Yeast Extract Broth Research Articles

Bioactivity of a dihydroxy flavone produced by Penicillium EU0013 against the wilt pathogen of tomato

Bioassay-guided isolation of a dihydroxy flavone from the cell extract of Penicillium EU0013 was carried out using preparative LCMS and the structure was interpreted using 1D and 2D NMR spectra. The fungus was fermented in three different nutrient media i.e. Glucose Nutrient Broth, Glucose Peptone Yeast Broth, and Yeast Extract Broth and the cell extract was obtained using organics. Comparative LCMS profiles of the fungus confirmed the enhanced production of dihydroxy flavone in GPYB media. In this study, the pathogenicity of the dihydroxy flavone is reported for the first time against Fusarium oxysporum f.sp. lycopersici, wilt causing pathogen of a tomato plant. In MIC experiments, the compound inhibited the pathogen at 80 µg mL−1. Molecular docking studies of the compound with Fol tomatinase showed five binding interactions. These docking studies may help explore dihydroxy flavones as a lead for developing new fungicides to prevent wilt disease in tomatoes.

Pesticidal activity of extra cellular metabolites extracted from the green muscardian fungi Metarhizium anisopliae

AbstractThe present study was undertaken to screen the pesticidal activity of extra cellular metabolites derived from entomopathogenic fungus Metarhizium anisopliae against Spodoptera litura and Aphis crassivora an important aphid pest under laboratory and pot culture condition. Fungal strain used in the study was isolated from the soil by serial dilution technique. Isolated fungal strain was mass multiplied in a liquid medium sabouraud maltose yeast extract broth under optimum condition (28°C). Culture filtrate and the collected culture derived from the medium was used for the source of metabolites. The culture filtrate was extracted with ethyl acetate, acetone and the concentrated extract was screened for pesticidal activity against Spodoptera litura and Aphis crassivora adopting leaf disc and pot culture assay respectively. Extracted metabolites showed pesticidal activity against both insect pests. However, notable effect was recorded in ethyl acetate derived metabolites. The ethyl acetate extract treatment recorded 91.2% mortality at a concentration of 100 μg against Spodoptera litura and 100% mortality at a concentration of 100 μg against Aphis crassivora. Phytotoxicity was determined by measuring the germination index (GI) of groundnut seeds by exposing it to the crude metabolites extracted in ethyl acetate and acetone. The results showed that the germination index was high on ethyl acetate extract which was recorded to be 98.0% and germination index of acetone extract was found to be 90.0%. Production of insecticidal metabolites from M. anisopliae under laboratory condition proposed the possible utilization of metabolites as an effective, safe pesticidal agent against economic important insect pests. No impact on the seedling emergence revealed non phytotoxic effect of the metabolites.

Veillonella faecalis sp. nov., a propionic acid-producing bacterium isolated from the faeces of an infant.

A strictly anaerobic, Gram-stain-negative, catalase-negative, cocci-shaped, and propionate-producing bacterial strain, named Ds1651T was isolated from the fecal sample collected from a South Korean infant. Through a comparison of 16S rRNA gene sequences, it was revealed that Ds1651T had the highest phylogenetic affinity with Veillonella nakazawae KCTC 25297T (99.86%), followed by Veillonella infantium KCTC 25370T (99.80%), and Veillonella dispar KCTC 25309T (99.73%) in the family Veillonellaceae. Average nucleotide identity values between Ds1651T and three reference species were 95.48% for Veillonella nakazawae KCTC 25297T, 94.46% for Veillonella infantium KCTC 25370T, and 92.81% for Veillonella dispar KCTC 25309T. The G + C content of Ds1651T was 38.58mol%. Major fermentation end-products were acetic and propionic acids in Trypticase peptone glucose yeast extract broth with 1% (v/v) sodium lactate. The predominant cellular fatty acids that account for more than 10% were summed in Feature 8 (C17:1 ω8c and/or C17:2) and C13:0. Based on the findings from phylogenetic, genomic, phenotypic, and chemotaxonomic studies, we propose that the type strain Ds1651T (= KCTC 25477T = GDMCC 1.3707T) represents a novel bacterial species within the genus Veillonella, with the proposed name Veillonella faecalis sp. nov.

Genetic characterization and pathogenicity in a mouse model of newly isolated bat-originated mammalian orthoreovirus in South Korea.

Mammalian orthoreoviruses (MRVs) infect a wide range of hosts, including humans, livestock, and wildlife. In the present study, we isolated a novel Mammalian orthoreovirus from the intestine of a microbat (Myotis aurascens) and investigated its biological and pathological characteristics. Phylogenetic analysis indicated that the new isolate was serotype 2, sharing the segments with those from different hosts. Our results showed that it can infect a wide range of cell lines from different mammalian species, including human, swine, and non-human primate cell lines. Additionally, media containing trypsin, yeast extract, and tryptose phosphate broth promoted virus propagation in primate cell lines and most human cell lines, but not in A549 and porcine cell lines. Mice infected with this strain via the intranasal route, but not via the oral route, exhibited weight loss and respiratory distress. The virus is distributed in a broad range of organs and causes lung damage. In vitro and in vivo experiments also suggested that the new virus could be a neurotropic infectious strain that can infect a neuroblastoma cell line and replicate in the brains of infected mice. Additionally, it caused a delayed immune response, as indicated by the high expression levels of cytokines and chemokines only at 14 days post-infection (dpi). These data provide an important understanding of the genetics and pathogenicity of mammalian orthoreoviruses in bats at risk of spillover infections.IMPORTANCEMammalian orthoreoviruses (MRVs) have a broad range of hosts and can cause serious respiratory and gastroenteritis diseases in humans and livestock. Some strains infect the central nervous system, causing severe encephalitis. In this study, we identified BatMRV2/SNU1/Korea/2021, a reassortment of MRV serotype 2, isolated from bats with broad tissue tropism, including the neurological system. In addition, it has been shown to cause respiratory syndrome in mouse models. The given data will provide more evidence of the risk of mammalian orthoreovirus transmission from wildlife to various animal species and the sources of spillover infections.

Thermal inactivation kinetics of Listeria monocytogenes in milk under isothermal and dynamic conditions

Thermal processing is a widely used method to ensure the microbiological safety of milk. Predictive microbiology plays a crucial role in quantifying microbial growth and decline, providing valuable guidance on the design and optimization of food processing operations. This study aimed to investigate the thermal inactivation kinetics of Listeria monocytogenes in milk under both isothermal and dynamic conditions. The thermal inactivation of L. monocytogenes was conducted under isothermal and non-isothermal conditions in sterilized and pasteurized milk, with and without background microbiota, respectively. Furthermore, a secondary model was developed between the shoulder effect and temperature, which was then integrated into the dynamic model. The results showed that L. monocytogenes grown in Tryptic Soy Yeast Extract Broth (TSBYE) prior to thermal inactivation exhibited higher heat resistance compared to cells grown in sterilized milk at isothermal temperatures of 60.0, 62.5, and 65℃. Moreover, the presence of background microbiota in milk significantly enhanced the heat resistance of L. monocytogenes, as evidenced by the increased D-values from 1.13 min to 2.34 min, from 0.46 min to 0.53 min, and from 0.25 min to 0.34 min at 60.0, 62.5, and 65 °C, respectively, regardless of whether the background microbiota was inactivated after co-growth or co-inactivated with L. monocytogenes. For non-isothermal inactivation, the one-step dynamic model based on the log-linear with shoulder model effectively described the microbial inactivation curve and exhibited satisfactory model performance. The model developed contributes to improved risk assessment, enabling dairy processors to optimize thermal treatment and ensure microbiological safety.

Utilization of the sugar fraction from Arabica coffee pulp as a carbon source for bacteria producing cellulose and cytotoxicity with human keratinocyte

Coffee pulp (CP), a by-product of coffee production, is an underutilized resource with significant potential value. CP contains monosaccharides that can serve as an ideal carbon source for bacterial cultivation, enabling the production of value-added components such as medical-grade cellulose. Herein, we extracted the sugar fraction from Arabica CP and used it as a supplement in a growing media of a bacteria cellulose (BC), Komagataeibacter nataicola. The BC was then characterized and tested for cytotoxicity. The CP sugar fraction yielded approximately 7% (w/w) and contained glucose at 4.52 mg/g extract and fructose at 7.34 mg/g extract. Supplementing the sugar fraction at different concentrations (0.1, 0.3, 0.5, 0.7, and 1 g/10 mL) in sterilized glucose yeast extract broth, the highest yield of cellulose (0.0020 g) occurred at 0.3 g/10 mL. It possessed similar physicochemical attributes to the BC using glucose, with some notable improvements in fine structure and arrangement of the functional groups. In cytotoxicity assessments on HaCaT keratinocyte cells, bacterial cellulose concentrations of 2–1000 µg/mL exhibited viability of ≥ 80%. However, higher concentrations were toxic. This research innovatively uses coffee pulp for bacterial cellulose, aligning with the principles of a bio-circular economy that focuses on sustainable biomass utilization.

Mass multiplication of Trichoderma asperellum on conifer needles and weed foliage

The impact of hazard by chemical fungicides has necessitated the development of safe and eco-friendly bio-fungicides. Trichoderma asperellum is one of the potent biocontrol agents and for its cost-effectiveness large scale production on several agricultural and industrial wastes has been tested so far. Forest fire due to highly inflammable pine needles and invasion of weeds are considered as a major cause of biodiversity loss in forests of NW Himalaya. Therefore, in the present work, the potential of forest wastes like needles of five conifer species and foliage of three noxious weeds along with some commonly used substrates were evaluated for the mass production of T. asperellum. Amongst all the screened substrates, the highest spore count was recorded on Pinus roxburghii needle powder supplemented with yeast extract and potato dextrose broth. Therefore, authors recommend the use of P. roxburghii needles for the mass multiplication of Trichoderma species as a replacement for useful agro-wastes.

Establishment of a modified opsonophagocytic killing assay for anti-pneumococcal surface protein A antibody

Streptococcus pneumoniae (pneumococcus) is a pathogenic gram-positive bacterium that causes pneumonia, meningitis, and sepsis. Pneumococcal surface protein A (PspA) induces antibodies that protect against lethal infections by pneumococci. PspA is a choline-binding protein present on the cell surface of almost all pneumococcal strains and is a non-capsular polysaccharide vaccine candidate. For research and development of PspA-based vaccines, an in-vitro test system to measure the activity of functional antibodies capable of killing pneumococci is essential. The opsonophagocytic killing (OPK) assay is used to evaluate the opsonic activity of functional antibodies induced by capsular polysaccharide (CPS)-based vaccines (standard OPK assay). Despite the potential of anti-PspA antibodies to protect against lethal infections in mice, the standard OPK assay fails to evaluate anti-PspA antibodies. Using a pneumococcal surface protein C-deficient strain and extending the incubation time of opsonized bacteria, complement, and HL-60 cells reportedly results in enhanced bactericidal activity (modified OPK assay). We aimed to measure the bactericidal activity of anti-PspA antibodies in intact pneumococcal strains. We optimized the pneumococcal culture method used in the OPK assay to increase the efficiency of anti-PspA antibody-mediated phagocytosis of HL-60 cells. As thick capsules hinder phagocytosis, we attempted to obtain pneumococci with thin capsules through an improved culture method. As pneumococci attached to cells exhibit thin capsules, pneumococci cultured in Todd Hewitt yeast extract (THY) broth were spread on blood agar plates and incubated for 4 h. cpsA mRNA transcript levels in pneumococci cultured on blood agar were lower than those in pneumococci cultured in THY broth. OPK activity against pneumococci expressing PspA of clades 1–5 was reasonably well detected using pneumococci cultured on blood agar in the modified OPK assay. The modified OPK assay for anti-PspA antibody using pneumococci cultured on blood agar represents a useful assay to determine the killing activity of functional anti-PspA antibodies against pneumococci.

Molecular Characterization of Multi-Drug Resistant Fungal Isolates Obtained from Raw Cow Milk in Bowen University, Nigeria

Cow milk is a highly nutritious food but many factors predispose it to microbial contamination. There is paucity of information on antifungal-resistant pathogens. Hence the study investigated the fungi from fresh cow milk samples and their resistance to some antifungals. Twenty-seven (27) fungi were isolated from forty-three (n=43) milk samples on PDA (Potato dextrose agar) media, re-cultured in glucose yeast extract broth, and incubated for 24 hours at 25°C. Taxonomic characterization on the isolates was done using photomicrography. Percentage occurrence of the isolates was determined. Molecular characterization was carried out on some isolates which were 100% resistant to antifungals; ketoconazole, amphotericin B, and clotrimazole using ITS1 and ITS4 primers. Isolate sequences were subjected to BLAST analysis and compared with representatives in GenBank. Kruskal-Wallis and Mann-Whitney Tests were used to analyze the data. Phylogenetic analysis and morphological characterization identified isolates as Rhodotorula paludigena, Candida sp and Candida tropicalis which had occurrence of 31.8%, 31.8% and 36.6% respectively. Level of resistance to ketoconazole (100%) and amphotericin B (92%) was significantly higher than clotrimazole (59%) (p< 0.05), while between ketoconazole and amphotericin B, there was no significant (p> 0.05) difference. Unprocessed raw milk is a potential source of drug-resistant pathogenic fungi. Pasteurization of raw milk is highly encouraged before consumption.

The inhibitory effect of copper, zinc, and manganese on Legionella longbeachae and other Legionella spp. in vitro.

Legionella longbeachae is an important cause of Legionnaires' disease in Australasia and is associated with exposure to potting soils. Our aim was to identify ways to reduce the load of L. longbeachae in potting soils. Inductively-coupled plasma optical emission spectrometry (ICP-OES) of an all-purpose potting mix showed copper (Cu) concentrations (mg/kg) range from 15.8 to 23.6. Zinc (Zn) and manganese (Mn) were significantly higher than Cu ranging from 88.6-106 to 171-203, respectively. Minimal inhibitory and bactericidal concentrations of 10 salts used in the horticultural industry were determined for Legionella species in buffered yeast extract (BYE) broth. For L. longbeachae (n=9) the median (range) minimum inhibitory concentration (MIC) (mg/L) of copper sulfate was 31.25 (15.6-31.25), zinc sulfate 31.25 (7.81-31.25), and manganese sulfate 31.25 (7.81-62.5). The MIC and minimum bactericidal concentration (MBC) were within one dilution of each other. Susceptibility to Cu and Zn salts increased as the concentration of pyrophosphate iron in the media decreased. The MIC values for these three metals against Legionella pneumophila (n=3) and Legionella micdadei (n=4) were similar. Combinations of Cu, Zn, and Mn were additive. Legionella longbeachae has similar susceptibility to Cu and other metal ions in comparison to L. pneumophila.

Effective combination of nisin and sesamol against Listeria monocytogenes

Nisin (NS) has been considered as a food preservative against Listeria monocytogenes, an important foodborne pathogen that causes serious public health problems. However, the effectiveness of NS is also affected due to its instability and resistance of L. monocytogenes to NS. Combining NS with other antimicrobial substances is an excellent strategy to improve the effect. Sesamol (SE) has been reported to have the ability to inhibit the growth of L. monocytogenes. Thus, in this study, based on the determination of the fractional inhibitory concentration indices, the in vitro synergistic antibacterial activity of NS and SE was confirmed using standard L. monocytogenes ATCC19112 and L. monocytogenes isolated from food and clinical sources. The inhibition of NS and SE combination on the growth of L. monocytogenes was higher than that of NS or SE alone in tryptic soy yeast extract broth (TSB-YE) and pasteurized milk. The changes and damages of the cell structure and the cell morphology in the combination group were more significant than those in the single use of NS or SE. These results indicate that the combined application of NS and SE may be a promising alternative to NS for controlling the contamination of L. monocytogenes in food.

First Report of Pseudomonas syringae Causing Bacterial Leaf Spot on Winterberry Holly (Ilex verticillata) in Ohio.

Winterberries (Ilex verticillata and hybrids) are deciduous species of holly whose branches bearing colorful fruit are cut in late Fall to be used for seasonal decorations. The annual wholesale value of the woody cuts is $1.5 million nationally (NASS, 2019). In June 2021, approximately 80% of the 45 Ilex verticillata 'Maryland Beauty' potted plants, which were maintained in a container yard at The Ohio State University research farm in Columbus, OH, presented leaves with irregular necrotic lesions surrounded by a chlorotic halo. No other symptoms were present on the plants. Bacterial streaming was observed from the lesions using a compound microscope and isolations were performed after surface disinfesting small sections of leaf tissue from the border of the lesions by soaking in 10% bleach for 30 sec, rinsing twice in sterile water, macerating in sterile water, and streaking the suspension on nutrient broth yeast extract agar. Creamy white, circular, smooth, and convex colonies were recovered after incubation at 28°C for 48 h. Bacterial identification of one representative isolate was initially pursued from single colonies of a purified culture using five discriminative phenotypic tests (i.e., LOPAT: "L", levan production; "O", oxidase activity; "P", pectinolytic activity; "A", arginine dehydrolase production; "T", tobacco hypersensitive reaction), which resulted in the L+ O- P- A- T+ profile consistent with the description of Pseudomonas syringae (Lelliott et al. 1996). Molecular identification was performed based on rpoD marker amplification and sequencing using primers PsrpoD FNP1/PsrpoDnprpcr1 (Parkison et al. 2011). NCBI GenBank BLASTn comparison of the rpoD sequence (GenBank Acc. No. OP221440) shared 99.12% identity to P. syringae pv. passiflorae (AB163366.1). Whole genome sequence analysis was conducted to strengthen the classification of the isolate species. To this extent, DNA was sequenced with an iSeq 100 Illumina benchtop sequencer using Illumina DNA Prep kit and iSeq 100 i1 Reagent v2 (Illumina, Inc, REF: 20060060 and 20031371). Illumina Local Run Manager software was used for base calling, demultiplexing, and trimming of the raw reads. Unicycler v0.5.0 was used for de novo assembly of the genome (Wick et al. 2017). The assembled genome size was 5.9 Mb with 959 contigs and 10× coverage (NCBI GenBank Biosample No. SAMN30281368; Acc. No. JANQCB010000000). Average nucleotide identity (ANI) analysis was performed on the server MiGA online (Rodriguez-R et al. 2018). Subgroup identification was inconclusive (p>0.05), positioning this isolate between P. syringae pv. actinidiae (96.45% ANI) and pv. viburni (96.65% ANI) (Rodriguez-R & Konstantinidis, 2016). Both these pathovars cause leaf spots on woody plants such as kiwi and viburnum (Donati et al. 2020; Garibaldi et al. 2005). To confirm pathogenicity, three separate branches on each of two I. verticillata 'Maryland Beauty' potted plants were selected, and 5-7 individual young leaves (>2 weeks from emergence) on each branch were infiltrated with a bacterial suspension (108 CFU/mL) in sterile water (SW) using a needleless syringe by delivering 30-50 µL of suspension per infiltration point. One additional branch per plant was infiltrated with SW to serve as control. Plants were covered with a plastic bag for two days post-inoculation (DPI) and maintained in the laboratory at an average of 23°C. All inoculated leaves showed necrotic lesions two DPI while control leaves remained asymptomatic. To fulfill Koch's postulates, the bacterium was re-isolated from the symptomatic leaves six DPI and confirmed to be identical to the original isolate based on rpoD gene sequencing. To the best of our knowledge, this report signifies the first instance of P. syringae causing bacterial leaf spot on winterberry worldwide. Ornamental plant sales are based primarily on visual appeal; therefore, identification and monitoring of emerging pathogens is essential to ensure the health of the industry.

Conidia Production of Beauveria Bassiana in Solid Substrate Fermentation Using a Biphasic System

Beauveria bassiana is an important entomopathogenic fungus that is widely used as a bioinsecticide around the world. Conidia production is a critical step in the production of high-quality bioinsecticide. This study investigated three liquid culture mediums and five combinations of solid substrates to enhance conidia production by B. bassiana. The fungus was isolated from infected insects in the cocoa plantation of PT. Perkebunan Nusantara XII in Kediri, East Java, Indonesia. The three culture mediums were malt extract broth (MB), potato dextrose broth (PDB), and yeast and malt extract broth (YMB). Five combinations of solid substrate were used: 100% rice, 100% maize, 75%:25% rice:maize, 50%:50% rice:maize, and 25%:75% rice:maize. The biphasic system was used in this study, in which the fungus was first grown under submerged conditions and then was allowed to conidiate in solid-state conditions. The data showed that PDB was the optimum culture medium to produce blastophore and beauvericin, the active compound that acts as a mycoinsecticide. In the selection test, 100% rice was the optimum solid substrate to produce high amounts of conidia, and the consistency and production tests yielded the same results, with conidia counts of 1.93x109, 1.78x109, and 2.08x109, respectively. In a rice storability test, B. bassiana conidia numbers remained stable for up to 105 days of storage at room temperature.&#x0D; Keywords: Beauveria bassiana, culture medium, solid-substrate, conidia, biphasic system

Suppression of Cephalosporium maydis by the resistance inducer beta-sitosterol

Late wilt, a vascular disease caused by the fungus of Cephalosporium maydis, is considered one of Egypt’s most severe maize threats. The purpose of this study was to investigate the suppressive effect of the resistance inducer beta-sitosterol on C. maydis, as well as its involvement in reducing the incidence of late wilt infection under greenhouse and field conditions. In in vitro studies on potato dextrose yeast extract agar (PDYA) and/or potato dextrose yeast extract broth (PDYB) with doses of 50, 100, 150, 200, and 250 ppm, beta-sitosterol significantly reduced colony diameter and spore germination of C. maydis. The efficiency of beta-sitosterol increased with concentration, with 250 ppm being the most efficient, reducing colony development by 100% and spore germination by 77.3%. Experiments were conducted in greenhouse and field trials using the split-plot design with three beta-sitosterol 250 ppm application methods (maize grain dipping, maize foliar spraying, and maize grain dipping with foliar spraying) and two maize cultivars (a land race and the cultivar fine seed 1005). In both trials, the combination treatment of maize grain dipping and foliar spraying with beta-sitosterol 250 ppm was most effective. Under greenhouse conditions, beta-sitosterol treatments significantly improved the growth parameters (plant height, plant fresh weight, and plant dry weight) of the two maize cultivars. Under similar conditions, beta-sitosterol significantly increased the activity of protective enzymes (peroxidase, polyphenoloxidase, and chitinase) and the levels of chlorophyll, total phenols, and flavonoids in the two maize cultivars. When compared to the untreated control, beta-sitosterol application reduced the incidence of late wilt disease under greenhouse and field conditions. The ear yield of the two maize cultivars was significantly increased in plots treated with beta-sitosterol 250 ppm in a field trial. The findings showed that beta-sitosterol inhibited C. maydis growth in vitro and improved maize plant resistance to late wilt infection in vivo. As a result, this plant resistance inducer could be used to improve the resistance of maize cultivars to late wilt disease.

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.

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.

First Report of Pectobacterium polaris Causing Soft Rot and Black Leg of Potato in Russia.

Blackleg and soft rot of potato (Solanum tuberosum) were monitored in the Central European part of Russia within a period of 2012- 2019. Symptoms included decay of tubers, blackening of stem vascular bundles, and partial yellowing of leaves. The disease causes serious potato yield losses in the field and storage. Pectobacterium parmentieri, P. brasiliense, P. versatile (syn. Ca. Pectobacterium maceratum), P. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are considered as main causal agents of soft rot and blackleg disease in Russia (Voronina et al. 2019, Ngoc Ha et al., 2019, Shirshikov et al. 2018, Kornev et al. 2012). Potato plant samples collected in commercial fields in routine plant health assay were used for bacteria isolation on crystal violet pectate agar (CVP) (Helias et al. 2012) as described previously (Voronina et al. 2019). Bacterial colonies producing pitting on CVP were re-isolated and purified on nutrient broth yeast extract medium. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (Cigna et al. 2017) followed by sequencing. DNA sequence alignment showed that the isolates F099, F100, F106, F109, and F118 were identical (deposited as part of NCBI Ref.Seq. for F109 NZ_RRYS01000004.1, locus KHDHEBDM_RS06360) and grouped together with the type strain Pectobacterium polaris NIBIO1006T (CP017481), a new species described as a potato pathogen (Dees et al. 2017). These strains were negative in diagnostic PCR assays using specific primers Y45/Y46 for the detection of P. atrosepticum, Br1f and L1r for P. brasiliense (Duarte et al. 2004), and ADE1/ADE2 for Dickeya sp. (Nassar et al. 1996). To further validate the identification, strain F109 of P. polaris was selected for genome sequencing. The genome of P. polaris strain F109, (NCBI Reference Sequence NZ_RRYS00000000.1) reveals >99% sequence similarity with type strain P. polaris IPO_1606 (GenBank accession GCA_902143345.1). The strain F109 was deposited to All-Russian Collection of Microorganisms under number VKM V-3420. Thus, the characterization of five isolates provided evidence that a previously unreported pathogen was present in the surveyed fields. The isolates were uniform in genetic and physiological properties; they were gram negative, facultative anaerobes with pectinolytic activity, negative for oxidase, urease, indole production, gelatin liquefaction. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl, unable to utilize malonate and citrate. All the isolates grew at 37°C. All isolates caused soft rot symptoms on 10 inoculated potato tubers. They produced typical black leg rot symptoms in young potato plants inoculated with 107 CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The bacteria were re-isolated successfully from symptomatic potato and pathogen confirmed by gapA sequencing to complete Koch's postulates. To our knowledge, this is the first report of blackleg and soft rot caused by P. polaris on potato in the Russian Federation. According to the data of commercial diagnostic laboratory "PhytoEngineering" (Moscow region), P. polaris occurred in 5% potato seed stocks harvested in 2017-2019 in the Moscow region. This finding may indicate that new Pectobacterium strains have adapted to a diverse environment, which is consistent with widespread distribution of commercial seed potatoes. The author(s) declare no conflict of interest. Funding: This work was supported by Russian Science Foundation grant #16-16-00073.

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.

Novel potential yeast strains for the biotechnological production of xylitol from sugarcane bagasse

AbstractThis study aimed to screen the xylitol‐producing strains of Cyberlindnera xylosilytica and Wickerhamomyces rabaulensis for the bioconversion of the hemicellulosic fraction of sugarcane bagasse. Among the 16 strains tested, two of each species (C. xylosilytica UFMG‐CM‐Y309 and UFMG‐CM‐Y409, and W. rabaulensis UFMG‐CM‐Y3716 and UFMG‐CM‐Y3747) were selected on the basis of xylitol yield (Yp/s) and xylitol volumetric productivity (Qp) in YPX broth (yeast extract, 10 g L−1; peptone, 20 g L−1; and d‐xylose, 60 g L−1). These strains were cultivated in sugarcane bagasse hemicellulosic hydrolysate (SCHH) supplemented with ammonium sulfate, rice bran extract, and yeast extract. The xylitol yield was the highest in the strain C. xylosilytica UFMG‐CM‐Y‐309 with a production of 14.06 g L−1; xylitol yield, 0.63 g g−1; xylitol volumetric productivity, 0.20 g L−1 h−1. Xylitol yields ranged from 14% to 74% above those of other xylitol‐production yeast species. This work demonstrates the potential of biotechnological application of these yeasts for xylitol production. © 2021 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Detection of Legionella pneumophila and Legionella dumoffii Biochemically in Water Samples in Baghdad City, Iraq

Background: Legionellae can be isolated from various sources of water. Legionella pneumophila istransmitted via contaminated water and caused many diseases like pneumonia; lungs abscesses and fever.Objectives: Isolation and identification of L. pneumophila from environmental samples; study the factorsthat affect the frequency of L. pneumophila; and some oxidizing substances on their growth.Materials and Method: A total of one hundred water samples were collected from Cooling tower; Watertanks; Tap water and Swab from inner tap water. All samples were cultured on Buffered charcoal yeast extract(BCYE) agar and Buffered charcoal yeast extract (BCYE) broth to isolate Legionella pneumophila andLegionella dumoffii. Effect of pH; temperature; chlorine; Iron and effect of soluble zinc on L. pneumophilagrowth were studied.Results: The isolates gave positive results for the tests of catalase, oxidase, gelatin degradation, nitratereduction and beta-lactamase. Iron; Soluble Zinc; Chloride; Calcium hardness (CaCO3); Turbidity and silverwere ? 3ppmas; 1-3 ppmas; 0.2-0.5 ppm; 600 ppm; ? 0.3 and 3 ppmas respectively. The distribution of L.pneumophila in the water samples were studied.Conclusion: FatalL. pneumophila may be transmitted by drinking contaminated water and led to deathbecause they caused pneumonia.