Sporulating Cells Research Articles

Heat Shock Improves Random Spore Analysis in Diverse Strains of Saccharomyces cerevisiae.

Random spore analysis (RSA) is a classic method in yeast genetics that allows high-throughput purification of recombinant haploid spores following specific crosses. RSA typically involves a number of steps to induce sporulation, purge vegetative cells that fail to sporulate, and disrupt the ascus walls of sporulated cells to release haploid spores. These steps generally require expensive chemicals and/or enzymes that kill diploid cells but have few effects on spores. In the fission yeast Schizosaccharomcyes pombe, heat shock has been reported as an effective addition to RSA protocols, but to our knowledge heat shock has not been used for this purpose in the budding yeast Saccharomyces cerevisiae. Here, we evaluate the effects of heat shock on vegetative and sporulated cultures of four diverse yeast strains: a European wine strain (DBVPG6765), a Japanese sake strain (Y12), a West African palm wine strain (DBVPG6044) and a North American strain isolated from the soil beneath an oak tree (YPS128). We characterize this phenotype under multiple combinations of temperature and incubation time, and find specific conditions that lead to the exclusion of vegetative cells and an enrichment in spores, which differ by strain. We also collected genome sequence data from a recombinant population that experienced multiple rounds of RSA, including one round with a heat shock treatment. These data suggest that when incorporated into an RSA protocol, heat shock leads to increased genetic diversity among the cells that survive and mate. Ultimately, our work provides evidence that short heat treatments can improve existing RSA protocols, though in a strain-specific manner. This result informs applications of high-throughput RSA protocols, such as QTL mapping and experimental evolution research.

EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin.

Dectin-1 and ephrin type-A receptor 2 (EphA2) receptors recognize β-glucan present in the fungal cell wall. Inhibition of Dectin-1 with the monoclonal 2a11 antibody was shown to reduce internalization of conidia of the human pathogen Aspergillus fumigatus into epithelial cells. In this study, we investigated the role of the EphA2 receptor present on A549 epithelial type II lung cells in the interaction with A. fumigatus conidia. We assessed whether EphA2 is involved in association and internalization of conidia by receptor inhibition by an antibody or by using the kinase inhibitor dasatinib. A 50% reduction of internalization of conidia was observed when this receptor was blocked with either the EphA2-specific monoclonal antibody or dasatinib, which was similar when Dectin-1 was inhibited with the 2a11 monoclonal antibody. Inhibition of both receptors reduced the internalization to 40%. EphA2 inhibition was also assessed in a hydrophobin deletion strain (ΔrodA) that exposes more β-glucan and a dihydroxynaphthalene (DHN)-melanin deletion strain (ΔpksP) that exposes more glucosamine and glycoproteins. The ΔrodA strain behaved similar to the wild-type strain with or without EphA2 inhibition. In contrast, the ΔpksP mutant showed an increase in association to the A549 cells and a decrease in internalization. Internalization was not further decreased by EphA2 inhibition. Taken together, the presence of DHN-melanin in the spore cell wall results in an EphA2-dependent internalization of conidia of A. fumigatus into A549 cells.

Influence of Nonthermal Atmospheric Plasma-Activated Water on the Structural, Optical, and Biological Properties of Aspergillus brasiliensis Spores

Plasma-activated water (PAW) has emerged as a platform for sterilizing fungal pathogens. In this study, we investigated the influence of PAW on black melanized spores of Aspergillus brasiliensis to explore the mechanism of fungal spore inactivation. PAW was prepared by activating deionized water with a nonthermal atmospheric pressure air plasma jet (soft plasma jet). The concentrations of H2O2 and NOx in the PAW treated by the soft plasma jet for 3 min were 50 μM and 1.8 mM, respectively, and the pH of the PAW was 3.10. The reactive oxygen and nitrogen species (RONS) in the PAW increased with longer plasma activation time. After being treated for 30 min in the PAW with a plasma activation time of 3 min, the spore viability dramatically dropped to 15%. The viabilities of 0.3% H2O2- and 0.3% HNO3-treated spores were 22% and 42%, respectively. The breakage of the spore cell wall by the PAW was revealed in scanning electron microscope images and flow cytometry measurements. Disruption of cell wall integrity provides a path for intracellular components to escape and RONS of the PAW can attack intracellular components directly. Degradation of high molecular genomic DNA was also observed by agarose gel electrophoresis. These results suggest that long-lived reactive species generated in the PAW play an important role in the inactivation of melanized fungal spores. Consequently, PAW produced by a soft plasma jet can be applied to sterilize bioprotective walled fungal spores in a relatively large volume.

Bioactivity studies of porphyrinoids against microsporidia isolated from honeybees

Microsporidian infections are dangerous to honeybees due to the absence of an efficient treatment for nosemosis. In the present work, the abilities of several porphyrins to directly inactivate microsporidia derived from Nosema-infected honeybees were studied in vitro. Amide derivatives of protoporphyrin IX (PPIX) conjugated with one and two amino acid moieties were synthesized, and their activities were compared with those of two cationic porphyrins, TMePyP and TTMePP. The most active porphyrins, PP[Lys-Asp]2, PP[Lys-TFA]2, PP[Asp(ONa)2]2 and PP[Lys-Lys]2 at concentrations as low as 10–50 µM exerted significant effects on microsporidia, reducing the number of spores by 67–80% compared to the control. Live-cell imaging of the spores treated with porphyrins showed that only 1.6% and 3.0% of spores remained alive after 24 h-incubation with 50 µM PP[Asp(ONa)2]2 and PP[Lys-Asp]2, respectively. The length of the amino acid side chains and their identity in the PPIX molecules affected the bioactivity of the porphyrin. Importantly, the irradiation of the porphyrins did not enhance their potency in destroying Nosema spores. We showed that the porphyrins accumulated inside the living spores but not inside dead spores, thus the destruction of the microsporidia by non-metallated porphyrins is not dependent on photosensitization, but is associated with their active transport into the spore cell. When administered to honeybees in vivo, PPIX[Lys-TFA]2 and PPIX[Lys-Lys]2 reduced spore loads by 69–76% in infected individuals. They both had no toxic effect on honeybees, in contrast to zinc-coordinated porphyrin.

Atomic oxygen radical‐induced intracellular oxidization of mould spore cells

AbstractThe inactivation mechanism of mould spores using plasma‐generated neutral reactive oxygen species (ROS) was investigated in this paper. Typical cell viability using a counting of colony‐forming unit assay indicated a major state of the spore cells. Both intracellular and extracellular damages have been investigated by a couple of well‐established cell visualization techniques: confocal fluorescence emission microscopy, scanning electron microscopy, and transmission electron microscopy. It is revealed in this paper that the combination of the spore cell viability with those visualization results strongly suggested an early stage of the cell inactivation when ROS can induce intracellular lipid peroxidation through both nanometer‐thick lipid cell membrane and less‐damaged several hundred nanometer‐thick cell wall.

Formulation of Bacillus and Azotobacter Consortia in Liquid Cultures: Preliminary Research on Microbes-Coated Urea

The spore-forming Bacillus and cysts forming Azotobacter are Plant Growth Promoting Rhizobacteria which has been used as biofertilizer in sustainable agriculture since they tolerant to dried soil. Drought resistant microbes will be useful to coat urea in order to reduce the lost of nitrogen. The objectives of this preliminary study were to study the effect of molasse based liquid media on the population of Bacillus spore and Azotobacter vegetative cell and to determine the composition of four bacterial species in liquid formula. In the first experiment The Bacillus subtilis, B. megaterium, A. chroococcum and A. vinelandii were grown separately in 1% cane molasses enriched with 0.1% NH4Cl. As control treatment, The Bacillus and Azotobacter were grown in Nutrient Broth and Ashby’s mannitol broth respectively. In the second experiment, different composition of said Bacillus and Azotobacter were grown in molasses based liquid media prior to count the spore and vegetative cell. The results showed that molasses-based media supported bacterial growth and initial ratio 1:1:1:1 of liquid inoculant was effective to increase bacterial growth. This experiment suggested that the use of organic based media was useful practice of liquid biofertilizer formulation for granule urea coating.

Characterization of a fungal competition factor: Production of a conidial cell-wall associated antifungal peptide.

Competition is one of the fundamental driving forces of natural selection. Beauveria bassiana is a soil and plant phylloplane/root fungus capable of parasitizing insect hosts. Soil and plant environments are often enriched with other fungi against which B. bassiana competes for survival. Here, we report an antifungal peptide (BbAFP1), specifically expressed and localized to the conidial cell wall and is released into the surrounding microenvironment inhibiting growth of competing fungi. B. bassiana strains expressing BbAFP1, including overexpression strains, inhibited growth of Alternaria brassicae in co-cultured experiments, whereas targeted gene deletion of BbAFP1 significantly decreased (25%) this inhibitory effect. Recombinant BbAFP1 showed chitin and glucan binding abilities, and growth inhibition of a wide range of phytopathogenic fungi by disrupting membrane integrity and eliciting reactive oxygen species (ROS) production. A phenylalanine residue (F50) contributes to chitin binding and antifungal activity, but was not required for the latter. Expression of BbAFP1 in tomato resulted in transgenic plants with enhanced resistance to plant fungal pathogens. These results highlight the importance of fungal competition in shaping primitive competition strategies, with antimicrobial compounds that can be embedded in the spore cell wall to be released into the environment during the critical initial phases of germination for successful growth in its environmental niche. Furthermore, these peptides can be exploited to increase plant resistance to fungal pathogens.

Inhibitory effect of fidaxomicin on biofilm formation in Clostridioides difficile

Clostridioides difficile infection results from a disturbance of the normal microbial flora of the colon, allowing proliferation of C. difficile and toxin production by toxigenic strains. Fidaxomicin, a macrocyclic antibiotic that prevents RNA synthesis in C. difficile and inhibits spore formation, toxin production, and cell proliferation, is clinically effective in treating C. difficile infection. As recent studies have suggested that biofilm formation influences C. difficile colonization and infection in the colon, we undertook the present study to determine the effects of fidaxomicin on C. difficile biofilm formation. Sub-minimum inhibitory concentrations (MICs) of fidaxomicin inhibited biofilm formation by C. difficile UK027 and delayed planktonic growth. Sub-MICs of vancomycin did not inhibit biofilm formation or affect planktonic growth. In C. difficile UK027 exposed to sub-MICs of fidaxomicin, mRNA expression of biofilm-related flagellin gene fliC was slightly increased compared with that of other biofilm-related genes (pilA1, cwp84, luxS, dccA, and spo0A). In conclusion, this study indicates that sub-MICs of fidaxomicin inhibit C. difficile UK027 biofilm formation by influencing cell growth and fliC transcription.

Time-lapse single-cell transcriptomics reveals modulation of histone H3 for dormancy breaking in fission yeast

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial ‘wake-up’ trigger at dormancy breaking.

BACTERIA SURVIVAL STRATEGIES IN CONTACT WITH ANTIBIOTICS

Bacteria survival in the conditions of antimicrobial therapy is the global problem of health care. This review highlights the complexity and diversity of mechanisms used by bacteria to neutralize antibiotics. To analyze the problem, the search was made using PubMed database, Russian scientific electronic library eLIBRARY, search system of World Health Organization and European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Based on the analysis of survival strategies in the conditions of antibiotics action we propose new classification of resistant bacteria. Classification criteria include the ability to divide under antibiotics action, the survival strategies application as a species trait, the presence of specialized genes determining the transition to the state with reduced/stopped metabolism. Two main groups are resistant bacteria and bacteria with reduced/stopped metabolism, which survive but do not divide in the presence of antibiotic. The first group includes two subgroups: bacteria with intrinsic and adaptive resistance. The second group includes (1) bacteria with specialized genes responsible for cell transformation to the state with reduced/stopped metabolism, (2) bacteria transforming to the state with reduced/stopped metabolism without involvement of special genes, and (3) cell forms with special morphology - spores, cysts and cyst-like cells. We described the usefulness of proposed classification including improved understanding of the correlation between bacteria survival in the presence of antibiotics and molecular mechanism of cell metabolism inhibition, presence or absence of targets for using molecular-genetic methods of bacteria resistant variant determination, the possibility for development of rational antimicrobial therapy methods.

Effects of Chitosan on Clostridium perfringens and Application in the Preservation of Pork Sausage

The effects of chitosan with 95% deacetylation degree (DD95) on the spore germination, cell proliferation, and heat resistance of Clostridium perfringens CCRC 10,648 and CCRC 13,019 were investigated, and its application on pork sausage with sodium nitrite reduction was also evaluated. DD95 chitosan can strongly reduce the heat resistance of both strains. The D80 and D100 values for strain CCRC 13,019 decreased from 40.98 and 4.64 min to 39.21 and 3.26 min, respectively, as a result of adding 250 ppm DD95; meanwhile, addition of chitosan decreased the D80 and D100 values for CCRC 10,648 from 41.15 and 6.46 min to 39.52 and 3.78 min, respectively. In pork sausage, addition of 3000 ppm DD95 chitosan considerably slowed down the bacterial proliferation and volatile basic nitrogen production. There were no significant differences in color (L* and b* values), shearing force, and hardness in the pork sausages with or without DD95 chitosan during storage at 4 and 25 °C. However, the addition of DD95 chitosan in pork sausage significantly retarded the decrease of the a* value. Therefore, DD95 chitosan could reduce the concentration of sodium nitrite required in pork sausages for color retention.

STUDY OF THE MASS-SPECTORS’ FEATURES OF SPORES AND VEGETATIVE FORMS OF BACILLUS ANTHRACIS BY THE METHOD OF TIME OF FLIGHT MASS-SPECTROMETRY

Aim. Investigation of the features of the protein profiles of the spore and vegetative form of the anthrax microbe by the MALDI-TOF MS method using the resources of the Mass-Up program and the package for the statistical software with open source code R. Materials and methods. Spores and vegetative forms of 32 strains of B. anthracis from the collection of microorganisms of the Stavropol Research Institute for Plague Control, including 8 strains isolated from an outbreak of anthrax in Yamal in 2016. Protein profiling was carried out on the Microflex MALDI-TOF mass spectrometer «Bruker Daltonics». Results. The alternative bioinformational-statistical approach used to analyze the MALDI-TOF mass spectra of the causative agent of anthrax made it possible to differentiate the spores and vegetative forms of the microbe based on the identification of the corresponding groups of biomarkers. Conclusion. A comparison of vegetative and spore cells of typical and atypical strains of anthrax causative agent on the basis of MALDI-TOF MS data was made. It has been experimentally confirmed that the protein profiles of cultures of Bacillus anthracis of the spore and vegetative form differ significantly, and this difference can be used to search for potential markers of each of the forms.

Live cell imaging of cell movement and transdifferentiation during regeneration of an amputated multicellular body of the social amoeba Dictyostelium discoideum

The regeneration of lost body parts is a fascinating phenomenon exhibited by some multicellular organisms. In social amoebae, such as Dictyostelium discoideum, the pseudoplasmodium is a temporary migratory multicellular structure with high regeneration ability. It consists of future stalk cells (prestalk cells) at the anterior end and future spore cells (prespore cells) at the posterior end, and if amputated, the remaining cells can rapidly regenerate the lost portion within several hours. Details of this regeneration event have been extensively documented; however, little is known about the behavior of individual cells involved in this process. In this study, we performed live cell imaging of cell behavior during regeneration of the excised anterior prestalk region. We used cells that specifically express GFP in the prestalk cell lineage to examine how the prestalk region is regenerated after this region is excised. The current model of prestalk regeneration suggests that the progenitors of prestalk cells, known as anterior-like cells (ALCs), which are sparsely distributed in the prespore region, are redistributed to form the new prestalk region. However, we found that the regenerated prestalk region was formed mainly by the transdifferentiation of prespore cells surrounding the excised anterior end, with little clustering of pre-existing ALCs. Furthermore, the movement of randomly distributed labeled cells during regeneration revealed that although the posterior end was deformed and rounded in shape, the relative position of cells along the anterior-posterior axis remained largely unchanged. These results suggest that the original anterior-posterior axis is maintained in posterior bodies and that prespore cells at the anterior side transdifferentiate and regenerate the prestalk region.

Spore Viability and Cell Wall Integrity of Cordyceps pruinosa Treated with an Electric Shock-Free, Atmospheric-Pressure Air Plasma Jet

Atmospheric-pressure A r plasma jets are known to be detrimental to Cordyceps pruinosa spores. However, it is not clear what kinds of reactive species are more effective with regard to fungal cell death. Herein, we study which reactive species plays pivotal roles in the death of fungal spores using an electric shock-free, atmospheric-pressure air plasma jet, simply called soft plasma jet. Plasma treatment significantly reduced the spore viability and damaged fungal DNA. As observed from the circular dichroism spectra, scanning electron microscope images, and flow cytometric measurements, cell wall integrity was decreased by reactive oxygen and nitrogen species (RONS) from the plasma itself and the plasma-activated water. Consequently, degradation of the spore cell wall allows RONS from the plasma to reach the intracellular components. Such plasma-induced intracellular RONS can attack spore DNA and other intracellular components, as confirmed by electrophoresis analysis and phosphorylated histone measurement. In addition, weakening of the spore cell wall allowed for the loss of intracellular components, which can lead to cell death. Plasma radicals were investigated by measuring the optical emission spectrum of the soft plasma jet, and intracellular reactive oxygen species were confirmed by measuring the fluorescence of 2′, 7′-dichlorodihydrofluorescein-diacetate ( H 2 D C F - D A )-stained spores. The soft plasma jet generated considerable amounts of H 2 O 2 and N O x but a very small number of O H radicals as compared to the atmospheric-pressure A r plasma jet; this indicates that plasma-induced long-lived reactive species ( H 2 O 2 and N O x ) play an important role in the weakening of spore cell walls and cell death.

Controlling Heterogeneity and Increasing Titer from Riboswitch-Regulated Bacillus subtilis Spores for Time-Delayed Protein Expression Applications.

Sporulated cells have potential as time-delayed expression chassis of proteins for applications such as "on-demand" biologics production, whole cell biosensors, or oral vaccines. However, the desired attributes of high expression rates and low product variances are difficult to maintain from germinated spores. In this work, we study the effect of an integrating vs theta-replicating plasmid in a wild-type Bacillus subtilis and two PolY mutants. The cells were engineered to produce a fluorescent reporter protein (RFP) under the control of a riboswitch activated by theophylline. This allowed for greater sensitivity to point mutations. The fluorescence and cell-growth curves were fit with a custom kinetic model, and a peak kinetic rate (LKPmax) was extracted for each clonal population (n = 30 for all cell, vector, and growth combinations). Plasmid-based expression yields higher (8.7×) expression rates because of an increased copy number of the expression cassette (10× over integrated). The variance of LKPmax values increased 2.1× after sporulation for the wild-type strain. This increase in variance from sporulation is very similar to what is observed with UV exposure. This effect can be partially mitigated by the use of PolY knockouts observed in suspended cell growths and adherent biofilms.

Effect of thermal and non-thermal techniques for microbial safety in food powder: Recent advances.

Food powders are appreciated worldwide, as it enables food to be preserved for an extended period without significant loss of quality, even under the ambient storage condition. However, it is evidenced that the development of resistant microbial spore and viable microbial cells is a matter of concern even in low moisture foods like food powders. For microbial inactivation, the strategy generally applied is the implication of conventional preservation methods, such as heat treatment which is greatly accompanied by degradation of nutritional organoleptic properties. To overcome the shortcomings of conventional thermal processing, a set of advanced or emerging technologies are being developed which can inactivate the microbial spores and viable microbial cells capable of surviving with maximum retention in the nutritional or organoleptic profile. The examples include infrared heating, microwave heating, radiofrequency heating, instant control pressure drop technology, high-pressure processing, pulsed electric field, pulsed light, ozone processing, and cold plasma. In this review, the potential of different advanced thermal and non-thermal technologies towards the inactivation of spores and viable cells of microorganisms in food powders has been highlighted precisely along with their mechanism of action. The summary of the literature encompassing the use of different processing techniques will help the readers to understand the underlying mechanism of microbial inactivation associated with each processing techniques applied to powders. Eventually, this information will help them to select the suitable technique (individual or in combination with another counterpart) to inactivate spores and viable cells in a specific food powder.

Dependence of the Bacillus subtilis biofilm expansion rate on phenotypes and the morphology under different growing conditions.

Biofilms are communities of tightly associated bacteria encased in an extracellular matrix and attached to surfaces of various objects, such as liquid or solid surfaces. Here we use the multi-channel wide field stereo fluorescence microscope to characterize growth of the Bacillus subtilis biofilm, in which the bacterial strain was triple fluorescence labeled for three main phenotypes: motile, matrix producing and sporulating cells. We used the feature point matching approach analyzing time lapse experimental movies to study the biofilm expansion rate. We found that the matrix producing cells dominate the biofilm expansion, at the biofilm edge, the expansion rate of matrix producing cells was almost the same as the velocity of the whole biofilm; however, the motile and sporulating cells were nearly rest. We also found that the biofilm expansion rate evolution relates to cell differentiation and biofilm morphology, and other micro-environments can influence the biofilm growth, such as nutrient, substrate hardness and colony competition. From our work, we get a deeper understanding of the biofilm growth, which can help us to control and to further disperse the biofilm.

Epigenetic Regulation of Plant Gametophyte Development.

Unlike in animals, the reproductive lineage cells in plants differentiate from within somatic tissues late in development to produce a specific haploid generation of the life cycle—male and female gametophytes. In flowering plants, the male gametophyte develops within the anthers and the female gametophyte—within the ovule. Both gametophytes consist of only a few cells. There are two major stages of gametophyte development—meiotic and post-meiotic. In the first stage, sporocyte mother cells differentiate within the anther (pollen mother cell) and the ovule (megaspore mother cell). These sporocyte mother cells undergo two meiotic divisions to produce four haploid daughter cells—male spores (microspores) and female spores (megaspores). In the second stage, the haploid spore cells undergo few asymmetric haploid mitotic divisions to produce the 3-cell male or 7-cell female gametophyte. Both stages of gametophyte development involve extensive epigenetic reprogramming, including siRNA dependent changes in DNA methylation and chromatin restructuring. This intricate mosaic of epigenetic changes determines, to a great extent, embryo and endosperm development in the future sporophyte generation.

Enhancement of antifungal and mycotoxin inhibitory activities of food-grade thyme oil nanoemulsions with natural emulsifiers

Thyme oil-in-water nanoemulsions stabilized by three different sources of natural emulsifiers (bovine serum albumin, quillaja saponins, and soy lysolecithin) were prepared as potential natural antifungal agents. The thyme oil nanoemulsion containing bovine serum albumin or quillaja saponins with mean particle diameters <200 nm showed high physical stability during 28 days’ storage. The antifungal and mycotoxin inhibitory activities of thyme oil nanoemulsions stabilized with three natural emulsifiers against Fusarium graminearum isolates were then investigated. Mycelial growth, spore germination, and mycotoxin production results indicated that the antifungal activity was mainly dominated by thyme oil rather than emulsifier to alter mycelial and spore cell membrane integrity. The mycotoxin inhibitory efficacy of thyme oil was enhanced considerably in nanoemulsion form as compared to bulk oil. Among the three natural emulsifiers, bovine serum albumin stabilized thyme oil nanoemulsion exhibited higher mycotoxin inhibitory activity due to a joint function of thyme oil and emulsifier.

Fengycin produced by Bacillus subtilis NCD-2 is involved in suppression of clubroot on Chinese cabbage

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most important diseases of Brassicaceae family. Bacillus spp. that produce lipopeptide antibiotics are often used as biocontrol agents to suppress soilborne diseases. B. subtilis NCD-2 isolated from the cotton rhizosphere has been found to inhibit pathogenic fungi through the production of fengycin-type cyclopeptides. In this study, the biocontrol efficacy of B. subtilis NCD-2 against P. brassicae and the role of fengycin in disease suppression were evaluated on Chinese cabbage in glasshouse. B. subtilis NCD-2 reduced clubroot disease incidence and severity up to 47.7% and 51.6%, respectively, while the fengycin defective mutant ΔfenC and a PhoP/PhoR two-component regulatory system mutant, ΔphoP, were unable to prevent this disease. In vitro, P. brassicae resting spore cells were lysed when treated with standard fengycin and crude fengycin extracts of the wild type B. subtilis NCD-2, but not of its mutant ΔfenC. Moreover, the population dynamics of B. subtilis and P. brassicae was determined using quantitative real-time PCR (qPCR). The results were surprising that the wild type B. subtilis NCD-2 and its mutant ΔfenC colonized at similar rates in the rhizosphere, while levels of P. brassicae DNA in the rhizosphere and root tissue were reduced following B. subtilis NCD-2 treatment, but there was no effect of the mutant ΔfenC. Thus, the production of fengycins in B. subtilis NCD-2 may play a promising role in the suppression of P. brassicae in the rhizosphere and reduction of incidence of clubroot disease in Chinese cabbage.