High Antagonistic Activity Research Articles

The Potential of Novel Bacterial Isolates from Natural Soil for the Control of Brown Rot Disease (Monilinia fructigena) on Apple Fruits

Monilinia fructigena is one of the most important fungal pathogens causing brown rot on apple and is heavily affecting fruit production. The main objective of this study was to screen for potential bacterial isolates with higher antagonistic activity against M. fructigena. Our study focused on the identification of potential bacterial isolates capable of reducing both the mycelial growth of M. fructigena and the disease severity using in vitro and in planta trials, respectively. To achieve this goal, thirteen bacteria, isolated from natural soil, were evaluated for their abilities to produce lytic enzymes (amylase, cellulase and protease), hydrocyanic acid (HCN) and lipopeptides (bacillomycin, fengycin, iturin and surfactin). Further, results from the dual culture method, volatile and bacterial free-cell filtrate bioassays indicated that tested isolates showed a fungicidal activity against the mycelial growth of M. fructigena. Thus, out of the 13 isolates tested, 12 exhibited significant mycelial inhibition (more than 70%) against M. fructigena, while remaining the last isolate displayed only a partial inhibition (up to 43%). Further, 12 of the bacteria isolates displayed an amylase production, 10 showed cellulase production, 11 revealed protease production, while only 2 displayed HCN production. In addition, most bacterial isolates were found to have genes encoding for different lipopeptides: bacillomycin (10), fengycin (3), iturin (11) and surfactin (1). Interestingly, two bacterial isolates, Bacillus amyloliquefaciens B10W10 and Pseudomonas sp. B11W11 were found to be the most effective and displayed the lowest disease severity in planta trial. These two bacteria reduced the brown rot incidence compared to the synthetic fungicide in a semi-commercial large-scale trial. Therefore, our findings suggest that these two later bacterial isolates provide apple protection against M. fructigena via direct and indirect mechanisms. These isolates may be used, therefore, as potential biological control agents (BCAs) in preventive treatment to control brown rot disease on apple fruits.

Antagonistic activity of strains of lactic acid bacteria isolated from Carpathian cheese

A promising area for improving probiotics is the search for new sources of strains and the development of complex preparations which would include different types of bacterial cultures that complement each other. Sources of selection may be traditional dairy products, in particular, cheeses made from raw milk. Wild strains can be endowed with antibacterial properties. The antagonistic action of lactic acid bacteria (LAB) has long attracted the attention of researchers and scientists. The aim of the study was to investigate the antagonistic activity against pathogenic and opportunistic microorganisms of LAB strains isolated from traditional Carpathian cheese. Three samples of cheese were selected for the research – one sample of brynza and budz (brynza before salting), made in the highlands of the Carpathians, and one sample of budz, made in the foothills. LAB were identified using classic microbiological and modern molecular genetic methods (RAPD-PCR, RFLP-PCR, 16S rRNA gene sequencing). The objects of our studies were five strains of LAB: Lactococcus lactis IMAU32258, L. garvieae JB2826472, Enterococcus durans FMA8, E. faecium L3-23, E. faecium IMAU9421. Technological parameters such as acid-forming activity of milk fermentation, resistance to high concentrations of NaCl and temperature optimums of cultivation were taken as the main criteria for assessing the suitability of LAB for inclusion in fermentation preparations. Antagonistic activity was determined by agar diffusion (agar well method) and optic density of test cultures using a Multiscan FC microplate reader (Thermo scientifiс, USA) at the wave of 620 nm. There were four reference strains of pathogenic and opportunistic microorganisms were test cultures: Listeria monocytogenes PCM 2191, Staphylococcus aureus PCM 458, Escherichia coli PCM 2208, Salmonella typhimurium PCM 2182. Strains of the test cultures were received from the collection of microorganisms of the Institute of Biology and Biotechnology the (University of Rzeszów, Poland). According to the ability of LAB strains to form lactic acid, L. lactis IMAU32258 was the best acid-forming agent with an acid-forming energy of 94 °T. E. faecium was characterized by moderate levels of active and titratable acidity. Less pronounced acid-forming ability was determined for the species E. durans and L. garvieae. Cultures of the genus E. faecium, L. garvieae and E. durans were the most resistant to high concentrations of NaCl (6.5%). Regarding temperature optimums, we found that strains of E. faecium and E. durans species grew both at temperatures of 10, 15 and 45 °C, whereas no growth of L. lactis IMAU32258 and L. garvieae JB282647 2 was observed at 45 °C. Among the studied bacteria, the strains of E. durans FMA8 and E. faecium L3-23 were characterized by the highest antagonistic activity in producing the largest zones of growth inhibition and optic density of pathogenic and opportunistic microorganisms. The strain L. garvieae JB282647 2 exhibited the lowest level of antagonistic activity against pathogenic and opportunistic microorganisms.

Efficacy of some Trichoderma Species on Management of Sunflower Head-Rot

The efficacy of different Trichoderma species were evaluated in vitro and under field conditions against five pathogenic of sunflower head-rot fungi (HRF). Bioagents were applied by three application methods during 2015and2016 growing seasons. In vitro, six species of Trichoderma harzinum, T.virid T.hamatum, T.koningii, T.reesei and T.polysporum were selected for field studies.Both of T. harzianum and T. viride recordid the highest antagonistic potential against almost of the pathogenic fungi followed by T.reesei. In field experiments, the tested bioagents were studied in series of experiments using different three application methods, i.e. foliar spray [F.S.], soil drench[S.D.] and foliar spray with soil drench[F.S.+ S.D.].These bioagents were applied at three stages i.e. 30, 45 and 60 days after sowing. Head diameter, stem diameter, leaf area, seed number per head, 1000 seed weight , seed yield per plant, yield per plot and yield per feddan were determined as crop parameters. Results indicated that the three application methods gave the lowest values of disease incidence and increased crop parameters compared with untreated plants. The highest antagonistic activity was achieved with T.harzinum when applied as foliar spray which gave the highest percentage of disease reduction and increased crop parameters during both seasons, followed by strain of T.viride when applied as soil drench and T. reesei when applied as (soil drench and foliar spray), respectively.

Antagonistic Potential of Novel Endophytic Bacillus Strains and Mediation of Plant Defense against Verticillium Wilt in Upland Cotton.

Verticillium wilt caused by Verticillium dahliae is a threatening disease of cotton, causing economic loss worldwide. In this study, nine endophytic Bacillus strains isolated from cotton roots exhibited inhibitory activity against V. dahliae strain VD-080 in a dual culture assay. B. altitudinis HNH7 and B. velezensis HNH9 were chosen for further experiments based on their high antagonistic activity. The secondary metabolites of HNH7 and HNH9 also inhibited the growth of VD-080. Genetic marker-assisted detection revealed the presence of bacillibactin, surfactin, bacillomycin and fengycin encoding genes in the genome of HNH7 and HNH9 and their corresponding gene products were validated through LC-MS. Scanning electron microscopy revealed mycelial disintegration, curling and shrinkage of VD-080 hyphae after treatment with methanolic extracts of the isolated endophytes. Furthermore, a significant reduction in verticillium wilt severity was noticed in cotton plants treated with HNH7 and HNH9 as compared to control treatments. Moreover, the expression of defense-linked genes, viz., MPK3, GST, SOD, PAL, PPO and HMGR, was considerably higher in plants treated with endophytic Bacillus strains and inoculated with VD-080 as compared to control.

Biodiversity of meatborne Listeria spp. in Himachal Pradesh and their interaction with indigenous probiotics.

This study determined the anti-listerial activity of indigenous probiotics from traditional fermented foods of Western Himalaya against meat borne Listera monocytogens isolates from Himachal Pradesh. One hundred samples of meat and meat products like chicken (n = 25), chevon (goat meat, n = 20), fish (n = 20) and pork (n = 30) were collected and were analyzed for the presence of Listeria spp. by recommended culture and biochemical methods. L. monocytogens isolates were confirmed by PCR targeting the virulence gene hlyA (haemolysin A) and by16S rRNA sequencing. Anti-listerial activity of probiotic bacteria isolated from indigenous fermented foods of Himachal Pradesh was determined by well diffusion method using Lactobacillus rhamnosus GG (ATCC 53103) as the reference strain. Five percent of tested samples were found positive for L. monocytogens with incidence of 8.0% in chicken (2/25), 10.0% in fish (2/20) and 4.0% in chevon meat (1/25). None of the tested pork samples were found contaminated with L. monocytogenes. Among 11 indigenous probiotics used in this study, highest antagonistic activity was exhibited by Lactobacillus plantarum (ADF 10) and Enterococcus faecium (ADF1) which was equivalent to the reference strain.

Properties of formed compositions of probiotic strains isolated from Carpathian bryndza

In order to give the product functional properties, promising for the industry strains of cultures of lactic acid bacteria L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18, isolated from traditional Carpathian bryndza. It was found that under conditions of co-cultivation, the studied strains were compatible, ie did not show interspecific antagonism. Based on these probiotic strains, five compositions of L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18 in the ratios,%: composition № 1 – 50:25:25; № 2 – 33:33:33; № 3 – 50:40:10; № 4 – 50:10:40 and № 5 – 70:15:15 respectively. When determining the rate of biomass accumulation, it was found that at a temperature of 32 ° C the highest growth intensity was registered in the cultivation of compositions № 1, № 2 and № 4, it exceeded the control by 19.5–20.1 times (P <0.001). At a temperature of 37 °C for 24 h, the highest change in the optical density of the nutrient medium was found in the cultivation of compositions № 1, № 2 and № 3, in particular, it increased, respectively, in 17.9, 17.8 and 18.1 (P < 0.001) times compared to control. When determining the ability of different compositions of the consortium of lactic acid bacteria to reduce the pH of the medium, it was found that for 24 h of cultivation at 32 ° C the best acid-forming properties showed compositions № 3, № 4 and № 5 because they reduced the pH of the medium by 32.8, 33.3 and 32.8 %, respectively, to 4.87–4.91 units. At a temperature of 37 °C formed by us compositions were able to reduce the pH of the medium to 5.03-5.12 units, while the best acid-forming properties showed compositions № 1, № 3 and № 5. In determining the antagonistic properties of the studied compositions of microorganisms when cultured at temperature 32 °C was found to have moderate antagonistic activity against Escherichia coli, Enterobacter aerogenes, Proteus mirabilis, Salmonella enteritidis, Staphylococcus aureus and Pseudomonas aeruginosa, ie against all cultures of pathogenic and opportunistic microorganisms. Compared with other compositions, composition № 3 showed the highest antagonistic activity against Staphylococcus aureus, as well as quite high against Salmonella enteritidis, Pseudomonas aeruginosa and Enterobacter aerogenes. When culturing the studied compositions of a consortium of microorganisms at a temperature of 37 °C, all compositions showed moderate antagonistic activity against all test cultures of pathogenic and opportunistic microorganisms. Composition № 3 showed slightly higher antagonistic activity against Staphylococcus aureus, Salmonella enteritidis, Enterobacter aerogenes, as well as at a temperature of 32 °C, as well as against Pseudomonas aeruginosa. Therefore, isolated from traditional Carpathian cheese L. lactis ssp. lactis IMAU 32258, Lb. plantarum KLDS 1.0728 and E. durans SB18 have a high degree of biocompatibility and are suitable in different ratios for use in one composition, as indicated by the ability to accumulate biomass, lower pH and antagonistic activity to pathogenic and opportunistic microflora.

Antimicrobial Potential of Bioactive Metabolites and Silver Nanoparticles from Bacillus spp. and of Some Antibiotics Against Multidrug Resistant Salmonella spp.

The synthesis of nanoparticles using microorganisms and their metabolites is of increasing interest because they are potential producers of biocompatible and environmental friendly nanoparticles. Their nanoparticles can serve as potent alternatives to antibiotics against multidrug resistant (MDR) bacteria. The antibacterial potential of Bacillus spp. metabolites, their silver nanoparticles (SNPs), and some antibiotics against MDR Salmonella spp. was evaluated. The antimicrobial potential of metabolites and SNPs biosynthesized from Bacillus spp. was characterized, the effect of physicochemical parameters on SNP biosynthesis, the antimicrobial activity of the SNPs, and combination of SNPs and antibiotics against MDR Salmonella strains were evaluated. The bioactive metabolites of the Bacillus spp. exhibited varied antimicrobial potential against the tested MDR Salmonella spp. The metabolites were able to bioreduce silver nitrate (AgNO3) to Ag+ for SNP biosynthesis. Change in color from whitish to darkish brown and a surface plasma resonance peak of 600-800 nm were observed. The SNPs were aggregated, rods, and crystalline in shape and their sizes were 15 μm, 16 μm, and 13 μm. Carboxylic acid, amino acid, alcohol, esters, and aldehydes were the functional groups found in the biosynthesized SNPs. The antibacterial activity of BAC1-SNPs, BAC7-SNPs, and BAC20-SNPs against MDR Staphylococcus aureus 9 (MDRSA9) and MDRSA18 was 6.0-22 mm and 11-20 mm. SNPs biosynthesized at pH 7 and 10 mM AgNO3 had the highest antagonistic activity. Combination of SNPs and antibiotics exhibited the best antagonistic potential. The metabolites and SNPs from Bacillus spp. exhibited antagonistic effects against MDR Salmonella spp. The combined SNPs and antibiotics had better antimicrobial activity.

Identification of New Biocontrol Agent against Charcoal Rot Disease Caused by Macrophomina phaseolina in Soybean (Glycine max L.)

Controlling agricultural pests using suitable biocontrol agents has been considered the best strategy for sustainable agriculture. Charcoal rot caused by a necrotrophic fungus Macrophomina phaseolina is responsible for a 30–50% annual reduction in soybean yield worldwide. Little is known about the role of Bacillus clausii in reducing charcoal rot disease severity in the soybean crop. In this study, we investigated plant growth promoting and antagonistic potential of Pseudomonas putida (MT604992) and Bacillus clausii (MT604989) against charcoal rot disease incidence in soybean. Among twenty bacteria isolated from soil and water samples of two different hot springs of Gilgit-Baltistan, Pakistan, 80% were siderophore positive; 65% were hydrogen cyanide (HCN) positive; 55%, 30%, and 75% were phosphate, potassium, and zinc solubilizers, respectively. Based on higher antagonistic activities and plant growth promoting traits five strains were selected for in vitro screening. Out of all tested strains, Pseudomonas putida and Bacillus clausii showed a significant increase in germination, growth, and disease suppression in soybean. These strains produced a pronounced increase in relative water content, photosynthetic pigments, membrane stability, proline, antioxidant enzymes status, phytohormones content (Salicylic acid, and Jasmonic acid), and disease suppression in comparison to control plants. Bacillus clausii mitigated the disease by 97% with a marked increase in the proline content (73% and 89%), superoxide dismutase (356% and 208%), peroxidase (439% and 138.6%), catalase (255.8% and 80.8%), and ascorbate peroxidase (228% and 90%) activities in shoots and roots, respectively. Infected plants showed an increase in salicylic acid and jasmonic acid content which was further increased with the application of the selected strains to increase resistance against pathogens. To our knowledge, this is the first study showing a rise in salicylic acid and jasmonic acid in Macrophomina phaseolina infected plants. These two strains are suggested as a cost-effective, eco-friendly, and sustainable alternative to chemical fungicides. However, there is a need to explore the field testing and molecular mechanisms leading to disease suppression by these strains.

Phylogenetic Diversity of Trichoderma Strains and Their Antagonistic Potential against Soil-Borne Pathogens under Stress Conditions.

Trichoderma species are known as excellent biocontrol agents against soil-borne pathogens that cause considerable crop losses. Eight strains of Trichoderma were isolated from five Egyptian regions. They identified based on translation elongation factor-1α (TEF1) sequencing as four different Trichoderma species: Trichoderma asperellum, Trichoderma harzianum, Trichoderma viride, and Trichoderma longibrachiatum. Optimal growth conditions (temperature and media), and the phosphate solubilization capability of Trichoderma strains were evaluated in vitro. Further, the ability of these strains to antagonize Fusarium solani, Macrophomina phaseolina, and Fusarium graminearum was also evaluated. The results revealed that Trichoderma harzianum (Th6) exhibited the highest antagonistic ability against F. solani, M. phaseolina and F. graminearum with inhibition rates of 71.42%, 72.97%, and 84.61%, respectively. Trichoderma viride (Tv8) exhibited the lowest antagonism against the same pathogens with inhibition rates of 50%, 64% and 69.23%, respectively. Simple-sequence repeats (SSRs) and random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic variability of the Trichoderma strains. The results revealed that of 45 RAPD amplified bands, 36 bands (80%) were polymorphic and of SSRs amplified 36 bands, 31 bands (86.11%) were polymorphic. The amplification of calmodulin and β-1,3-endoglucanase was noted at 500 bp and 230 bp, respectively. Data indicated that T. viride (Tv8) had the highest phosphate solubilization index (10.0 mm), while T. harzianum (Th6) had the lowest phosphate solubilization index (4.0 mm). In conclusion, T. harzianum (Th6) had the highest antagonistic activity in dual culture assay along with the growth rate; while T. viride (Tv8) had the highest phosphate solubilization activity. There are still gaps in obtaining new formulations, selecting potent Trichoderma strains to confirm disease control in planta. For improving Trichoderma recommendation in the organic agricultural system and sustaining the fertility of the soil, the field application of highly antagonistic biocontrol agents in different types of soil and plant species will be the first approach toward bio-pesticide treatments along with bio-fertilizer inoculation. Furthermore, secondary metabolites will be investigated for the most promising strains with the combination of different pathogens and application timing.

ANTAGONISTIC ACTIVITY OF A NEW STRAIN OF TRICHODERMA VIRIDE AND ITS EFFECT ON MICROMYCETES POPULATIONS IN THE ROOT ZONE OF CORN PLANTS

Goal. To investigate the antagonistic activity of a new strain of Trichoderma viride F-100076and its effect on the formation of micromycetes populations in the root zone of corn plants underfield conditions. Methods. The antagonistic activity of T. viride F-100076 was studied by the method of mixed (counter) cultures on wort agar using phytopathogenic fungi, which were isolated andidentified in the Laboratory of Plant-Microbial Interactions. The appearance and type of relationship were registered using a scale modified by Symonian and Mamikonian. The number of micromycetes was determined by the method of soil dilutions. Isolation, accounting and cultivation of fungiwas carried out according to conventional methods. Micromycetes were identified according to thedeterminants appropriate for a specific systematic group of micromycetes. Results. It was foundthat T. viride IMB F-100076 is characterized by high antagonistic activity against a wide range ofphytopathogenic fungi, showing hyperparasitism as early as on the eighth day. The highest antagonistic activity of the strain was found against: Alternaria radicina, Acremonium strictum, Acremonium сucurbitacearum, Fusarium oxysporum var. orthoceras, Fusarium moniliforme var. lactis, Torula expansa (5 points on the corresponding Symonian and Mamikonian scale). Data from the mycological analysis of the sod-podzolic soil of the corn rhizosphere showed that the mycocenosis ofthe sod-podzolic soil of the corn rhizosphere was formed by micromycetes belonging to the generaAcremonium Link, Cladosporium Corda, Fusarium Link:Fr, Gliocladium Corda, Mucor Mich, Penicillium Link:Fr, Rhizopus Ehrenb, Trichoderma Hers, among which the most represented were micromycetes of the genus Penicillium (59 %). The total number of fungi in the control variant was291.00 ± 79.67 thousand CFU/g of soil. The introduction of straw affected both the total number ofmicromycetes and the genus composition of fungi. The total number of fungi in the variant withstraw increased 2.6 times and amounted to 744.00 ± 114.67 thousand CFU/g of soil. The number ofrepresentatives of all studied genera of micromycetes also increased. In addition, the introductionof straw provoked the development of fungi of Bipolaris and Fusarium genera, which can be considered a negative outcome since representatives of these species are commonly recognised as pathogens of root diseases. Application of the fungus antagonist T. viride IMB F-100076 to the soilalong with straw did not significantly affect the total number of micromycetes. At the same time, a displacement of fungi of the genus Bipolaris and Fusarium from the rhizosphere of corn was registered.The number of fusaria decreased from 96.00 ± 5.44 to 23.00 ± 2.32 thousand CFU/g of soil or almost4 times and reached the level of the control variant. Fungi of the genus Bipolaris in the variant withthe introduction of trichoderma were not detected. Conclusion. The antagonist fungus T. virideF-100076, introduced into the soil along with straw, strikes root in the soil and exhibits antagonisticactivity against micromycetes of the genera Bipolaris and Fusarium, which are commonly represented by root rot pathogens of many crops. Thus, the new strain T. viride F-100076 allows increasing theantagonistic potential of the rhizosphere soil of corn and protecting plants from pathogens.

Antagonistic Activity of Fungal Endophytes Isolated from Garcinia atroviridis against Colletotrichum gloeosporioides

The extensive use of synthetic fungicides in controlling plant disease generates detrimental impacts on the environment and human health. In response to this problem, an alternative method was developed, known as biological control using antagonistic microorganisms. Since investigation on fungal endophytes of Garcinia atroviridis is still unclear, it was chosen for the study. The aim of the present work was to evaluate biocontrol potential of endophytic fungi against Colletotrichum gloeosporiodes, a phytopathogen that caused anthracnose disease. A total of 92 endophytic fungi were isolated from different tissue parts of Garcinia atroviridis including leaves, petioles, branches, and fruits. Results demonstrated that, most of endophytic fungal isolates showed some inhibitory action over the growth of C. gloeosporiodes during dual culture growth. Endophyte isolate F14 showed the highest antagonistic activity against Colletotrichum gloeosporiodes with 67.38% percentage inhibition radial growth (PIRG). However, 7 out of 92 isolates showed no inhibitory effect against Colletotrichum gloeosporiodes. In conclusion, endophytic fungi isolated from G. atroviridis indicate the potential as biocontrol agents. It is hoped that the finding of isolated endophytic fungi in this study with antagonistic activity against anthracnose pathogen may be used in biocontrol programmes of plant disease in the region.

Isolation and Identification of New Strains of Lactic Bacteria from The Microbiom of Young Carp Fish

Isolation and screening of new strains of lactic acid bacteria from the gut microbiome of carp and carp fingerlings were carried out. Morphological and cultural properties of isolates were studied. All isolates revealed high antagonistic activity against such opportunistic bacteria as Shewanella ximenensis AU 2R-1 B-RKM 0724, Pseudomonas taiwanensis CB 2R-1 B-RKM 0726, Pseudomonas aeruginosa G13 B-RKM 0427, Aeromonas punctata G30 B-RKM 0287, Staphylococcus aureus В-RKM 0470, Escherichia coli В-RKM 0447. Based on the analysis of the nucleotide sequences of the 16S rRNA gene fragment, 6 most active LAB cultures were identified, which belong to representatives of the genera Pediococcus, Lactobacillus and Leuconostoc. The procedure of depositing all strains in the Central Museum of Microorganisms of the Republican Collection of Microorganisms was carried out.

The Effectiveness of Potential Probiotics Lactobacillus rhamnosus Vahe and Lactobacillus delbrueckii IAHAHI in Irradiated Rats Depends on the Nutritional Stage of the Host.

Several species of eukaryotic organisms living in the high mountain areas of Armenia with naturally occurring levels of radiation have high adaptive responses to radiation. We speculate on the role of the gastrointestinal microbiota in this protection against radiation. Therefore, seventeen microorganisms with high antagonistic activities against several multi-drug-resistant pathogens were isolated from the human and animal gut microbiota, as well as from traditional Armenian fermented products. These strains were tested in vivo on Wistar rats to determine their ability to protect the eukaryotic host against radiation damages. The efficiency of the probiotics' application and the dependence on pre- and post-radiation nutrition of rats were described. The effects of Lactobacillus rhamnosus Vahe, isolated from a healthy breastfed infant, and Lactobacillus delbrueckii IAHAHI, isolated from the fermented dairy product matsuni, on the survival of irradiated rats, and their blood leucocyte and glucose levels, were considered to be the most promising, based on this study's results.

ANTIMICROBIAL ACTIVITY OF LACTOBACILLI AGAINST STAPHYLOCOCCI ISOLATED FROM BIOFILMS FROM PATIENTS WITH ACNE VULGARIS

Aim. Determine the effectiveness of the antimicrobial activity of Lactobacillus isolates against biofilm producing staphylococci in vitro. Methods. There were used 15 strains of biofilm producing staphylococci isolated from purulent pustules of patients with acne vulgaris and 7 strains of lactobacilli. L. fermentum (n = 3) were obtained from oropharyngeal mucus, L. plantаrum (n = 3) from the feces of practically healthy individuals, probiotic strain L. plantarum 8P-A3 from the drug "Lactobacterin" (Biopharma, Kyiv).The antimicrobial activity of bacterial cultures of lactobacilli and filtered supernatant liquid of lactobacilli were studied using biofilm producing S. aureus strains by agar diffusion method after 12, 24 and 48 hours. Results. The antagonistic activity of lactobacilli was determined by 15 strains of S. aureus in which icaA and icaD genes were detected. For all strains of L. plantаrum (12 h), high antagonistic activity was established and an average antagonistic activity for L. fermentum was established. In 24-h, in the studied lactobacilli cultures, there was observed a tendency of decreasing their activity. After 48 hours, a significant decrease in staphylococci growth inhibition zones was observed in comparison with the results of 12-hour cultures – on average 7.9 ± 0.2 mm versus 12.5 ± 0.7 mm, respectively. Filtration of the supernatant liquid of lactobacilli (12 h) led to a sharp decrease in their activity by 2.8 times compared with the rates of native cultures of lactobacilli. Conclusions. The antibacterial activity of native bacterial cultures of lactobacilli against biofilm producing S. aureus reaches a maximum level after 12 hours. The minimal inhibitory effects of the filtered supernatant liquid of lactobacilli against biofilm producing strains of S. aureus were established. Isolates of L. plantarum showed higher antagonistic activity against staphylococci than L. fermentum, and may be proposed as an option for the development of combination therapy of acne.

Discovery of ORN0829, a potent dual orexin 1/2 receptor antagonist for the treatment of insomnia

Here, we present the design, synthesis, and SAR of dual orexin 1 and 2 receptor antagonists, which were optimized by balancing the antagonistic activity for orexin receptors and lipophilicity. Based on the prototype compound 1, ring construction and the insertion of an additional heteroatom into the resulting ring led to the discovery of orexin 1 and 2 receptor antagonists, which were 3-benzoyl-1,3-oxazinane derivatives. Within these derivatives, (−)-3h enabled a high dual orexin receptor antagonistic activity and a low lipophilicity. Compound (−)-3h exhibited potent sleep-promoting effects at a po dose of 1 mg/kg in a rat polysomnogram study, and optimal PK properties with a rapid Tmax and short half-lives in rats and dogs were observed, indicating a predicted human half-life of 0.9–2.0 h. Thus, (−)-3h (ORN0829; investigation code name, TS-142) was selected as a viable candidate and is currently in clinical development for the treatment of insomnia.

SALMONELLA INFECTION LEVEL IN CHICKEN POPULATIONS VERSUS ANTAGONISTIC ACTIVITY OF LACTOBACILLACEAE AND ENTEROCOCCACEAE AGAINST SALMONELLA ENTERICA

The antagonistic activity of lactobacilli in the intestine in relation to various enteropathogenic microorganisms can vary within wide limits, including depending on the species composition of the lactobiota of the intestine. The purpose of this work was to determine the antagonistic activity of representatives of the order Lactobacillales isolated from chickens in poultry farms with different levels of Salmonella infection. The test object was the chickens of the parent herd and broiler chickens of crosses Ross 308 and Hubbard F - 15 from five poultry farms. Three poultry farms were characterized by a low level of salmonella infection in birds (less than 5% for cloacal swabs in PCR and the absence of salmonella isolation from food products). Two poultry farms were characterized by a high level of Salmonella infection (poultry infection by cloacal swabs of more than 10% and official salmonellosis disadvantage due to isolation of Salmonella cultures in food products). The level of infection was evaluated by real-time PCR after preliminary subculture of cloacal swabs on Shadler’s broth. The antagonistic activity of lactobacilli and related bacterial species isolated from the same chickens was carried out in co-cultivation tests on the Shadler broth with subsequent identification of salmonella on the RVS broth. Poultry farms with low Salmonella infection were characterized by the presence of L. reuteri as a major component of intestinal lactobiota and had a higher antagonistic activity against more Salmonella cultures (odds ratio (OR) 17.33 (CI 95 = 5.99-50.07776))

A new species of Trichoderma and gliotoxin role: A new observation in enhancing biocontrol potential of T. virens against Phytophthora capsici on chili pepper

Phytophthora capsici is a phytopathogen causing a destructive pepper blight. To control this disease, 77 isolates of Trichoderma spp. were isolated from rhizosphere soil and 15 isolates showed high antagonistic activity. Based on the analysis of the rDNA internal transcribed spacers (ITS), translation elongation factor 1-α (TEF1) and RNA polymerase II subunit B (RPB2) gene sequence data and morphological traits, the isolates were identified as T. brevicompactum, T. atroviride, T. afroharzianum, T. koningiopsis, T. citrinoviride, T. asperellum, T. harzianum, T. virens and a new species. A new species was described as T. dorothopsis. The in vitro antagonistic assay and analysis of metabolite fractions indicate that the T. virens HZA14 could cause colony collapse and degradation of P. capsici. A high activity compound was identified as gliotoxin by spectrometric analysis. The biocontrol tests demonstrated that the T. virens HZA14 delayed the occurrence of chili pepper blight and significantly reduced the disease incidence and severity by 62.64% and 64.20%, respectively. Hence isolate HZA14 could be considered for developing potential biocontrol agent for management of P. capsici in pepper.

Isolation of Weissella cibaria from Pacific White Shrimp (Litopenaeus vannamei) Gastrointestinal Tract and Evaluation of Its Pathogenic Bacterial Inhibition

Objective: The present study reported the isolation of lactic acid bacteria from the shrimp gastrointestinal tract and evaluation of its antagonistic activity against pathogenic bacteria. Methods/ statistical analysis: Lactic bacteria from the gastrointestinal tract of alive healthy shrimp were screened on sterilized MRS medium. Vibrio sp. inhibition by isolates was conducted by the agar-well diffusion assay. The isolates were identified by 16S rRNA sequencing. Antibacterial activity was investigated against 25 Vibrio sp. isolates, Staphylococcus aureus ATCC 25023, and Escherichia coli ATCC 85922. Hemolytic activity, salt, and antibiotic resistance were also evaluated. Findings: Total of 22 isolates exhibited Vibrio parahaemolyticus inhibition with antagonistic activities ranged from 220 AU/ml to 460 AU/ml. Sequence analysis of 16S rRNA indicated the 5 isolates belong to Weissella cibaria species, namely HN02, HN03, HN04, and HN06 AND HN07. Among these, W. cibaria HN05 had highest antagonistic activity and exhibited the broad inhibition spectrum against Vibrio sp. as well as E. coli ATCC 85922 but not S. aureus ATCC 25023. β-hemolytic and salt resistance assays showed that W. cibaria HN05 had no β-hemolytic activity and grew well in medium containing 5% of salt. Antibiotic resistance analysis indicated W. cibaria HN05 was suppressed by ampicillin, chloramphenicol, oxytetracycline hydrochloride, and chlortetracycline hydrochloride. Application/improvements: This is the first study on isolation and evaluation pathogenic bacteria including Vibrio sp. inhibition by W. cibaria, which is isolated from shrimp gastrointestinal tract. Keywords: Antagonistic Activity, Gastrointestinal Tract, Shirmp, Weissella cibaria, Vibrio sp.

Antifungal, plant growth-promoting, and mycotoxin detoxication activities of Burkholderia sp. strain XHY-12.

A bacterial strain named XHY-12 was isolated from corn soil samples and identified as Burkholderia sp. based on 16S rDNA sequencing, it displayed high antagonistic activity against 12 fungal pathogens and the common fungal contaminant in grain Aspergillus flavus. Plate experiment showed that XHY-12 fermentation broth reduced the incidence of S. sclerotiorum on detached rape leaves (Brassica campestris L.) by 100%, and a greenhouse experiment showed that it could promote the growth of rape seedlings with significant increases in plant height, root length, and fresh weight. Furthermore, a novel funding was the reduction of aflatoxin B1 and B2 by over 85% in 60h, and the decomposition enzymes should be extracellular. The results suggest that XHY-12 has a potential for commercial applications as biocontrol, mycotoxin detoxification agent or biofertilizer.

Long-Term Fipronil Treatment Induces Hyperactivity in Female Mice.

Fipronil is an insecticide widely used for veterinary and agricultural purposes. While its insecticidal properties mostly rely on its high affinity antagonistic activity on insect γ aminobutyric acid (GABA) receptors, fipronil and its main metabolite fipronil sulfone nevertheless display non-negligible affinity for mammalian GABAA receptor. As several environmental toxicants have been shown to raise the risk of developing various neurodegenerative disorders, the aim of this study was to evaluate whether long-term low dose administration of fipronil could lead to cognitive deficiencies. Our results indicate that long-term fipronil treatment leads to behavioral perturbations in mice, indicating an accumulative effect of sustained exposure to low dose of fipronil. Although no memory impairment was observed during the course of our study, we noticed a significant hyperlocomotion behavior after 43 weeks of weekly fipronil administration, which is consistent with its direct effect on the GABAergic system.