aspergillus versicolor

The fungus Aspergillus versicolor is generally found on food products and produces sterigmatocystin, a carcinogenic and hepatotoxic mycotoxin. This study reports the usefulness of polar and non-polar fractions of methanolic extract of hemp (Cannabis sativa L.) leaves against this fungus. Dried leaves of hemp were soaked in methanol for two weeks. After filtration and evaporation, water was added to the residual and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol. Different concentrations of each fraction were prepared which ranged from 1.562 to 200 mg mL-1. Antifungal activity was carried out in malt extract broth medium. In general, all the concentrations of the four organic solvent fractions significantly controlled the growth of A. versicolor. There was 71–82%, 59–82%, 65–80% and 69–82% decline in biomass of A. versicolor due to n-hexane, chloroform, ethyl acetate and n-butanol fractions, respectively. It is concluded that different fractions of leaf extract of C. sativa has remarkable potential in controlling growth of A. versicolor.

First Report of Rhodococcus fascians Causing Fasciation of Lilies (Lilium longiflorum Thunb.) in South Korea.

Rhodococcus fascians is a bacterium that causes growth abnormalities such as leafy galls, fasciation, and shoot proliferation in many plants, including ornamental plants. In February 2020, the Animal and Plant Quarantine Agency of South Korea detected 492,000 contaminated lily bulbs using an in-house PCR test based on the R. fascians fasD gene, and subsequently 1.3 million imported bulbs were destroyed. Because no pathogen isolation was associated with this diagnosis, there has been great cultivator demanded for bacterial isolation evidence of lily bulb infection with pathogenic R. fascians. To isolate the causal bacterium of the PCR tests, we sampled leaf, stem, and bulb tissues from 130 lilies with growth abnormality symptoms, collected from 24 South Korean mass production lily farms from June to August 2020. Supernatants of the homogenized samples were spread on mD2 medium (Kado and Heskett 1970) and incubated at 28°C for 10 days. Yellow to orange colonies were isolated into pure culture on mD2. Total DNA was extracted from cultures grown in yeast extract broth (YEB) at 28°C for 24 hours with Wizard DNA prep kit (Promega, Madison, WI, USA). PCR was performed to test for pathogenicity genes fas (A,D, and R) and att (A and R) (Putnam and Miller 2007). Colonies that produced at least one amplicon from these pathogenicity genes were analyzed by partial 16s rRNA gene sequencing to determine the corresponding species. Three strains that were isolated from the bulbs of fasciated lilies from Wanju (35°56´22.1˝N; 127°08´52.0˝E), Gwacheon (37°26´51.6˝N; 127°00´11.8˝E), and Yeongwol (37°18´45.8˝N; 128°11´05.6˝E), or W1, G3, and Y5 strains, yielded PCR products of the expected size for fas and att genes with the primer sets published in Serdani et al. (2013) and developed in this study (attAF: 5'-CCCGGCTACACGCATTCGC-3', attAR: 5'-CGAACGCGGTGTGCAGGT-3' and attRF: 5'-AGTGTCCCGTCGGCGAG-3', attRR: 5'-CGCGGCAGATCGAAGTCCT-3'). Sequences of the three strains were deposited in Genbank for fasA (accession MW122940-942), fasD (G3:MW122935 and 936), and fasR (MW122937-939); all shared 98.3 - 100% nucleotide identity to corresponding sequences from phytopathogenic R. fascians A25f (CP049745.1 Protein_ID fasA:QII09280.1, fasD:QII09282.1, and fasR:QII09277.1). The attA and attR products were only present in G3 (attA: MW122943 and attR: MW122944) and resulted in 100% identity to those of A25f (CP049745.1 Protein_ID attA:QII09269.1, attR:QII09267.1). Partial 16s rRNA gene sequences were obtained (MW064131-133) and clustered with phytopathogenic R. fascians strains D188, A21d2, and A25f. Thus we concluded that strains (W1, G3, and Y5) corresponded to R. fascians. To test the pathogenicity of these three strains, 10 seeds of garden peas for each strain were inoculated at 108 CFU/ml according to Nikolaeva et al. (2009), and the length of the main stem of each seedling was calculated 22 days post-inoculation. Seedlings inoculated with G3 and Y5 resulted in a stunted phenotype with up to 40% height reduction (p ≤ 0.001) compared to non-inoculated seedlings. As for the seedlings inoculated with W1, they exhibited as much as 15% height reduction (p ≤ 0.001). Colonies were recovered from the inoculated seedlings, identity was confirmed through colony PCR for fas and att genes. To our knowledge, this is the first report of phytopathogenic R. fascians in lilies cultivated in South Korea.

Microbiological study in a gneissic cave from Sri Lanka, with special focus on potential antimicrobial activities

The emergence of antibiotic resistance is a global health crisis, thus the search for novel antimicrobial compounds has become a continuous necessity. Underexplored and extreme environments, such as cave ecosystems, have been identified as a promising potential source for the discovery of novel microorganisms with novel antimicrobial compounds (AMC). This study presents the first cave microbiological investigation in Sri Lanka, with a special preference for bioprospecting of novel AMC. The cave sediment characterization demonstrated the presence of close to strong acidic conditions (pH 3.1 – 3.3) and thus indicates the possibility of isolating acidophilic microorganisms. Eight cave wall/ceiling fungal strains were isolated from Sthreepura Cave - Kuruwita and identified using both morphological and ribosomal Internal Transcribed Spacer (ITS) region sequence analysis. Interestingly, four fungal isolates (Penicillium panissanguineum, P. cremeogriseum, Aspergillus bertholletius and Trichoderma yunnanense) were found to be the first records in Sri Lanka. Of these eight isolates, three showed antimicrobial activity (AMAs) against at least one of the five tested human pathogens in preliminary screening, while A. fumigatus (SKW 404) strain showed the highest AMA against Staphylococcus aureus (ATCC 11778) assessed by agar culture plug method on Muller Hinton Agar (MHA). Crude Ethyl Acetate (EtOAc) fraction of both mycelial and Potato Dextrose Broth (PDB) extracts of A. fumigatus demonstrated similar bioactive metabolic profiles with four corresponding chemical fractions [Rf = 0.47, 0.56, 0.65, 0.82; EtOAc: Hexane (4:1, v/v)] in TLC: agar overlay bioassay. The present study indicates that there is potential for discovering novel Sri Lankan deep cave microorganisms and bioprospecting of their novel bioactive compounds. Hence, further island-wide in-depth cave microbiological investigations are required for a better understanding of the Sri Lankan cave microbiology.

Isolation, Antimicrobial activities, Antioxidant Scavenging activities and Trace Element Investigation of Isolated Actinomycetes spp. in Kalar, Iraqi Kurdistan Region

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The poor prognosis of the most microbial pathogenic diseases, due to acquiring resistance against the most convenient antimicrobial medication, encourages researchers to find out a new approach to obtain a novel medication. The current study was aimed to obtain new actinomycetes strains or their novel bioactive metabolites. Eleven isolates were found from 45 distinct non-farming soils in Kalar, Iraqi Kurdistan Region. The non-farming soil 2 (NFS2) isolate was demonstrated as the most effective isolate among all other isolates when tested for primary antimicrobial screening. In addition, in case of crude extract, the NFS2 confers great antimicrobial activities particularly against Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATTC 9341, as well as, against Candida albicans ATCC 10231, with inhibition zones diameters, 25.5, 33.5 and 30.5 mm respectively. However, the isolate has no any antibacterial responses against Gram-negative bacteria, using all types of extracts including crude extract and organic solvent extracts. The crude extract exhibited the powerful antioxidant and H2O2 scavenging activities recording 93.46% with 30mg/ml and 43.94% with 1.25 mg/ml respectively. Investigating the trace elements concentration in broth cultures yeast malt extract broth (ISP2) by inductively coupled plasma (ICP), for confirming the value of these elements particularly (Ca, Fe, K, Na, P, Sr, Mg, and Zn). Primarily, these elements were either exhausted or produced during the fermentation process. Furthermore, manipulating the trace elements concentration will play a crucial role in enhancing or diminishing the final secondary metabolites products in actinomycets isolates.

Sensitivity of Molds From Spoiled Dairy Products Towards Bioprotective Lactic Acid Bacteria Cultures.

Fungal spoilage of dairy products is a major concern due to food waste and economical losses, some fungal metabolites may furthermore have adverse effects on human health. The use of lactic acid bacteria (LAB) is emerging as a potential clean label alternative to chemical preservatives. Here, our aim was to characterize the growth potential at three storage temperatures (5, 16, and 25°C) of a panel of molds (four Mucor and nine Penicillium strains) isolated from dairy products, then investigate the susceptibility of the molds toward 12 LAB cultures. Fungal cell growth and morphology in malt extract broth was monitored using oCelloScope at 25°C for 24 h. Mucor plumbeus 01180036 was the fastest growing and Penicillium roqueforti ISI4 (P. roqueforti ISI4) the slowest of the tested molds. On yogurt-agar plates, all molds grew at 5, 16, and 25°C in a temperature-dependent manner with Mucor strains growing faster than Penicillium strains regardless of temperature. The sensitivity toward 12 LAB cultures was tested using high-throughput overlay method and here all the molds except P. roqueforti ISI4 were strongly inhibited. The antifungal action of these LAB was confirmed when spotting mold spores on agar plates containing live cells of the LAB strains. However, if cells were removed from the fermentates, the inhibitory effects decreased markedly. The antifungal effects of volatiles tested in a plate-on-plate system without direct contact between mold and LAB culture media were modest. Some LAB binary combinations improved the antifungal activity against the growth of several molds beyond that of single cultures in yogurt serum. The role of competitive exclusion due to manganese depletion was examined as a possible antifungal mechanism for six Penicillium and two Mucor strains. It was shown that this mechanism was a major inhibition factor for the molds tested apart from the non-inhibited P. roqueforti ISI4 since addition of manganese with increasing concentrations of up to 0.1 mM resulted in partly or fully restored mold growth in yogurt. These findings help to understand the parameters influencing the mold spoilage of dairy products and the interactions between the contaminating strains, substrate, and bioprotective LAB cultures.

Iron Coordination Properties of Gramibactin as Model for the New Class of Diazeniumdiolate Based Siderophores.

Gramibactin (GBT) is an archetype for the new class of diazeniumdiolate siderophores, produced by Paraburkholderia graminis, a cereal‐associated rhizosphere bacterium, for which a detailed solution thermodynamic study exploring the iron coordination properties is reported. The acid‐base behavior of gramibactin as well as its complexing ability toward Fe3+ was studied over a wide range of pH values (2≤pH≤11). For the latter the ligand‐competition method employing EDTA was used. Only two species are formed: [Fe(GBT)]− (pH 2 to 9) and [Fe(GBT)(OH)2]3− (pH≥9). The formation of [Fe(GBT)]− and its occurrence in real systems was confirmed by LC‐HRESIMS analysis of the bacteria culture broth extract. The sequestering ability of gramibactin was also evaluated in terms of the parameters pFe and pL0.5. Gramibactin exhibits a higher sequestering ability toward Fe3+ than EDTA and of the same order of magnitude as hydroxamate‐type microbial siderophores, but smaller than most of the catecholate‐type siderophores and much higher than the most known phytosiderophores.