Sporulation Conditions Research Articles

Influence of Sporulation Temperature on Germination and Growth of B. weihenstephanensis Strains in Specific Nutrients and in an Extended Shelf-Life Refrigerated Matrix Under Commercial Pasteurization and Storage Conditions

Extended shelf-life (ESL) refrigerated ready-to-eat foods are thermally pasteurized to ensure food safety and stability. However, surviving psychrotrophic Bacillus cereus spores can still pose a challenge. Studies predicting their behavior often overlook sporulation conditions. This study investigated the effect of sporulation temperature on germination of three Bacillus weihenstephanensis strains in specific nutrients (inosine and/or amino acids) with or without prior heat activation (80 °C, 10 min). Sporulation temperature variably affected germination, with stronger effects in moderately responsive strains and nutrients. Heat activation strongly stimulated germination, particularly in nutrients with poorer responses, mitigating differences induced by sporulation temperature. The influence of sporulation temperature on germination and growth in an ESL matrix at refrigeration temperatures (4 °C or 8 °C) in vacuum packaging after heat activation or commercial pasteurization (90 °C, 10 min) was also studied. The latter treatment increased germination rates of surviving spores; however, some strains suffered damage and lost viability upon germination at 4 °C but recovered and grew at 8 °C. These findings highlight the need to account for variability in spore recovery and outgrowth during quantitative risk assessments for psychrotrophic B. cereus in ESL foods.

Novel characterisation of Curvularia geniculata associated with leaf blight on Costus igneus in India

Costus igneus Nak, commonly referred as insulin plant or spiral flag, is a perennial herb belonging to the family Costaceae. Owing to its ability to regulate the blood sugar level, it is considered as a boon plant for diabetic patients, hence popularly known as the “sugar plant”. Although few foliar diseases have been documented on C. igneus, they generally occur in minor frequency under Indian conditions. However, in May 2022, a moderate to severe incidence of leaf blight was observed on insulin plants at University of Agricultural Sciences, GKVK, Bangalore, and nearby medicinal gardens. The affected leaves exhibited light brown lesions coalesced to form papery blight, gradually leading to complete drying of leaves. The current study aimed to identify, characterize, and document the fungal pathogen responsible for the disease, with a focus on its morphological, molecular, and biological characteristics to ensure accurate identification. Additionally, the study sought to determine optimal conditions for pathogen growth and sporulation to support the development of effective disease management strategies. The associated fungus was isolated, purified and identified as Curvularia geniculata through morphological and multigene phylogenetic analysis, using internal transcribed spacer (ITS) and large sub unit (LSU) regions of rDNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Biological characterization revealed that cornmeal agar (CMA) at 30 °C and pH of 7 were ideal for better growth and abundant sporulation. These findings, highlighting the emergence of C. geniculata as a novel fungal pathogen infecting C. igneus in India, are crucial for developing effective disease management strategies to safeguard the medicinal properties of the plant.

Role of water activity on sporulation traits and resistance to 915 MHz microwave in the emetic type of Bacillus cereus on rice

The objective of this study is to investigate the influence of water activity on the sporulation of emetic strains of Bacillus cereus and the subsequent susceptibility of sporulated B. cereus to 915 MHz microwave treatment. Water activity levels were manipulated in the sporulation medium by adjusting glycerol concentrations to 0 %, 3 %, 7.5 %, and 10 %, resulting in corresponding water activities of 0.996, 0.981, 0.971, and 0.960, respectively and sporulated at 30℃. These changes in water activity were intended to simulate the variations in water activity that can occur during the cultivation and processing of rice, where B. cereus is commonly found. Sporulation rates increased with higher water activity, achieving over 90 % of total cells after 3, 5, 5, and 6 days of incubation at water activities of 0.996, 0.981, 0.971, and 0.960, respectively. Resistance to microwave treatment increased with higher water activity levels during sporulation. Microwave treatment for 5 min yielded a reduction of 3.03 log CFU/g at a water activity of 0.960 and 1.98 log CFU/g at 0.996 during sporulation. Lower water activity led to higher % dipicolinic acid (DPA) release during microwave treatment, as measured using a spectrometer, indicating greater membrane damage. To investigate the morphology of spores, transmission electron microscopy was used. Spores produced at lower water activity levels were observed to be enveloped by the mother cell, whereas those sporulated at higher water activity levels were distinct from the mother cell. Additionally, spores sporulated at higher water activity levels exhibited higher wet density in Percoll gradient. The levels of germination were also compared after sporulation under different water activity medium using L-alanine. The spores evolved under low water activity exhibited higher germination level compared to those from high water activity conditions. These findings suggested that water activity conditions during sporulation induce variations in morphology, density, germination level, and resistance to 915 MHz microwave. Our experimental results indicated that, even with the same sporulation percentage, there can be differences in the maturation stage, indicating that the level of 915 MHz microwave treatment should be adjusted according to the sporulation conditions of B. cereus.

Characteristics of Corynespora cassiicola, the causal agent of tobacco Corynespora leaf spot, revealed by genomic and metabolic phenomic analysis

Corynespora cassiicola is a highly diverse fungal pathogen that commonly occurs in tropical, subtropical, and greenhouse environments worldwide. In this study, the isolates were identified as C. cassiicola, and the optimum growth and sporulation were studied. The phenotypic characteristics of C. cassiicola, concerning 950 different growth conditions, were tested using Biolog PM plates 1–10. In addition, the strain of C. cassiicola DWZ from tobacco hosts was sequenced for the using Illumina PE150 and Pacbio technologies. The host resistance of tobacco Yunyan 87 with different maturity levels was investigated. In addition, the resistance evaluation of 10 common tobacco varieties was investigated. The results showed that C. cassiicola metabolized 89.47% of the tested carbon source, 100% of the nitrogen source, 100% of the phosphorus source, and 97.14% of the sulfur source. It can adapt to a variety of different osmotic pressure and pH environments, and has good decarboxylase and deaminase activities. The optimum conditions for pathogen growth and sporulation were 25–30 °C, and the growth was better on AEA and OA medium. The total length of the genome was 45.9 Mbp, the GC content was 51.23%, and a total of 13,061 protein-coding genes, 202 non-coding RNAs and 2801 and repeat sequences were predicted. Mature leaves were more susceptible than proper mature and immature leaves, and the average diameter of diseased spots reached 17.74 mm at 12 days. None of the tested ten cultivars exhibited obvious resistance to Corynespora leaf spot of tobacco, whereby all disease spot diameters reached > 10 mm and > 30 mm when at 5 and 10 days after inoculation, respectively. The phenotypic characteristics, genomic analysis of C. cassiicola and the cultivar resistance assessment of this pathogen have increased our understanding of Corynespora leaf spot of tobacco.

Isolation, identification, biological characteristics, and pathogenicity of an entomogenous fungus against the Egyptian mealybug, Icerya aegyptiaca (J.) (Hemiptera: Monophlebidae)

BackgroundIn this study, an entomogenous, fungus was isolated from the Egyptian mealybug, Icerya aegyptiaca (J.) (Hemiptera: Monophlebidae) on the parasol leaf tree, Macaranga tanarius, in China where evaluated as a biocontrol fungus to reduce the population of the target insect. The strain was identified as Aspergillus parasiticus by morphological and phylogenetic analysisand named ZHKUAP1. The biological characteristics, pathogenicity, and field control effect of the strain were determined.ResultsThe most suitable medium for the mycelial growth of strain ZHKUAP1 was PPDA medium, with an optimum temperature of 30 °C and pH 7, in addition to glucose and peptone as carbon and nitrogen sources. The optimum sporulation conditions were the PPDA medium at 30 °C and pH 6, using the soluble starch and beef extract as carbon and nitrogen sources. The mycelial growth and spore production of strain ZHKUAP1 were stopped at 70 °C and above, indicating that it was not resistant to high temperatures. High concentrations of spore suspension, against young insect age, resulted high corrected mortality, as well as decreased the median lethal time. When the spore concentration was 1 × 108 cfu/ml, the corrected mortality of the second nymph was 88.33%, and the LT50 was 0.66 day. After 10 days of inoculation, the LC50 of the second instar nymph was the smallest, reaching 4.07 × 104 cfu/ml. On the 10th day of the field experiment, the corrected mortality was 76.45%, indicating that the A. parasiticus strain ZHKUAP1 had strong pathogenicity on I. aegyptiaca population.ConclusionsThe indoor toxicity of the strain to I. aegyptiaca was determined, and the field control effect of the pathogen was explored on this basis. The results have important application prospects in the biological control of I. aegyptiaca.

Biological, physical and morphological factors for the programming of a novel microbial hygromorphic material

The urgency for energy efficient, responsive architectures has propelled smart material development to the forefront of scientific and architectural research. This paper explores biological, physical, and morphological factors influencing the programming of a novel microbial-based smart hybrid material which is responsive to changes in environmental humidity. Hygromorphs respond passively, without energy input, by expanding in high humidity and contracting in low humidity. Bacillus subtilis develops environmentally robust, hygromorphic spores which may be harnessed within a bilayer to generate a deflection response with potential for programmability. The bacterial spore-based hygromorph biocomposites (HBCs) were developed and aggregated to enable them to open and close apertures and demonstrate programmable responses to changes in environmental humidity. This study spans many fields including microbiology, materials science, design, fabrication and architectural technology, working at multiple scales from single cells to ‘bench-top’ prototype.Exploration of biological factors at cellular and ultracellular levels enabled optimisation of growth and sporulation conditions to biologically preprogramme optimum spore hygromorphic response and yield. Material explorations revealed physical factors influencing biomechanics, preprogramming shape and response complexity through fabrication and inert substrate interactions, to produce a palette of HBCs. Morphological aggregation was designed to harness and scale-up the HBC palette into programmable humidity responsive aperture openings. This culminated in pilot performance testing of a humidity-responsive ventilation panel fabricated with aggregated Bacillus HBCs as a bench-top prototype and suggests potential for this novel biotechnology to be further developed.

Optimization of Bacillus velezensis S26 sporulation for enhanced biocontrol of gray mold and anthracnose in postharvest strawberries

The growing demand for organic and pesticide-free food has prompt the adoption of biological strategies for controlling postharvest diseases. While strawberries are appreciated for their sensory characteristics and nutritional value, the pseudofruit are highly susceptible to fungal infections, leading to potential contamination with chemical residues. Bacillus spp. are endospore-forming bacteria that offer an alternative for managing various plant diseases. The present study aimed to investigate the effectiveness of both Bacillus velezensis S26 endospores and endospore-rich inoculant in controlling anthracnose and gray mold in strawberries. Initially, the sporulation process was fine-tuned, followed by the utilization of vegetative cells and endospores for in vitro assays. Subsequently, in vivo assays were performed using an endospore-rich inoculant on strawberries. The optimized sporulation conditions led to the production of 10.3 log10 spores mL−1 after 16 h of cultivation (sporulation yield of 96%). Both the vegetative cells and endospores of B. velezensis S26 inhibited mycelial growth and conidial germination of Botrytis cinerea and Colletotrichum spp., which are the causal agents of gray mold and anthracnose, respectively. While the cell viability of endospores decreased after storage, fresh and stored inoculants proved effective in controlling anthracnose and gray mold during storage. Consequently, the endospores of B. velezensis S26 demonstrated their viability and antagonistic potential against postharvest diseases in strawberries, thereby contributing to an extended bacterial shelf life.

Analysis of growth dynamics in five different media and metabolic phenotypic characteristics of Piriformospora indica.

Piriformospora indica is an important endophytic fungus with broad potential for alleviating biotic and abiotic stress on host plants. This study monitored the growth dynamics of P. indica on five commonly used artificial media for microorganisms and analyzed its metabolic characteristics using Biolog Phenotype Microarray (PM) technology. The results showed that P. indica grew fastest on Potato Dextrose Agar (PDA), followed by Kidney Bean Agar (KBA), Alkyl Ester Agar (AEA), Oatmeal Agar (OA), and Luria-Bertani Agar (LB), and the most suitable medium for spore production was OA. Using Biolog PM1-10, 950 metabolic phenotypes of P. indica were obtained. P. indica could metabolize 87.89% of the tested carbon sources, 87.63% of the tested nitrogen sources, 96.61% of the tested phosphorus sources, and 100% of the tested sulfur sources. P. indica displayed 92 kinds of tested biosynthetic pathways, and it could grow under 92 kinds of tested osmotic pressures and 88 kinds of tested pH conditions. PM plates 1-2 revealed 43 efficient carbon sources, including M-Hydroxyphenyl acid, N-Acetyl-D-Glucosamine, Tyramine, Maltotrios, α-D-Glucosine, I-Erythritol, L-Valine, D-Melezitose, D-Tagatose, and Turanose. PM plates 3,6-8 indicated 170 efficient nitrogen sources, including Adenosine, Inosine Allantoin, D, L-Lactamide, Arg-Met, lle-Trp, Ala-Arg, Thr-Arg, Trp-Tyr, Val-Asn, Gly-Gly-D-Leu, Gly-Gly-Phe, and Leu-Leu-Leu. This study demonstrates that P. indica can metabolize a variety of substrates, such as carbon and nitrogen sources, and has a wide range of environmental adaptability. The growth dynamics on artificial culture media and metabolic phenotypes of P. indica can be used to investigate its biological characteristics, screen for more suitable growth and sporulation conditions, and elucidate the physiological mechanisms that enhance the stress resistance of host plants. This study provides a theoretical basis for its better application in agriculture.

Coniolariella gamsii Causes Poplar Leaf Spot Disease in Xinjiang, China

Populus laurifolia is one of the most valuable tree species in the world and an important silvicultural tree species in the Xinjiang Uygur Autonomous Region, China. In July 2017, an unreported brown leaf spot disease was observed on P. laurifolia in Altay City, Xinjiang. The causal agent of this leaf spot disease was isolated, and Koch’s postulates were performed to confirm its pathogenicity. Based on a morphological characterization and phylogenetic analyses, the causal organism was identified to be a fungal species, Coniolariella gamsii. The optimum mycelial growth conditions for C. gamsii are on PLPDA (Populus leaves potato dextrose agar) medium, at 28 °C, in the dark. The sporulation time when using PLPDA medium (12 days) is much less than that for PDA medium (25 days). Pathogenicity tests revealed that C. gamsii can also infect two other Populus species (P. bolleana and P. tomentosa). This is the first report of C. gamsii causing brown leaf spot disease on P. laurifolia, and the optimum culture and sporulation conditions have also been optimized for the first time. This study provides a theoretical basis for the diagnosis of this disease and the monitoring of the disease’s occurrence and epidemic status.

Seasonal Periodicity of the Airborne Spores of Fungi Causing Grapevine Trunk Diseases: An Analysis of 247 Studies Published Worldwide.

Grapevine trunk diseases (GTDs) are among the most devastating grapevine diseases globally. GTDs are caused by numerous fungi belonging to different taxa, which release spores into the vineyard and infect wood tissue, mainly through wounds caused by viticultural operations. The timing of operations to avoid infection is critical concerning the periodicity of GTD spores in vineyards, and many studies have been conducted in different grape-growing areas worldwide. However, these studies provide conflicting and fragmented information. To synthesize current knowledge, we conducted a systematic literature review, extracted quantitative data from published papers, and used these data to identify trends and knowledge gaps that need to be addressed in future studies. Our database included 26 papers covering 247 studies and 3,529 spore sampling records concerning a total of 29 fungal taxa responsible for Botryosphaeria dieback (BD), Esca complex (EC), and Eutypa dieback (ED). We found a clear seasonality in the presence and abundance of BD spores, with a peak from fall to spring, more in the northern hemisphere than in the southern hemisphere, but not for EC and ED. Spores of these fungi were present throughout the growing season in both hemispheres, possibly because of higher variability in spore types, sporulation conditions, and spore release mechanisms in EC and ED fungi than in BD. Our analysis has limitations because of knowledge gaps and data availability for some fungi (e.g., basidiomycetes, which cause EC). These limitations are discussed to facilitate further research.

Genomic Approaches and Analyses of Slow-Growing Obligate Iron-Metabolizing Microbes

The biogeochemical cycling of iron is a vastly important process that has been a major factor defining life on Earth both before and after the Great Oxidation Event. While both abiotic and biotic factors contributing to the iron cycle have been studied for many years, the bulk of studies on iron metabolizing organisms has focused on a select few, easily manipulated model organisms. Recent discoveries have identified several unique and difficult to work with organisms from iron rich environments that survive solely on iron as either an electron acceptor or donor. The Fe(III)-reducing, Gram-positive Firmicute Metallumcola ferriviriculae MK1 was recently isolated from the Soudan Underground Mine in northern Minnesota from brine waters that intersect 2.7 Ga banded iron formations within the Canadian Shield. M. ferriviriculae MK1, which grows anaerobically using Fe(III)-citrate as its sole electron acceptor, is also mesophilic, spore-forming, culturable, and rich in multiheme cytochromes. Multiheme cytochromes are a well-established mechanism for Fe(III) reduction among the model Gram-negative microbes, such as Shewanella oneidensis and Geobacter sulfurreducens, but is poorly studied in Gram-positives. While the slow growth times of M. ferriviriculae MK1 make it difficult to study in the laboratory, the genome encodes homologs to multiheme cytochromes utilized by G. sulfurreducens for Fe(III) reduction. Specifically, two gene clusters (MK1_2258-2259 and MK1_2264-2265) each encode proteins homologous to the b-type cytochrome domain (60% and 61% sequence similarity, respectively) and c-type cytochrome domain (44.9% and 47.14%, respectively) of cbcL, which is used for reduction of mid-range redox potential acceptors in G. sulfurreducens and MK1_1670 is homologous to imcH (54.3% sequence similarity), which is used for higher redox potential acceptors. Additionally, the MK1 genome contains genes associated with sporulation, including genes encoding the master sporulation regulator spo0A, the peptidoglycan remodeling enzymes spoIID, spoIIP, and spoIIM, the spore morphogenesis protein spoIVA, and the small, acid-soluble spore proteins sspA, sspB, sspC, sspD, and sspF. Extraction and isolation of MK1 spores will facilitate evaluation of sporulation and germination conditions to shed light on a crucial preservation mechanism from an organism found in an environment with limited nutrients. Further evaluation into this novel organism can also give us insights into the microbial impacts on the iron cycle in the deep terrestrial biosphere. Another microbe of interest that was isolated from an iron-rich environment is the obligate Fe(II)-oxidizing Mariprofundus ferrooxydans PV-1, which was enriched from iron-rich mats associated with hydrothermal vents at the Kama’ehuakanaloa Seamount (previously Lo’ihi) in Hawaii. M. ferrooxydans PV-1 is a stalk-forming Fe(II)-oxidizing microbe and the first Zetaproteobacterium characterized. While M. ferrooxydans PV-1 can reliably be grown (doubling time of ~12 hours) in liquid medium traditional genetic methods are challenging because it does not make colonies on agar plates. To identify essential and non-essential genes, we developed a conjugation method using E. coli and successfully generated a transposon library in M. ferrooxydans. Libraries were grown under kanamycin selection with Fe(II)-chloride as the sole electron donor and samples isolated for analysis at two different timepoints. Deep sequencing of the mutant population at these timepoints was performed and the reads were mapped back to the M. ferrooxydans PV-1 genome to identify transposon insertion sites. Initial evaluation of the reads has identified 31 transposon insertion sites found within both the first- and second-generation populations, with 21 hits representing stable insertions in non-essential genes, including a predicted malate dehydrogenase (SPV1_772) and a predicted phospholipase (SPV1_8286), that may be useful future targets for gene insertion sites. Further evaluation comparing the read depth between the generations should identify genes that were selected for or against during the subculturing. These studies and approaches are providing insights into the roles these challenging, non-model organisms are performing in iron-rich environments, which provides a better context for the biogeochemical cycling of iron in relevant biospheres.

Factors Affecting Spore Formation of Carrot Leaf Blight Caused by Alternaria dauci In Vitro

In order to examine the pathogenicity of <i>Alternaria dauci</i>, the causal agent of carrot leaf blight, it is necessary to standardize sporulation conditions to ensure the optimal quantity and quality of spore inoculum. Therefore, in this study, the effects of the growth medium, aeration treatment, and UV treatment with 12-hr photoperiod on the sporulation of <i>A. dauci</i> KACC42997 were investigated. <i>A. dauci</i> KACC42997 was pre-cultured for 7 days in a potato dextrose agar medium at 25oC in the dark condition. When the pre-culture, after removing aerial mycelia, was re-incubated for 5 days, with simultaneous aeration treatment and 12-hr cycle UV treatment at 20oC, the largest number of spores was produced. One hundred seventy isolates of <i>A. dauci</i> were isolated from major carrot growing regions such as Pyeongchang, Gumi and Jeju and tested for sporulation under the conditions established in this study. Except for 20 strains, all strains produced spores. Statistically significant differences in the extent of sporulation were found among local populations of <i>A. dauci</i> isolates, but no difference was observed in their pathogenicity on carrots.

Sporulation conditions influence the surface and adhesion properties of Bacillus subtilis spores.

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca2+ or Mg2+ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca2+ or Mg2+ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.

Rie1 and Sgn1 form an RNA-binding complex that enforces the meiotic entry cell fate decision.

Budding yeast cells have the capacity to adopt few but distinct physiological states depending on environmental conditions. Vegetative cells proliferate rapidly by budding while spores can survive prolonged periods of nutrient deprivation and/or desiccation. Whether or not a yeast cell will enter meiosis and sporulate represents a critical decision that could be lethal if made in error. Most cell fate decisions, including those of yeast, are understood as being triggered by the activation of master transcription factors. However, mechanisms that enforce cell fates posttranscriptionally have been more difficult to attain. Here, we perform a forward genetic screen to determine RNA-binding proteins that affect meiotic entry at the posttranscriptional level. Our screen revealed several candidates with meiotic entry phenotypes, the most significant being RIE1, which encodes an RRM-containing protein. We demonstrate that Rie1 binds RNA, is associated with the translational machinery, and acts posttranscriptionally to enhance protein levels of the master transcription factor Ime1 in sporulation conditions. We also identified a physical binding partner of Rie1, Sgn1, which is another RRM-containing protein that plays a role in timely Ime1 expression. We demonstrate that these proteins act independently of cell size regulation pathways to promote meiotic entry. We propose a model explaining how constitutively expressed RNA-binding proteins, such as Rie1 and Sgn1, can act in cell fate decisions both as switch-like enforcers and as repressors of spurious cell fate activation.

Comparative study on the impact of equally stressful environmental sporulation conditions on thermal inactivation kinetics of B. subtilis spores

Control of bacterial spores continues to be one of the main challenges for the food industry due to their wide dissemination and extremely high resistance to processing methods. Furthermore, the large variability in heat resistance in spores that contaminate foods makes it difficult to establish general processing conditions. Such heterogeneity not only derives from inherent differences among species and strains, but also from differences in sporulation environments that are generally ignored in spores encountered in foods. We evaluated heat inactivation kinetics and the thermodependency of resistance parameters in B. subtilis 168 spores sporulated at adverse temperatures, water activity (aw), and pH, applying an experimental approach that allowed us to quantitatively compare the impact of each condition. Reduction of incubation temperature from the optimal temperature dramatically reduced thermal resistance, and it was the most influential factor, especially at the highest treatment temperatures. These spores were also more sensitive to chemicals presumably acting in the inner membrane. Reducing sporulation aw increased heat resistance, although the magnitude of that effect depended on the solute and the treatment temperature. Thus, changes in sporulation environments varied 3D100°C values up to 10.4-fold and z values up to 1.7-fold, highlighting the relevance of taking such a source of variability into account when setting heat processing conditions. UV-C treatment and sodium hypochlorite efficiently inactivated all spore populations, including heat-resistant ones produced at low aw.

Comparison of sporulation and germination conditions for Clostridium perfringens type A and G strains.

Clostridium perfringens is a spore forming, anaerobic, Gram-positive bacterium that causes a range of diseases in humans and animals. C. perfringens forms spores, structures that are derived from the vegetative cell under conditions of nutrient deprivation and that allows survival under harsh environmental conditions. To return to vegetative growth, C. perfringens spores must germinate when conditions are favorable. Previous work in analyzing C. perfringens spore germination has produced strain-specific results. Hence, we analyzed the requirements for spore formation and germination in seven different C. perfringens strains. Our data showed that C. perfringens sporulation conditions are strain-specific, but germination responses are homogenous in all strains tested. C. perfringens spores can germinate using two distinct pathways. The first germination pathway (the amino acid-only pathway or AA) requires L-alanine, L-phenylalanine, and sodium ions (Na+) as co-germinants. L-arginine is not a required germinant but potentiates germination. The AA pathway is inhibited by aromatic amino acids and potassium ions (K+). Bicarbonate (HCO3-), on the other hand, bypasses potassium-mediated inhibition of C. perfringens spore germination through the AA pathway. The second germination pathway (the bile salt / amino acid pathway or BA) is more promiscuous and is activated by several bile salts and amino acids. In contrast to the AA pathway, the BA pathway is insensitive to Na+, although it can be activated by either K+ or HCO3-. We hypothesize that some C. perfringens strains may have evolved these two distinct germination pathways to ensure spore response to different host environments.

Effects of sporulation conditions on the growth, germination, and resistance of Clostridium perfringens spores

Clostridium perfringens can form metabolically dormant spores that can survive in meat preservation processes and cause food spoilage and human disease upon germination and outgrowth. The characteristics of spores in food products are closely related to the sporulation environment. To control or inactivate C. perfringens spores in food industry, the effects of sporulation conditions on the spores characteristics should be examined. This study aimed to investigate the effects of temperature (T), pH, and water activity (aw) on the growth, germination, and wet-heat resistance of C. perfringens C1 spores isolated from food product. The results showed that C. perfringens C1 spores produced at T = 37 °C, pH = 8, and aw = 0.997 had the highest sporulation rate and germination efficiency and lowest wet-heat resistance. A further increase in pH and sporulation temperature reduced the spore counts and germination efficiency, but enhanced spores' wet-heat resistance. By using air-drying method and Raman spectroscopy analysis, the water content, composition, and levels of calcium dipicolinic acid, proteins, and nucleic acids in spores produced under different sporulation conditions were determined. The results obtained revealed that sporulation conditions should be carefully considered during food production and processing, thus providing a novel insight into prevention and control of spores in food industry.

Artificial activation of both σH and Spo0A in Bacillus subtilis enforced initiation of spore development at the vegetatively growing phase.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σH are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σH -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σA. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σA expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σA did not change drastically throughout growth. Mechanisms that sequester σA from the core RNA polymerase and help σH to become active exist, but this has not yet been elucidated.

Optimization of Sporulation Conditions for Bacillus subtilis BSNK-5

Bacillus subtilis spores have important biological applications; however, high spore-cell densities and sporulation efficiencies in fermentation is poorly reported. This study systematically analyzed the spore densities and formation efficiency of B. subtilis BSNK-5 in different culture substrates. A response surface regression equation was established based on the results of single factor and Box–Behnken experimental designs. The optimal medium formulation, as predicted from the equation, consisted of soluble starch at 3 g·L−1, soybean flour at 12 g·L−1, and MgSO4 at 5 g·L−1. The spore yield reached 2.43 × 109 CFU·mL−1, and the sporulation rate was 83.3%, which was nearly three times higher than before optimization using an optimized medium at 36 °C and 200 rpm for 60 h.

Morpho-cultural, physiological and molecular characterisation of Colletotrichum nymphaeae causing anthracnose disease of walnut in China

Anthracnose disease has harmed walnut in recent years, resulting in yield losses in China. As a results, both morphological and molecular techniques must be used to confirm the etiology of anthracnose on walnut. In May 2020, walnut branches with indications of anthracnose were gathered in Ganquan, China (N33°56'/E105°44′). A strain named JT20 was isolated and morphologically characterized as a Colletotrichum specie. Pathogenicity tests further confirmed that the strain caused apparent anthracnose symptoms on walnut which were consistent with those seen in the field. On PDA, colonies were gray, cotton wool-like on the surface and pale gray to pale orange on the dorsal side. Conidia were aseptate, hyaline, fusiform to cylindrical with rounded to pointy ends and a length of 5.52–9.30 × 2.18–4.61 μm. PDA, lactose, yeast extract, pH 6–8, temperature of 25 °C and complete darkness were shown to be the optimum culture conditions for surface mycelium growth while PSA, lactose, urea, pH 9, temperature of 30 °C and complete darkness were found to be the best conditions for pathogen sporulation. The isolate was deduced as based on phylogenetic analysis with 3 genes (ribosomal DNA-ITS, ACT and GAPDH) as well as morphological characteristics and cultural features, the isolate was identified C. nymphaeae. This is the first report of C. nymphaeae causing walnut anthracnose in China.