A novel 96-well gel-based assay for determining antifungal activity against filamentous fungi

Troskie, Anscha Mari; Rautenbach, Marina

doi:10.1016/j.mimet.2012.09.025

Cited by 15 publications

(16 citation statements)

References 22 publications

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“…A commercial preparation of GS inhibited germination of B. cinerea conidiospores with distinct antifungal activity at 10 μM GS and complete inhibition of spore germination at 100 μM, similar to the results reported previously (Edwards and Seddon, 2001; Troskie et al, 2012). The direct application of A. migulanus cells to suspensions of B. cinerea spores proved that the strain Nagano was more effective than NCTC 7096 at reducing germination of B. cinerea spores, suggesting that factors other than quantities of GS-1141 produced were responsible for the antimicrobial effects observed.…”

Section: Discussionsupporting

confidence: 89%

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites

Alenezi

Rekik²,

Bouket

et al. 2017

Front. Microbiol.

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The soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a biocontrol agent against plant diseases. In contrast, A. migulanus NCTC 7096 proved less effective for inhibition of plant pathogens. Nagano strain exerts biocontrol activity against some gram-positive and gram-negative bacteria, fungi and oomycetes through the production of gramicidin S (GS). Apart from the antibiotic effects, GS increases the rate of evaporation from the plant surface, reducing periods of surface wetness and thereby indirectly inhibiting spore germination. To elucidate the molecular basis of differential biocontrol abilities of Nagano and NCTC 7096, we compared GS production and biosurfactant secretion in addition to genome mining of the genomes. Our results proved that: (i) Using oil spreading, blood agar lysis, surface tension and tomato leaves wetness assays, Nagano showed increased biosurfactant secretion in comparison with NCTC 7096, (ii) Genome mining indicated the presence of GS genes in both Nagano and NCTC 7096 with two amino acid units difference between the strains: T342I and P419S. Using 3D models and the DUET server, T342I and P419S were predicted to decrease the stability of the NCTC 7096 GS synthase, (iii) Nagano produced two additional GS-like molecules GS-1155 (molecular weight 1155) and GS-1169 (molecular weight 1169), where one or two ornithine residues replace lysine in the peptide. There was also a negative correlation between surface tension and the quantity of GS-1169 present in Nagano, and (iv) the Nagano genome had a full protein network of exopolysaccharide biosynthesis in contrast to NCTC 7096 which lacked the first enzyme of the network. NCTC 7096 is unable to form biofilms as observed for Nagano. Different molecular layers, mainly gramicidin secondary metabolite production, account for differential biocontrol abilities of Nagano and NCTC 7096. This work highlighted the basis of differential biological control abilities between strains belonging to the same species and demonstrates techniques useful to the screening of effective biocontrol strains for environmentally friendly secondary metabolites that can be used to manage plant pathogens in the field.

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Section: Discussionsupporting

confidence: 89%

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites

Alenezi

Rekik²,

Bouket

et al. 2017

Front. Microbiol.

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“…After one day, Bacillus strains or their cell free supernatants were placed 2cm away from the plug and plates were evaluated after one week. MIC assays were set up in 96 microtitre plates and dilution series of extracted cLP were incubated against R. solani at 288C for 3 days according to the protocol Troskie et al (2012). For hemolytic assays, 1 mL overnight cultures of Bacillus strains, cell free supernatants and lipopeptide fractions were tested on 10% sheep blood agar plates and incubated at 288C for 48 h. Surfactin A from B. subtilis (from Sigma) were included as positive control.…”

Section: Methodsmentioning

confidence: 99%

Surfactin variants mediate species‐specific biofilm formation and root colonization in Bacillus

Aleti

Lehner

Bacher

et al. 2016

Environmental Microbiology

107

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Cyclic lipopeptides (cLP) and especially surfactins produced by Bacillus spp. trigger biofilm formation and root colonization and are crucial for biocontrol activity and systemic resistance in plants. Bacillus atrophaeus 176s isolated from the moss Tortella tortuosa produces the cLP fengycins, iturins and surfactins, possesses antifungal activities and can protect tomato, lettuce and sugar beet against Rhizoctonia solani infection. In B. atrophaeus we identified for the first time the variant surfactin C, which differs from surfactin A produced by B. subtilis and B. amyloliquefaciens by an isoleucine instead of a leucine at position 7 of the lipopeptide backbone. The analysis of the complete surfactin gene clusters revealed that the dissimilarity is encoded in the adenylation domain of srfC and show that surfactin variations are distributed in a species-specific manner in bacilli. We demonstrate that the surfactin A and C with subtle structural differences have varying signal strengths on biofilm formation and root colonization and act specifically on the respective producing strain. This became evident as biofilm formation and root colonization but not swarming motility in surfactin biosynthesis mutants was restored differentially in the presence of exogenously supplemented cognate and non-cognate surfactin variants.

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“…Following the methods of Troskie et al . (). The agar was allowed to solidify after which 10 µl of spore suspension (2000 spores/well) was added.…”

Section: Methodsmentioning

confidence: 97%

“…About 70 ml PDA (Carl Roth) was pipetted into each well of a black 96 well plate (Greiner bio-one) using reverse pipetting to avoid contamination. Following the methods of Troskie et al (2012). The agar was allowed to solidify after which 10 ml of spore suspension (2000 spores/well) was added.…”

Section: Fungal Metabolic Activity Assaymentioning

confidence: 99%

Highly potent host external immunity acts as a strong selective force enhancing rapid parasite virulence evolution

Rafaluk

Yang

Mitschke

et al. 2017

Environmental Microbiology

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Virulence is often under selection during host-parasite coevolution. In order to increase fitness, parasites are predicted to circumvent and overcome host immunity. A particular challenge for pathogens are external immune systems, chemical defence systems comprised of potent antimicrobial compounds released by prospective hosts into the environment. We carried out an evolution experiment, allowing for coevolution to occur, with the entomopathogenic fungus, Beauveria bassiana, and the red flour beetle, Tribolium castaneum, which has a well-documented external immune system with strong inhibitory effects against B. bassiana. After just seven transfers of experimental evolution we saw a significant increase in parasite induced host mortality, a proxy for virulence, in all B. bassiana lines. This apparent virulence increase was mainly the result of the B. bassiana lines evolving resistance to the beetles' external immune defences, not due to increased production of toxins or other harmful substances. Transcriptomic analyses of evolved B. bassiana implicated the up-regulation of oxidative stress resistance genes in the observed resistance to external immunity. It was concluded that external immunity acts as a powerful selective force for virulence evolution, with an increase in virulence being achieved apparently entirely by overcoming these defences, most likely due to elevated oxidative stress resistance.

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A novel 96-well gel-based assay for determining antifungal activity against filamentous fungi

Cited by 15 publications

References 22 publications

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites

Increased Biological Activity of Aneurinibacillus migulanus Strains Correlates with the Production of New Gramicidin Secondary Metabolites

Surfactin variants mediate species‐specific biofilm formation and root colonization in Bacillus

Highly potent host external immunity acts as a strong selective force enhancing rapid parasite virulence evolution

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