A comparison of the inhibitory activities of Lactobacillus and Bifidobacterium against Penicillium expansum and an analysis of potential antifungal metabolites

2020 ◽  
Vol 367 (18) ◽  
Author(s):  
Nanzhen Qiao ◽  
Leilei Yu ◽  
Chengcheng Zhang ◽  
Chaozhi Wei ◽  
Jianxin Zhao ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Economic Loss ◽  
Microtiter Plate ◽  
Penicillium Expansum ◽  
Dual Culture ◽  
Phenyllactic Acid ◽  
Bifidobacterium Adolescentis ◽  
Antifungal Metabolites ◽  
Potent Inhibition ◽  
Well Assay

ABSTRACT The infection of fruits by Penicillium expansum (P. expansum) do not only cause economic loss but also potentially endanger human health, especially because few biocontrol agents against this fungus have been well studied yet. In this work, to verity the antifungal activity against P. expansum of 22 Bifidobacterium and 44 Lactobacillus, dual-culture overlay assay, microtiter plate well assay and agar spot assay were successively performed. One of the strain, Bifidobacterium adolescentis (B. adolescentis) CCFM1108 exhibited the most potent inhibition ability among all tested strains. Additionally, we showed that multiple antifungal compounds produced by tested strain synergistically inhibit the growth of P. expansum, including lactic acid, acetic acid, 3-phenyllactic acid and p-hydroxyphenyllactic acid. Those active compounds mentioned were detected in the cell-free supernatant and characterized by metabolomics analysis using GC-MS. Correspondingly, B. adolescentis CCFM1108 supernatant disrupted plasma membrane integrity of the P. expansum mycelial and drastically reduced patulin production in P. expansum. The inhibitive effects of B. adolescentis CCFM1108 were also confirmed with three other P. expansum strains. The active inhibitory properties of Bifidobacterium strains, especially B. adolescentis CCFM1108, indicate that B. adolescentis can be potentially used as a novel bioagent to prevent or delay fungal spoilage on fruit.

scholarly journals Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

2002 ◽  
Vol 68 (11) ◽  
pp. 5209-5216 ◽  
Author(s):  
Kaouther Ben Amor ◽  
Pieter Breeuwer ◽  
Patrick Verbaarschot ◽  
Frank M. Rombouts ◽  
Antoon D. L. Akkermans ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Salt Stress ◽  
Bile Salt ◽  
Cell Sorting ◽  
Physiological Activity ◽  
Membrane Integrity ◽  
Multiparameter Flow Cytometry ◽  
Bifidobacterium Adolescentis ◽  
Multiparametric Flow Cytometry ◽  
Plate Counts

ABSTRACT Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

Role of chitinase and β-1,3-glucanase activities produced by a fluorescent pseudomonad and in vitro inhibition of Phytophthora capsici and Rhizoctonia solani

2007 ◽  
Vol 53 (2) ◽  
pp. 207-212 ◽  
Author(s):  
Naveen Kumar Arora ◽  
Min Jeong Kim ◽  
Sun Chul Kang ◽  
Dinesh Kumar Maheshwari
Keyword(s):  
Rhizoctonia Solani ◽  
Phytophthora Capsici ◽  
Phytopathogenic Fungi ◽  
Culture Technique ◽  
Dual Culture ◽  
Fluorescent Pseudomonad ◽  
Antifungal Metabolites ◽  
Fluorescent Pseudomonas

A study was conducted to investigate the possibility of involvement of chitinase and β-1,3-glucanase of an antagonistic fluorescent Pseudomonas in growth suppression of phytopathogenic fungi, Phytophthora capsici and Rhizoctonia solani . Fluorescent Pseudomonas isolates GRC3 and GRC4 were screened for their antifungal potential against phytopathogenic fungi by using dual culture technique both on solid and liquid media. The percent inhibition was calculated. Various parameters were monitored for optimization of enzyme activities by fluorescent Pseudomonas GRC3. The involvement of chitinases, β-1,3-glucanases, and antifungal metabolites of nonenzymatic nature was correlated with the inhibition of P. capsici and R. solani. The results provide evidence for antibiosis as a mechanism for antagonism. The study also confirms that multiple mechanisms are involved in suppressing phytopathogens as evidenced by the involvement of chitinase and β-1,3-glucanase in inhibition of R. solani but not P. capsici by isolate GRC3.

scholarly journals Antifungal activity of indigenous bacillus sp. isolate Q3 against marshmallow mycobiota

2011 ◽  
pp. 111-120
Author(s):  
Dragana Josic ◽  
Radmila Pivic ◽  
Snezana Pavlovic ◽  
Sasa Stojanovic ◽  
Goran Aleksic ◽  
...  
Keyword(s):  
Hydrolytic Enzymes ◽  
Pathogenic Fungi ◽  
Phytopathogenic Fungi ◽  
Bacterial Suspension ◽  
Dual Culture ◽  
Seed Infection ◽  
Bacillus Sp ◽  
Antifungal Metabolites ◽  
Parasitic Fungi ◽  
Plant Growth Promoting

Marshmallow is a host of a number of saprophytic and parasitic fungi in Serbia. The seeds of marshmallow are contaminated with fungi from different genera, especially Alternaria and Fusarium, which significantly reduced seed germination and caused seedling decay. In this study we investigate antagnonism of indigenous Bacillus sp. isolate Q3 against marshmallow mycopopulation. Bacillus sp. Q3 was isolated from maize rhizosphere, characterized by polyphasic approch and tested for plant growth promoting treats. Bacillus sp. Q3 produced antifungal metabolites with growth inhibition activity against numerous fungi in dual culture: 61.8% of Alternaria alternata, 74.8% of Myrothecium verrucaria and 33.6% of Sclerotinia sclerotiorum. That effect could be caused by different antifungal metabolites including siderophores, hydrolytic enzymes, organic acids and indole acetic acid (IAA). Suppression of natural marshmallow seed infection by Q3 isolate was observed. The seeds were immersed in different concentrations of bacterial suspension during 2h and their infections by phytopathogenic fungi were estimated. The results showed significant reduction of seed infection by Alternaria spp. The presented results indicate possible application of this isolate as promising biological agent for control of marshmallow seed pathogenic fungi.

scholarly journals Metabolite Profiles of Lactic Acid Bacteria in Grass Silage

2007 ◽  
Vol 73 (17) ◽  
pp. 5547-5552 ◽  
Author(s):  
Anders Broberg ◽  
Karin Jacobsson ◽  
Katrin Ström ◽  
Johan Schnürer
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Pichia Anomala ◽  
Coumaric Acid ◽  
Phenyllactic Acid ◽  
Liquid Cultures ◽  
Antifungal Metabolites ◽  
Metabolite Profiles ◽  
Bioassay Guided Fractionation ◽  
Test Organisms

ABSTRACT The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml−1 for two of the three test organisms).

scholarly journals Purification and Characterization of Novel Antifungal Compounds from the Sourdough Lactobacillus plantarum Strain 21B

2000 ◽  
Vol 66 (9) ◽  
pp. 4084-4090 ◽  
Author(s):  
Paola Lavermicocca ◽  
Francesca Valerio ◽  
Antonio Evidente ◽  
Silvia Lazzaroni ◽  
Aldo Corsetti ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactobacillus Plantarum ◽  
Wheat Flour ◽  
Culture Filtrate ◽  
Bacterial Culture ◽  
Fungal Growth ◽  
Lactobacillus Brevis ◽  
Penicillium Expansum ◽  
Antifungal Compounds ◽  
Phenyllactic Acid

ABSTRACT Sourdough lactic acid bacteria were selected for antifungal activity by a conidial germination assay. The 10-fold-concentrated culture filtrate of Lactobacillus plantarum 21B grown in wheat flour hydrolysate almost completely inhibited Eurotium repens IBT18000, Eurotium rubrum FTDC3228,Penicillium corylophilum IBT6978, Penicillium roqueforti IBT18687, Penicillium expansum IDM/FS2,Endomyces fibuliger IBT605 and IDM3812, Aspergillus niger FTDC3227 and IDM1, Aspergillus flavus FTDC3226,Monilia sitophila IDM/FS5, and Fusarium graminearum IDM623. The nonconcentrated culture filtrate ofL. plantarum 21B grown in whole wheat flour hydrolysate had similar inhibitory activity. The activity was fungicidal. Calcium propionate at 3 mg ml−1 was not effective under the same assay conditions, while sodium benzoate caused inhibition similar toL. plantarum 21B. After extraction with ethyl acetate, preparative silica gel thin-layer chromatography, and chromatographic and spectroscopic analyses, novel antifungal compounds such as phenyllactic and 4-hydroxy-phenyllactic acids were identified in the culture filtrate of L. plantarum 21B. Phenyllactic acid was contained at the highest concentration in the bacterial culture filtrate and had the highest activity. It inhibited all the fungi tested at a concentration of 50 mg ml−1 except forP. roqueforti IBT18687 and P. corylophilumIBT6978 (inhibitory concentration, 166 mg ml−1). L. plantarum 20B, which showed high antimold activity, was also selected. Preliminary studies showed that phenyllactic and 4-hydroxy-phenyllactic acids were also contained in the bacterial culture filtrate of strain 20B. Growth of A. niger FTDC3227 occurred after 2 days in breads started with Saccharomyces cerevisiae 141 alone or with S. cerevisiae andLactobacillus brevis 1D, an unselected but acidifying lactic acid bacterium, while the onset of fungal growth was delayed for 7 days in bread started with S. cerevisiae and selectedL. plantarum 21B.

Biological control of apple blue mold withPseudomonas fluorescens

2005 ◽  
Vol 51 (7) ◽  
pp. 591-598 ◽  
Author(s):  
Hassan-Reza Etebarian ◽  
Peter L Sholberg ◽  
Kenneth C Eastwell ◽  
Ronald J Sayler
Keyword(s):  
Biological Control ◽  
Fluorescent Protein ◽  
Biological Control Agent ◽  
Major Effect ◽  
Penicillium Expansum ◽  
Postharvest Disease ◽  
Dual Culture ◽  
Blue Mold ◽  
Iron Chelate ◽  
Penicillium Spp

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 °C and by P. expansum and P. solitum after 25 d at 5 °C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 °C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 °C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.Key words: Penicillium expansum, P. solitum, postharvest disease, Malus, GFP.

High Throughput 96-Well Plate Assay for Receptor-Mediated Phosphatidylinositol Turnover

1997 ◽  
Vol 2 (2) ◽  
pp. 91-97 ◽  
Author(s):  
Ye Tian ◽  
Lan-Hsin Wu ◽  
Fu-Zon Chung
Keyword(s):  
Signal Transduction ◽  
High Throughput ◽  
High Throughput Screening ◽  
Inositol Phosphates ◽  
Microtiter Plate ◽  
Receptor Activation ◽  
Neurotransmitter Receptors ◽  
Functional Assay ◽  
Phosphatidylinositol Turnover ◽  
Well Assay

The G-protein coupled receptor family represents a large number of neurotransmitter receptors. Among the diverse signal transduction pathways mediated via G-proteins, phospholipase C mediated phosphatidylinositol hydrolysis represents one of the best characterized signal transduction mechanisms. Accordingly, the measurement of agonist-induced phosphatidylinositol turnover has been used as a convenient functional assay for receptor activation. Assays currently used for this purpose, however, are not suitable for high throughput screening. In this article, an improved technique using 96-well microtiter plate format for measuring phosphatidylinositol turnover is introduced. Anion exchange columns were prepared on fiber glass 96-well multiscreen filter plate. Separation and detection of released inositol phosphates were conducted in a 96-well format. Cells expressing certain neurotransmitter receptors were challenged with agonists and the receptor-mediated PI turnover was measured by the new technique and the results obtained were compared to that obtained from traditional assays. The results indicate that the 96-well assay is 10 to 20 times more efficient than the traditional method and is, furthermore, suitable for high throughput drug screening. Our data also indicate that this method is particularly useful for characterizing multiple antagonists by Schild analysis.

scholarly journals Synergistic Antibiofilm Effects of Ultrasound and Phenyllactic Acid against Staphylococcus aureus and Salmonella enteritidis

Foods ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2171
Author(s):  
Jiaojiao Zhang ◽  
Debao Wang ◽  
Jinyue Sun ◽  
Zhilan Sun ◽  
Fang Liu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Respiratory Chain ◽  
Laser Scanning ◽  
Salmonella Enteritidis ◽  
Membrane Integrity ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
Phenyllactic Acid ◽  
Cell Membrane Integrity

This study evaluated the effect of the combination of ultrasound and phenyllactic acid (PLA) on inactivating Staphylococcus aureus and Salmonella enteritidis biofilm cells and determined the possible antibiofilm mechanism. S. aureus and S. enteritidis biofilm cells were separately treated with ultrasound (US, 270 W), phenyllactic acid (PLA, 0.5% and 1%), and their combination (US + 0.5% PLA, and US + 1% PLA) for 5, 10, 20, 30, and 60 min. Biofilm inactivation, polysaccharide, and respiratory chain dehydrogenase assays were conducted. US and PLA had a synergistic effect on inactivating bacterial cells in S. aureus and S. enteritidis biofilms. The combination of US and PLA significantly decreased the contents of soluble and insoluble polysaccharides and the activity of respiratory chain dehydrogenase in the biofilm cells compared to the single treatment. Confocal laser scanning microscopy, scanning electron microscopy, and intracellular adenosine-triphosphate (ATP) analyses indicated that the combination of US and PLA seriously destroyed the cell membrane integrity of the S. aureus and S. enteritidis biofilms and caused the leakage of intracellular ATP. These findings demonstrated the synergistic antibiofilm effect of US combined with PLA and offered a research basis for its application in the food industry.

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

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1438
Author(s):  
Nadeem Hasan ◽  
Ayaz Farzand ◽  
Zhou Heng ◽  
Irfan Ullah Khan ◽  
Anam Moosa ◽  
...  
Keyword(s):  
Verticillium Wilt ◽  
Economic Loss ◽  
Antagonistic Activity ◽  
Dual Culture ◽  
Gene Products ◽  
Methanolic Extracts ◽  
Dual Culture Assay ◽  
Antagonistic Potential ◽  
Encoding Genes ◽  
Bacillus Strains

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.

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