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Microbial Pathogenesis Jun 2024The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries...
The antifungal activity of Serratia plymuthica CCGG2742, a bacterial strain isolated from grapes berries skin, against a phytopathogenic fungus isolated from blueberries was evaluated in vitro and in vivo. In order to characterize the wild fungal isolate, phylogenetic analysis using concatenated DNA sequences from the RPB2 and TEF1 genes and of the ITS region was performed, allowing the identification of the fungal isolate that was called Alternaria tenuissima CC17. Hyphae morphology, mycelium ultrastructure, conidia and reproductive structures were in agreement with the phylogenetic analysis. The antifungal activity of the S. plymuthica strain was dependent on the composition of the culture medium. The greatest inhibition of mycelial growth of A. tenuissima CC17 by S. plymuthica CCGG2742 was observed on YTS medium, which lacks of an easily assimilable carbon source. Fungal growth medium supplemented with 50 % of bacterial supernatant decreased the conidia germination of A. tenuissima CC17 up to 32 %. Preventive applications of S. plymuthica CCGG2742 to blueberries and tomato leaves at conidia:bacteria ratio of 1:100, protected in 77.8 ± 4.6 % and 98.2 ± 0.6 % to blueberries and tomato leaves from infection caused by A. tenuissima CC17, respectively. To the best of our knowledge, this is the first report on the antifungal activity of S. plymuthica against A. tenuissima, which could be used as a biological control agent of plant diseases caused by this fungal species. In addition, the results of this work could be a starting point to attribute the real importance of A. tenuissima as a pathogen of blueberries in Chile, which until now had been considered almost exclusively to A. alternata. Likewise, this research could be relevant to start developing highly effective strategies based on S. plymuthica CCGG2742 for the control of this important phytopathogenic fungus.
PubMed: 38906491
DOI: 10.1016/j.micpath.2024.106750 -
Polish Journal of Microbiology Jun 2024This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and...
This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and temperature, on the mycelial growth characteristics of . Employing single-factor experiments and response surface methodology within glass Petri dishes, the research identified that carrot powder, soybean powder, and ZnSO notably enhanced the proliferation of aerial mycelium, significantly augmenting the growth rate of mycelium. The resultant mycelium was observed to be dense, robust, and fluffy in texture. In particular, ZnSO markedly accelerated the mycelium growth rate. Furthermore, xanthan gum was found to effectively modulate the medium's viscosity, ensuring a stable suspension and facilitating nutrient equilibrium. The optimal cultivation temperature was determined to be 25°C, with mycelial growth ceasing below 5°C and mycelium perishing at temperatures exceeding 35°C. The optimal medium composition was established as follows: wheat starch 5 g/l, carrot powder 5 g/l, soybean powder 7.50 g/l, glucose 10 g/l, ZnSO 0.71 g/l, NHCl 0.68 g/l, xanthan gum 0.5 g/l, and agar 20 g/l. Under these optimized conditions, the mycelium of exhibited a rapid growth rate (1.04 ± 0.14 mm/day), characterized by a thick, dense, and well-developed structure. This investigation provides a theoretical foundation for the conservation, strain selection, and breeding of .
Topics: Mycelium; Culture Media; Temperature; Nitrogen; Carbon; Polysaccharides, Bacterial
PubMed: 38905279
DOI: 10.33073/pjm-2024-022 -
Frontiers in Cellular and Infection... 2024species comprise a ubiquitous pathogenic fungal genus responsible for causing candidiasis. They are one of the primary causatives of several mucosal and systemic...
species comprise a ubiquitous pathogenic fungal genus responsible for causing candidiasis. They are one of the primary causatives of several mucosal and systemic infections in humans and can survive in various environments. In this study, we investigated the antifungal, anti-biofilm, and anti-hyphal effects of six -substituted phthalimides against three species. Of the derivatives, -butylphthalimide (NBP) was the most potent, with a minimum inhibitory concentration (MIC) of 100 µg/ml and which dose-dependently inhibited biofilm at sub-inhibitory concentrations (10-50 µg/ml) in both the fluconazole-resistant and fluconazole-sensitive and . NBP also effectively inhibited biofilm formation in other pathogens including uropathogenic , , , and , along with the polymicrobial biofilms of and . NBP markedly inhibited the hyphal formation and cell aggregation of and altered its colony morphology in a dose-dependent manner. Gene expression analysis showed that NBP significantly downregulated the expression of important hyphal- and biofilm-associated genes, i.e., , , and , upon treatment. NBP also exhibited mild toxicity at concentrations ranging from 2 to 20 µg/ml in a nematode model. Therefore, this study suggests that NBP has anti-biofilm and antifungal potential against various strains.
Topics: Biofilms; Antifungal Agents; Phthalimides; Microbial Sensitivity Tests; Candida albicans; Hyphae; Candida; Candidiasis; Animals; Humans; Candida parapsilosis; Fungal Proteins; Fluconazole
PubMed: 38903941
DOI: 10.3389/fcimb.2024.1414618 -
BMC Microbiology Jun 2024Rhizoctonia solani is an important plant pathogen worldwide, and causes serious tobacco target spot in tobacco in the last five years. This research studied the...
BACKGROUND
Rhizoctonia solani is an important plant pathogen worldwide, and causes serious tobacco target spot in tobacco in the last five years. This research studied the biological characteristics of four different anastomosis groups strains (AG-3, AG-5, AG-6, AG-1-IB) of R. solani from tobacco. Using metabolic phenotype technology analyzed the metabolic phenotype differences of these strains.
RESULTS
The results showed that the suitable temperature for mycelial growth of four anastomosis group strains were from 20 to 30C, and for sclerotia formation were from 20 to 25C. Under different lighting conditions, R. solani AG-6 strains produced the most sclerotium, followed by R. solani AG-3, R. solani AG-5 and R. solani AG-1-IB. All strains had strong oligotrophic survivability, and can grow on water agar medium without any nitrutions. They exhibited three types of sclerotia distribution form, including dispersed type (R. solani AG-5 and AG-6), peripheral type (R. solani AG-1-IB), and central type (R. solani AG-3). They all presented different pathogenicities in tobacco leaves, with the most virulent was noted by R. solani AG-6, followed by R. solani AG-5 and AG-1-IB, finally was R. solani AG-3. R. solani AG-1-IB strains firstly present symptom after inoculation. Metabolic fingerprints of four anastomosis groups were different to each other. R. solani AG-3, AG-6, AG-5 and AG-1-IB strains efficiently metabolized 88, 94, 71 and 92 carbon substrates, respectively. Nitrogen substrates of amino acids and peptides were the significant utilization patterns for R. solani AG-3. R. solani AG-3 and AG-6 showed a large range of adaptabilities and were still able to metabolize substrates in the presence of the osmolytes, including up to 8% sodium lactate. Four anastomosis groups all showed active metabolism in environments with pH values from 4 to 6 and exhibited decarboxylase activities.
CONCLUSIONS
The biological characteristics of different anastomosis group strains varies, and there were significant differences in the metabolic phenotype characteristics of different anastomosis group strains towards carbon source, nitrogen source, pH, and osmotic pressure.
Topics: Rhizoctonia; Phenotype; Nicotiana; Plant Diseases; Temperature; Mycelium; Plant Leaves; Virulence
PubMed: 38902632
DOI: 10.1186/s12866-024-03363-9 -
Fungal Genetics and Biology : FG & B Jun 2024The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target...
The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.
PubMed: 38897560
DOI: 10.1016/j.fgb.2024.103910 -
Molecules (Basel, Switzerland) May 2024A specific feature of mushrooms (including those of the genus ) is their natural ability to absorb and accumulate many chemical substances present in their immediate...
A specific feature of mushrooms (including those of the genus ) is their natural ability to absorb and accumulate many chemical substances present in their immediate environment, which makes them an excellent natural sorption material. Hence, fruiting bodies of mushrooms have been recognized for years as excellent indicators of the environment, reflecting its current state. Nevertheless, mushrooms can accumulate both health-promoting substances, such as bioelements, and toxic substances, such as heavy metals and organic compounds, including bisphenol A (BPA). This organic chemical compound in the phenol group, although it has been withdrawn in the EU since 2010, is widely present in the environment around us. In the present experiment, we aimed to determine the effect of adding BPA to liquid media for in vitro cultures of spp. The biomass increases were determined. Moreover, the degrees of adsorption and desorption of BPA from the obtained freeze-dried biomass in two different environments (neutral and acidic) were determined as a function of time. This is the first study to determine the bioavailability of adsorbed BPA in obtained biomass by extracting the mycelium into artificial digestive juices in a model digestive system. BPA was added to the liquid Oddoux medium in the following amounts: 0.01, 0.5, and 0.5 g/250 mL of medium. The amounts of adsorbed and desorbed BPA were determined by flow injection analysis (FIA) with amperometric detection. The addition of BPA to the substrate reduced the biomass growth in each of the discussed cases. BPA adsorption by the mycelium occurred at over 90% and depended on the morphology of the mushroom (structure, surface development, and pore size). BPA desorption depended on the pH of the environment and the desorption time. Mushrooms are an excellent natural remedial material, but BPA is extracted into artificial digestive juices; therefore, consuming mushrooms from industrialized areas may have health consequences for our bodies.
Topics: Phenols; Pleurotus; Benzhydryl Compounds; Biomass; Adsorption; Flow Injection Analysis
PubMed: 38893397
DOI: 10.3390/molecules29112520 -
Molecules (Basel, Switzerland) May 2024is a crucial edible fungus used in tea fermentation. In the industrial fermentation process, the fungus experiences a low to high osmotic pressure environment. To...
is a crucial edible fungus used in tea fermentation. In the industrial fermentation process, the fungus experiences a low to high osmotic pressure environment. To explore the law of material metabolism changes during osmotic pressure changes, NaCl was used here to construct different osmotic pressure environments. Liquid chromatography-mass spectrometry (LC-MS) combined with multivariate analysis was performed to analyze the distribution and composition of under different salt concentrations. At the same time, the in vitro antioxidant activity was evaluated. The LC-MS metabolomics analysis revealed significant differences between three mycelium samples grown on media with and without NaCl concentrations of 8% and 18%. The contents of gibberellin A3, A124, and prostaglandin A2 related to mycelial growth and those of arabitol and fructose-1,6-diphosphate related to osmotic pressure regulation were significantly reduced at high NaCl concentrations. The biosynthesis of energy-related pantothenol and pantothenic acid and antagonism-related fluvastatin, aflatoxin, and alternariol significantly increased at high NaCl concentrations. Several antioxidant capacities of mycelia were directly related to osmotic pressure and exhibited a significant downward trend with an increase in environmental osmotic pressure. The aforementioned results indicate that adapts to changes in salt concentration by adjusting their metabolite synthesis. At the same time, a unique set of strategies was developed to cope with high salt stress, including growth restriction, osmotic pressure balance, oxidative stress response, antioxidant defense, and survival competition.
Topics: Aspergillus; Metabolomics; Chromatography, Liquid; Salt Stress; Antioxidants; Metabolome; Osmotic Pressure; Mycelium; Mass Spectrometry; Sodium Chloride; Liquid Chromatography-Mass Spectrometry; Sugar Alcohols
PubMed: 38893389
DOI: 10.3390/molecules29112513 -
Communications Biology Jun 2024Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual...
Utilizing a microfluidic chip with serpentine channels, we inoculated the chip with an agar plug with Neurospora crassa mycelium and successfully captured individual hyphae in channels. For the first time, we report the presence of an autonomous clock in hyphae. Fluorescence of a mCherry reporter gene driven by a clock-controlled gene-2 promoter (ccg-2p) was measured simultaneously along hyphae every half an hour for at least 6 days. We entrained single hyphae to light over a wide range of day lengths, including 6,12, 24, and 36 h days. Hyphae tracked in individual serpentine channels were highly synchronized (K = 0.60-0.78). Furthermore, hyphae also displayed temperature compensation properties, where the oscillation period was stable over a physiological range of temperatures from 24 °C to 30 °C (Q = 1.00-1.10). A Clock Tube Model developed could mimic hyphal growth observed in the serpentine chip and provides a mechanism for the stable banding patterns seen in race tubes at the macroscopic scale and synchronization through molecules riding the growth wave in the device.
Topics: Neurospora crassa; Hyphae; Temperature; Lab-On-A-Chip Devices; Gene Expression Regulation, Fungal; Biological Clocks
PubMed: 38890525
DOI: 10.1038/s42003-024-06429-6 -
Plant Disease Jun 2024Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in...
Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17 N, 110.18 E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned becoming necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n = 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed twice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical . They colonies were gray to brownish black. Conidiophores were branched, brown. Conidia were brown, long ellipsoid, had 4-12 transverse and 0-3 longitudinal septa; measured within 67.5-127.8 (average = 105.6) × 12.5-27.8 (average = 20.4) µm (n = 30). Apical beak was longer than conidia body. measured within 40.5-423.5 (average = 365.2) × 2.5-5.8 (average = 3.2) µm (n = 30). Based on morphological characteristics, the three isolates were identified as Alternaria crassa (Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor (TEF1) with primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Walther et al. 2013; Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524-ON430526; GAPDH, ON500656-ON500658; RPB2, ON500659-ON500661; TEF1, ON500662-ON500664). The sequences were 100% identical with those of Alternaria crassa strain CBS 116647 upon BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18 ). Thus, the fungus associated with leaf yellow spot on D. stramonium was identified as A. crassa. Pathogenicity tests were conducted in a greenhouse at 24 ℃-30 ℃ with 80% relative humidity using 3 isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated using three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelium on 5 mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated comtrols.. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and was morphologically identical to the original isolates, fulfilling Koch's postulates. A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing leaf yellow spot on D. stramonium in China. This pathogen possesses potential biocontrol properties on the invasive weed, while this study also provides an important reference for the control of the disease of the medicinal plant.
PubMed: 38885028
DOI: 10.1094/PDIS-08-23-1494-PDN -
Plant Disease Jun 2024Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province,...
Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guava scab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit the margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 µm (n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates were consistent with the description of Neopestalotiopsis clavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25 ℃ to 32 ℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.
PubMed: 38885025
DOI: 10.1094/PDIS-11-23-2357-PDN