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Community analysis Heterologous expression system in vivo model Microbe-host interactions Microbial biochemistry Microbial biofilm Microbial cell biology Microbial genetics Microbial metabolism Microbial physiology Microbial proteomics Microbial signaling Pathogen detectionEvaluating In Vivo Translation Inhibition via Puromycin Labeling in Botrytis cinerea Germlings Treated With Antifungal Peptides
Antimicrobial peptides are effective agents against various pathogens, often targeting essential processes like protein translation to exert their antimicrobial effects. Traditional methods such as puromycin labeling have been extensively used to measure protein synthesis in mammalian and yeast systems; however, protocols tailored for plant pathogenic filamentous fungi, particularly those investigating translation inhibition by antifungal peptides, are lacking. This protocol adapts puromycin labeling to quantify translation inhibition in Botrytis cinerea germlings treated with antifungal peptides. Optimizing the method specifically for fungal germlings provides a precise tool to investigate peptide effects on fungal protein synthesis, advancing our understanding of translation dynamics during pathogen–host interactions in filamentous fungi.
Competition Assays to Quantify the Effect of Biocontrol Yeasts against Plant Pathogenic Fungi on Fruits
Yeasts such as Aureobasidium pullulans are unicellular fungi that occur in all environments and play important roles in biotechnology, medicine, food and beverage production, research, and agriculture. In the latter, yeasts are explored as biocontrol agents for the control of plant pathogenic fungi (e.g., Botrytis cinerea, Fusarium sp.); mainly on flowers and fruits. Eventually, such yeasts must be evaluated under field conditions, but such trials require a lot of time and resources and are often difficult to control. Experimental systems of intermediate complexity, between in vitro Petri dish assays and field trials, are thus required. For pre- and post-harvest applications, competition assays on fruits are reproducible, economical and thus widely used. Here, we present a general protocol for competition assays with fruits that can be adapted depending on the biocontrol yeast, plant pathogen, type of assay or fruit to be studied.
Identification and Quantification of Secondary Metabolites by LC-MS from Plant-associated Pseudomonas aurantiaca and Pseudomonas chlororaphis
Increased antibiotic resistance of plants and human pathogens and continuous use of chemical fertilizers has pushed microbiologists to explore new microbial sources as potential antagonists. In this study, eight strains of Pseudomonas aurantiaca and Pseudomonas chlororaphis, have been isolated from different plant sources and screened for their antagonistic and plant growth promoting potential (Shahid et al., 2017). All strains were compared with reference strain PB-St2 and their secondary metabolites were isolated by the use of solvent partitioning and subjected to LC/ESI/MS for confirmation of compounds. The ESI-mass spectra obtained were used to characterize the surfactants ionization behavior and [M + H]+ and [M + Na]+ ions were monitored for phenazines, derivatives of lahorenoic acid and cyclic lipopeptide (WLIP). LC-MS and HPLC methods were developed to see the elution of dominant metabolites in a single run to avoid the labor and separate methods of detection for all compounds. The method was found suitable and distinctively separated the compounds at different retention times in gradient flow. This method can be helpful to explore the metabolome of Pseudomonas sp. overall and in identification and quantification of strain specific metabolites.
In vitro Cell Wall Stress Assay for Fusarium oxysporum
In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).
Antifungal and Zearalenone Inhibitory Activity of Ocimum sanctum L. Essential Oil on Fusarium graminearum Determined by UHPLC and RT-qPCR
Fusarium graminearum has been given special attention in the context of agricultural commodities due to its ability to grow in diverse climatic conditions, and to produce different mycotoxins including zearalenone (ZEA) and type-B trichothecenes, which cause ill health effects on humans, animals and plants. The application of synthetic antifungal agents for the control of F. graminearum result in negative health impacts in livestock and humans and the upsurge of resistant organisms as well. Therefore, there is a need to propose proper food grain management practices, including the application of herbal antifungal and mycotoxin controlling agents, to reduce the growth of toxigenic F. graminearum as well as the production of ZEA in agricultural commodities. Ocimum sanctum also known as Holy Basil or Tulsi is widely used as a medicinal plant in Ayurveda. The current protocol demonstrates to quantify the antifungal activity of O. sanctum L. essential oil (OSEO) as reflected by the decreased F. graminearum growth and ZEA production. Antifungal activities of OSEO are carried out by micro well dilution method and further validated quantitatively by scanning electron microscopic methods. Effects of OSEO on ZEA production is analysed by Quantitative reverse transcription PCR (RT-qPCR) and Ultra high performance liquid chromatography (UHPLC) methods from a broth culture of F. graminearum. Anti-mycotoxic efficacy of OSEO is assessed directly on F. graminearum inoculated maize grains. The protocol efficiently assessed the activity of OSEO as an herbal antagonistic agent against fungal infestation and ZEA production by F. graminearum. The protocol can be used to test a wide variety of herbal compounds for antifungal activity against F. graminearum or with modifications on other mycotoxigenic fungi, an important intervention in food safety and processing industries where the fungal infestation is a major concern.
XTT Assay of Antifungal Activity
XTT assay is a colorimetric method that uses the tetrazolium dye, 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT) to quantify cell-mediated damage to fungi. Actively respiring fungal cells convert the water-soluble XTT to a water-soluble, orange colored formazan product (Meshulam et al., 1995). Here, we describe the protocol that measures the ability of plasmacytoid dendritic cells (pDCs) to exert antifungal activity. This approach was first established with human polymorphonuclear cells (PMN) by Meshulam et al. (1995) and then adapted to pDC by Ramirez-Ortiz et al. (2011) and Loures et al. (2015). It can be modified for use with other effector cells and to test compounds for antifungal activity.
Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata
Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014).
Detection of Hog1 Phosphorylation in Candida albicans in Response to an Antifungal Protein
It is becoming increasingly apparent that stress signalling is important for tolerance of fungal species to antifungal chemicals and proteins. The high-osmolarity glycerol (HOG) pathway responds to a number of stressors including osmotic and oxidative stress. This protocol describes a method to detect activation of the Candida albicans (C. albicans) MAPK Hog1 by monitoring its phosphorylation in response to an antifungal protein.
Minimum Inhibitory Concentration (MIC) Assay for Antifungal Drugs
The Minimum Inhibitory Concentration (MIC) Assay is widely used to measure the susceptibility of yeasts to antifungal agents. In serial two-fold dilutions, the lowest concentration of antifungal drug that is sufficient to inhibit fungal growth is the MIC. Typically, 50% inhibitory (MIC50) or 80% inhibitory (MIC80) values are reported. To facilitate visualization of antifungal susceptibility data, heat maps are generated whereby optical density values are represented quantitatively with colour.