Sporulation Temperature 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.

Optimisation of Saccharomyces cerevisiae BRYC 501 ascospore formation and recovery for heat inactivation experiments

Why was the work done: Ascospores from Saccharomyces cerevisiae BRYC 501 are useful biological indicators for validating the pasteurisation of beer. Ascospores are formed by yeast in response to low nutrient conditions and are characterised by greater resistance to stressors including desiccation, freezing and, during pasteurisation, high temperatures. Accordingly, the sporulation temperature of yeast ascospores, their shelf life, heat resistance and recovery need to be investigated. How was the work done: The role of temperature in sporulation was investigated at 25, 27 and 30°C by measurement of the rate of sporulation over ten days. Shelf life of ascospores was established in deionised water at 1-5°C over 120 days by the measurement of heat resistance in McIlvaine's buffer. The recovery conditions of ascospores were determined using six agars after heat inactivation experiments in McIlvaine's buffer, alcohol free and lager beer. What are the main findings: The sporulation rate of S. cerevisiae ascospores was highest at 27°C, and lowest at 30°C. Heat resistance of ascospores formed at 30°C was low with a D60 value of <3 minutes, whereas at 25 and 27°C the D60 was 8.8 and 9.2 minutes. The shelf life of ascospores was 60 days after an initial 14 days of maturation at both 25 and 27°C. The variation of heat resistance for ascospores grown at 25°C was shown to be lower than those grown at 27°C. Use of yeast extract-peptone-glucose agar to recover heat injured cells was > 200% higher than with the control agar. Why is the work important: Sporulation should be performed at 25°C to produce the maximum number of spores for heat inactivation experiments. Spores can be maintained for 60 days, enabling several experiments to be performed. After heat inactivation experiments, ascospores should be recovered on YEPG agar for 10 days at 27°C

Resistance of Alicyclobacillus acidoterrestris spores to atmospheric cold plasma: Insights from sporulation temperature and mechanism analysis

Environmental conditions during sporulation, such as temperature, pH value, and mineral content, can significantly influence the formation production and resistance of spores. In this study, we investigated the sporulation capacity and resistance of Alicyclobacillus acidoterrestris (AAT) spores obtained at different temperatures (25 °C, 35 °C, 45 °C) to Dielectric Barrier Discharge Atmospheric cold plasma (DBD-ACP, 20 and 30 kV for 2–8 min). The results indicated that the efficiency of sporulation was highest at the optimum temperature (45 °C), deviation from optimal temperature would have a negative effect on sporulation. Otherwise, sporicidal efficiency of DBD-ACP increased with longer treatment time and lower sporulation temperatures, suggesting that AAT spores exhibit weaker resistance to DBD-ACP at lower sporulation temperature. Results from the release of DPA, and electron microscopic observations confirmed that DBD-ACP caused disruption of the cortex and membrane structure of AAT spores, leading to the release of intracellular contents and subsequent spore's death. Further analysis through proteomics revealed intriguing insights into the differences between spores formed at 25 °C and those at 45 °C. Specifically, spores formed at 25 °C exhibited lower expression levels of proteins related to sporulation, intracellular energy metabolism, membrane transport, and significant down-regulation of proteins involved in peptidoglycan and spore coat formation compared to those at 45 °C. These alterations likely contribute to the development of a thinner and looser spore structure, rendering it less effective in defending, i.e. less resistant to DBD-ACP treatment. Industrial relevanceAAT spores' resistance to pasteurization and persistence in NFC juices impact juice quality and safety. This study investigates the resistance of AAT spore to DBD-ACP treatment under different sporulation temperatures. Insights into sporicidal behavior of DBD-ACP and the effect of sporulation temperature on resistance provide essential knowledge for optimizing juice processing and enhancing microbial safety. The findings offer practical guidance for the juice industry to develop improved preservation strategies, ensuring longer shelf life and consumer satisfaction.

Impact of sporulation temperature on germination of Bacillus subtilis spores under optimal and adverse environmental conditions

Bacillus subtilis spores are important food spoilage agents and are occasionally involved in food poisoning. In foods that are not processed with intense heat, such bacterial spores are controlled by a combination of different hurdles, such as refrigeration, acidification, and low water activity (aw), which inhibit or delay germination and/or growth. Sporulation temperature has long been regarded as a relevant factor for the assessment of germination in chemically defined media, but little is known about its impact on food preservation environments. In this study, we compared germination dynamics of B. subtilis spores produced at optimal temperature (37 °C) with others incubated at suboptimal (20 °C) and supraoptimal (43 °C) temperatures in a variety of nutrients (rich-growth medium, L-alanine, L-valine, and AGFK) under optimal conditions as well as under food-related stresses (low aw, pH, and temperature). Spores produced at 20 °C had a lower germination rate and efficiency than those incubated at 37 °C in all the nutrients, while those sporulated at 43 °C displayed a higher germination rate and/or efficiency in response to rich-growth medium and mostly to L-alanine and AGFK under optimal environmental conditions. However, differences in germination induced by changes in sporulation temperature decreased when spores were activated by heat, mainly due to the greater benefit of heat for spores produced at 20 °C and 37 °C than at 43 °C, especially in AGFK. Non-heat-activated spores produced at 43 °C still displayed superior germination fitness under certain stresses that had considerably impaired the germination of the other two populations, such as reduced temperature and aw. Moreover, they presented lower temperature and pH boundaries for the inhibition of germination in rich-growth medium, while requiring a higher NaCl concentration threshold compared to spores obtained at optimal and suboptimal temperature. Sporulation temperature is therefore a relevant source of variability in spore germination that should be taken into account for the accurate prediction of spore behaviour under variable food preservation conditions with the aim of improving food safety and stability.

A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.

Identification and characterization of pathogens causing saffron corm rot in China.

Corm rot is the most important disease of saffron, for which fungi from several genus such as Fusarium spp. Penicillium spp. and Botrytis spp., have been previously reported to be the pathogens. In this research, we used a combination of amplicon sequencing and traditional isolation methods to identify the causal agents, main infection source. The diversity of microbial communities in diseased saffron corms and soil decreased significantly compared with healthy corms and soil. The contents of Penicillium and Botrytis in healthy and diseased corms were similarly high, indicating that them were not directly related to the occurrence of corm rot. But the relative abundance of Fusarium, Cadophora and Fusicolla were significantly higher in the diseased corms than healthy ones. The abundance of Fusarium increased, while the abundance of Oidiodendron, Paraphaeosphaeria and the endophytic beneficial bacteria Pseudomonas decreased, which may relate to the occurrence of the disease. The co-occurrence network diagram showed that the correlation between fungal and bacterial communities was mainly positive. Plant pathogens were relatively abundant in the diseased soil, according to functional gene prediction. At the same time, we also collected 100 diseased corms from the fields in Jiande, where is known as the "hometown of saffron." All isolated pathogenic strains were identified as Fusarium oxysporum through morphological observation and phylogenetic tree analysis of ITS, Tef-1α and β-tubulin. To better clarify the biological characteristics of F. oxysporum, we cultured the isolates at different temperatures and pH values. The optimum temperature for mycelial growth and sporulation was 25°C, pH 6，carbon sources sorbitol and nitrogen sources, peptone. In short, our results suggests that F. oxysporum was the pathogen causing corm rot in Jiande and corms other than soils are the main primary infection source. These new understanding of saffron corm rot will provide the theoretical basis for its better and efficiently management.

Isolation and identification of the endophytic fungus J2-3 and its disease-preventive and growth-promoting effects on cucumber.

There are many problems that result from the use of a large number of chemical pesticides to control plant diseases, including pathogenic bacteria resistance, environmental contamination, and human health effects. Recently, endophytic fungi have become a significant source of bioactive fungicide products and an invaluable resource for excavating microbial pesticides. In this study, endophytic fungi with biocontrol potential were isolated and screened from Mikania micrantha leaves, stems, and roots. Fifty endophytic fungi were isolated and their antagonistic activity was studied in vitro using the confrontation culture method. The J2-3 strains from stems exhibit broad-spectrum and high activity. The strain's biological characteristics were determined by various culture conditions, and it was identified as Fusarium proliferatum by both morphological and ITS sequence analysis. Biological characteristics of the J2-3 strain were also tested. The optimum temperature for mycelium growth and sporulation was 25°C and 30°C, respectively. For mycelium growth, starch was the optimum carbon source, and peptone was the optimum nitrogen source for sucrose, mycelium growth, and sporulation. Mycelium growth was killed by a temperature of 60°C, and sporulation was killed by a temperature of 55°C. The light aided mycelium growth, and the light alternated between light and dark cycles for sporulation. Further, pot experiments were conducted to determine the antagonistic and viable effects of highly antagonistic strains on cucumber. The spore suspension's final control efficacy on cucumber wilt disease was up to 62.79% and it also promoted cucumber growth significantly. The results show that the entophytic fungus J2-3 from M. micrantha can protect cucumbers from wilt disease and promote growth.

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.

CotG controls spore surface formation in response to the temperature of growth in Bacillus subtilis.

SummaryBacterial spores of the Bacillus genus are ubiquitous in nature and are commonly isolated from a variety of diverse environments. Such wide distribution mainly reflects the spore resistance properties but some Bacillus species can grow/sporulate in at least some of the environments where they have been originally isolated. Growing and sporulating at different conditions is known to affect the structure and the resistance properties of the produced spore. In B. subtilis the temperature of growth and sporulation has been shown to influence the structure of the spore surface throughout the action of a sporulation‐specific and heat‐labile kinase CotH. Here we report that CotG, an abundant component of the B. subtilis spore surface and a substrate of the CotH kinase, assembles around the forming spore but also accumulates in the mother cell cytoplasm where it forms aggregates with at least two other coat components. Our data suggest that the thermo‐regulator CotH contributes to the switch between the coat of 25°C and that of 42°C spores by controlling the phosphorylation levels of CotG that, in turn, regulates the assembly of at least two other coat components.

Insights into the Structure and Protein Composition of Moorella thermoacetica Spores Formed at Different Temperatures.

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores.

Geobacillus stearothermophilus STCC4517 spore suspensions showed survival curves with shoulder phenomena independent of sporulation temperature and pH, whose duration was an exponential function of treatment temperature.

Deviations in linearity in survival curves are common in inactivation kinetics during heat treatment. These might lead one to underestimate how effective thermal treatment is. In previous research we reported a relationship between decimal reduction time values (DT) and shoulder lengths (Sl) of survival curves which was characteristic of each microorganism. However, the impact of other factors such as sporulation temperature and pH of the treatment media on shoulder length is still not known. The objective of this research was to evaluate the effect of sporulation temperature (45, 55 and 65 °C) and pH (4.0, 5.0, 6.0 and 7.0) treatment has on the profile of survival curves and on the relationship between Sl/DT of G. stearothermophilus STCC 4517. The results obtained demonstrated that all the spore suspensions, independently of sporulation temperature and pH, showed survival curves with shoulder phenomena, whose duration was an exponential function of treatment temperature. Although both parameters had a significant effect on heat resistance, the relationship between the shoulder length and DT values was constant at all pHs for spores produced at the optimum sporulation temperature.

Warm wheat does not rust - how wheat resists stripe rust when temperatures rise.

Stripe rust is a destructive wheat disease that is estimated to cause 5.47 million tonnes of wheat loss per year (Beddow et al., 2015). The fungus that causes the disease, Puccinia striiformis f. sp. tritici (Pst), occurs in all wheat-growing regions of the world but causes more problems in cooler climates. One reason for this is that the fungus requires relatively low temperatures for spore germination, growth, and sporulation. But some wheat plants also have a defense mechanism against Pst that only operates under high temperatures. Two types of temperature-sensitive defense mechanisms are known: high-temperature seedling plant resistance and high-temperature adult plant resistance. When grown under high-temperature conditions, wheat varieties with such a defense mechanism are resistant against Pst. Because high-temperature resistance provides immunity to all Pst races, understanding the genetic basis of this defense mechanism might be valuable for breeding. Over the last decades, breeders have developed resistant cultivars by deploying race-specific resistance genes. Race-specific resistant cultivars have limited durability because pathogens evolve quickly. In the case of Pst, new races have overcome most of the known race-specific resistance genes (Fu et al., 2009). Therefore, breeding for non-race-specific resistance such as high-temperature resistance might be a more durable solution in the fight against stripe rust. Xiaoping Hu, Professor of Plant Pathology at Northwest Agricultural and Forestry University in Yangling, China, is trying to understand the molecular mechanisms involved in high-temperature resistance in seedlings. His research group performed transcriptomic analysis on the resistant cultivar Xiaoyan 6 that had been inoculated with Pst and grown under high temperatures. Among the differentially expressed genes, they found a gene encoding a cysteine-rich receptor-like kinase that they designated TaCRK10. In this issue of The Plant Journal, the researchers investigated the role of this gene (Wang et al., 2021). To investigate if TaCRK10 is involved in resistance to Pst, the authors performed virus-induced gene silencing (VIGS) in the Xiaoyan 6 cultivar and inoculated the TaCRK10-silenced plants with Pst. When plants started to show symptoms of stripe rust, they were divided into two groups. One group was placed under high-temperature conditions (20°C) for 24 h, whereas the other group was kept at normal temperatures (16°C). In the TaCRK10-silenced plants grown under high temperatures, there was abundant spore formation on the leaves, whereas non-silenced plants showed only limited sporulation. Under normal temperatures, control plants and silenced plants did not show differences. These results suggest that TaCRK10 is required for high-temperature seedling plant resistance to Pst in the Xiaoyan 6 cultivar. Furthermore, when they overexpressed TaCRK10 from Xiaoyan 6 in the susceptible wheat cultivar Fielder, then inoculated the overexpression lines with Pst, sporulation was much lower than in wild-type plants. Interestingly, the overexpression lines were resistant against Pst when grown at normal temperatures. Further analysis of these lines revealed expression of genes involved in the salicylic acid defense pathway as well as accumulation of reactive oxygen species (ROS), which are common signs of an immunity response. To deduce the molecular action of TaCRK10, the authors performed a yeast-two-hybrid assay using a library constructed from Pst-infected leaves of the Xiaoyan 6 cultivar grown at high temperature. They found that TaCRK10 interacted with the histone variant TaH2A.1. Because TaCRK10 contains a serine/threonine kinase-like domain, the authors wondered if TaCRK10 might phosphorylate histone TaH2A.1. To test this, the researchers produced recombinantly expressed proteins and mixed them in the presence of ATP. Using an electrophoresis technique that separates phosphorylated and non-phosphorylated proteins followed by Western blot analysis, they found that TaH2A.1 was indeed phosphorylated in the presence of TaCRK10. To find out if H2A.1 is involved in high-temperature seedling plant resistance to Pst, the authors used VIGS to silence the histone variant. Subsequent inoculation experiments showed that sporulation was higher on TaH2A.1-silenced plants at both normal and high temperatures than on non-silenced plants. Hence, TaH2A.1 appears to be involved in wheat resistance to Pst (Figure). Xiaoping Hu wants to better understand the TaCRK10-TaH2A.1 pathway by identifying the phosphorylation sites on H2A.1 on which CRK10 acts and to determine if phosphorylation of TaH2A.1 plays a role in high-temperature resistance to stripe rust in seedlings. He explains that the findings might be used to develop wheat varieties with a durable resistance against Pst. Overexpression of CRK10 from Xiaoyan 6 in the susceptible variety Fielder resulted in plants that were resistant to the pathogen grown at normal temperatures, showing that susceptible cultivars can become resistant in this way, but also that constitutive overexpression of the allele leads to resistance that is no longer dependent on temperature.

Effect of sporulation conditions following solid-state cultivation on the resistance of Geobacillus stearothermophilus spores for use as bioindicators testing inactivation by H2O2

Geobacillus stearothermophilus spores play an essential role in the pharmaceutical and food industries as bioindicators (BI) to evaluate inactivation effects of hydrogen peroxide in processing and packaging lines' sterilization. Unstandardized, inconsistent BI production, as is the case for currently available commercial BI, can lead to variable resistances and incorrect test results. The solution to this problem could be the standardization of BI spore production. This study assessed the effects of sporulation temperature and pH on spore resistance in the production of G. stearothermophilus ATCC 7953 spores on solid-state agar. For this purpose, spores were produced under different sporulation conditions (51–63 °C, pH 6–8), and their resistance was determined by the D-value in 35% liquid hydrogen peroxide at 25 °C. Based on these data, a mathematical equation describing the resistance as a function of the sporulation conditions was derived. The analysis showed a quadratic effect of the sporulation temperature and a linear effect of the examined domain's pH on the resistance, without interactions between these factors. We observed the highest D25C,35%H2O2-value of 309 s at sporulation conditions of 57 °C and pH 6.0.

Morphophysiological characterization of Ceratocystis fimbriata isolates from yerba mate

ABSTRACT: This study aimed to morphologically characterize the isolates of Ceratocystis fimbriata from yerba mate and to evaluate the effect of culture medium and temperature on mycelial growth and sporulation of C. fimbriata. For the morphological characterization of the 11 monosporic isolates of the fungus, slides were prepared to determine the dimensions of the sexual and asexual structures of the fungus. Four experiments were conducted to evaluate the mycelial growth and to evaluate the sporulation of C. fimbriata in different culture mediums and temperatures. The isolates of C. fimbriata from yerba mate showed perithecia with brown to black necks, divergent ostiolar hyphae, hatshaped hyaline ascospores, single-celled, cylindrical endoconidia, and globular to ovoid aleurioconidia. PDA and V8-agar media showed the highest mycelial growth. The average optimum temperature for mycelial growth and sporulation of isolates of C. fimbriata of yerba mate were 22.5 and 22.4 ºC, respectively.

Diversity of water-borne conidial fungi in some freshwater bodies of Kumaun Himalaya in district Nainital (Uttarakhand), India

The aquatic ecosystem harbours a variety of micro-organisms, among which water-borne conidial fungi occupy an important place. Their occurrence in freshwater habitat has great significance in the decomposition of submerged plant materials, nutrient release and productivity. Their occurrence and frequency to extreme temperatures and pH may have a profound effect on fungal community composition and metabolic activities. The present paper deals with the diversity of conidial aquatic fungi from different unexplored freshwater bodies flowing through different elevations (1000-1500 m) in district Nainital, Kumaun Himalaya. Comparative study of species composition in different seasons, sporulation temperatures and pH conditions was also carried out. In all, 18 species of conidial fungi belonging to 14 genera were recorded, out of which maximum species (11 species) were recorded in both rainy (July to September) and winter seasons (November to December). Anguillospora crassa, Beltrania rhombica, Campylospora chaetocladia, Cylindrocarpon aquaticum, Helicomyces roseus and Tetracladium setigerum were isolated only during rainy season; Alatospora acuminata, Clavariopsis aquatica, Clavatospora tentacula, Lemonniera pseudofloscula, L. terrestris and Tetrachaetum elegans were isolated only during winter season while Lunulospora curvula, L. cymbiformis, Setosynnema isthmosporum, Tetracladium marchalianum and Triscelophorus acuminatus were isolated in both rainy and winter seasons. The preferred pH and the sporulation temperature ranged from 6-7 and 15-20 °? respectively. The results of the present study are clearly indicating fungal species composition variations along pH, temperature, seasonal and altitudinal gradients and the sites selected for this exploratory investigation are being undertaken for the first time.

Characterization and biocontrol potential of a naturally occurring isolate of Metarhizium pingshaense infecting Conogethes punctiferalis

An entomopathogenic fungus was isolated from an infected larva of Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), a highly polyphagous pest recorded from more than 120 plants and widely distributed in Asia and Oceanic countries. The fungus was identified as Metarhizium pingshaense Q.T. Chen & H.L. Guo (Ascomycota: Hypocreales) based on morphological characteristics and molecular studies. Scanning electron microscopic studies were conducted to study the infection of C. punctiferalis by M. pingshaense. Bioassay studies with purified conidial suspension proved that the isolate was highly virulent to C. punctiferalis, causing more than 86 % mortality to fifth instar larvae at 1 × 108 spores/mL, under laboratory conditions. The median lethal concentration (LC50) of the fungus against late instar larvae was 9.1 × 105 conidia/mL and the median survival time (MST) of late instar larvae tested at the doses of 1 × 108 and 1 × 107 conidia/mL were 4.7 and 6.4 days, respectively. The optimal temperature for fungal growth and sporulation was found to be 25 ± 1 °C. This is the first report of M. pingshaense naturally infecting C. punctiferalis. Isolation of a highly virulent strain of this fungus holds promise towards development of a potential mycoinsecticide against this pest.

The temperature of growth and sporulation modulates the efficiency of spore-display in Bacillus subtilis

BackgroundBacterial spores displaying heterologous antigens or enzymes have long been proposed as mucosal vaccines, functionalized probiotics or biocatalysts. Two main strategies have been developed to display heterologous molecules on the surface of Bacillus subtilis spores: (i) a recombinant approach, based on the construction of a gene fusion between a gene coding for a coat protein (carrier) and DNA coding for the protein to be displayed, and (ii) a non-recombinant approach, based on the spontaneous and stable adsorption of heterologous molecules on the spore surface. Both systems have advantages and drawbacks and the selection of one or the other depends on the protein to be displayed and on the final use of the activated spore. It has been recently shown that B. subtilis builds structurally and functionally different spores when grown at different temperatures; based on this finding B. subtilis spores prepared at 25, 37 or 42 °C were compared for their efficiency in displaying various model proteins by either the recombinant or the non-recombinant approach.ResultsImmune- and fluorescence-based assays were used to analyze the display of several model proteins on spores prepared at 25, 37 or 42 °C. Recombinant spores displayed different amounts of the same fusion protein in response to the temperature of spore production. In spores simultaneously displaying two fusion proteins, each of them was differentially displayed at the various temperatures. The display by the non-recombinant approach was only modestly affected by the temperature of spore production, with spores prepared at 37 or 42 °C slightly more efficient than 25 °C spores in adsorbing at least some of the model proteins tested.ConclusionOur results indicate that the temperature of spore production allows control of the display of heterologous proteins on spores and, therefore, that the spore-display strategy can be optimized for the specific final use of the activated spores by selecting the display approach, the carrier protein and the temperature of spore production.

Effect of Sporulation Conditions Following Submerged Cultivation on the Resistance of Bacillus atrophaeus Spores against Inactivation by H2O2.

The resistance formation of spores in general and of Bacillus atrophaeus in particular has long been the focus of science in the bio-defense, pharmaceutical and food industries. In the food industry, it is used as a biological indicator (BI) for the evaluation of the inactivation effects of hydrogen peroxide in processing and end packaging lines’ sterilization. Defined BI resistances are critical to avoid false positive and negative tests, which are salient problems due to the variable resistance of currently available commercial BIs. Although spores for use as BIs have been produced for years, little is known about the influence of sporulation conditions on the resistance as a potential source of random variability. This study therefore examines the dependence of spore resistance on the temperature, pH and partial oxygen saturation during submerged production in a bioreactor. For this purpose, spores were produced under different sporulation conditions and their resistance, defined by the D-value, was determined using a count reduction test in tempered 35% liquid hydrogen peroxide. The statistical analysis of the test results shows a quadratic dependence of the resistance on the pH, with the highest D-values at neutral pH. The sporulation temperature has a linear influence on the resistance. The higher the temperature, the higher the D-value. However, these factors interact with each other, which means that the temperature only influences the resistance when the pH is within a certain range. The oxygen partial pressure during sporulation has no significant influence. Based on the data obtained, a model could be developed enabling the resistance of BIs to be calculated, predicted and standardized depending on the sporulation conditions. BI manufacturers could thus produce BIs with defined resistances for the validation of sterilization effects in aseptic packaging/filling lines for the reliable manufacture of shelf-stable and safe food products.

Occurrence and characterization of Alternaria species associated with leaf spot disease in rapeseed in Serbia

AbstractThe global demand for rapeseed makes it one of the fastest growing markets in crop production, with a need for increasing growing area and productivity, both of which depend on effective pathogen control strategies. Alternaria pathogens cause serious losses of brassica crops and occur in most rapeseed‐growing regions around the world. In this study, morphological, molecular, and pathogenic analyses of 113 isolates collected from nine important rapeseed‐growing areas in Serbia identified four pathogens: Alternaria brassicae, A. brassicicola, A. japonica, and A. alternata, causing leaf spot disease. Molecular analyses of ITS, GAPDH, Alt a1, and ATP sequences revealed one multilocus haplotype for A. brassicae and A. japonica isolates, whereas for A. brassicicola and A. alternata three and five haplotypes were distinguished, respectively. Pathogenicity tests showed that A. brassicicola was the most virulent while A. brassicae and A. japonica exhibited the same level of pathogenicity. The A. alternata population was generally weakly pathogenic with one nonpathogenic, genetically separated but closely related group of isolates, suggesting that pathogenicity is more unstable in this phylogenetic lineage. The data recorded on rate of growth and sporulation of isolates at 0, 5, 10, 15, 20, 25, 30, 35, and 40 °C revealed significant differences in evolutionary strategies among species, as A. alternata had the widest optimum range and the fastest growth rate, A. brassicicola showed the highest sporulation intensity, and A. brassicae expressed lower optimum temperatures for sporulation compared to other groups. All species indicated the potential for cross‐infection of cabbage, and some haplotypes of A. brassicicola were previously isolated from horseradish in Serbia, suggesting the presence of one persistent Alternaria population on multiple brassica hosts in the region. This report describes the first detailed study of Alternaria spp. in rapeseed in Serbia.

Assessing the Role of Temperature, Inoculum Density, and Wounding on Disease Progression of the Fungal Pathogen Ceratocystis fimbriata Causing Black Rot in Sweetpotato.

In 2014, Ceratocystis fimbriata, causal agent of black rot in sweetpotato, reemerged and inflicted large financial losses on growers in the United States. Black rot continues to damage sweetpotatoes and has become a priority to the industry since then. In contrast, little is known about the biology of C. fimbriata and the epidemiology of sweetpotato black rot. In this study, effects of environmental factors such as inoculum density, RH, and temperature on sweetpotato black rot were determined. Cured sweetpotatoes were wounded with a toothpick to simulate puncture wounds, inoculated with different spore suspensions (inoculum density) (104, 105, or 106 spores/ml), and incubated under different RH (85.53, 94.09, or 97.01%) and temperature (13, 18, 23, 29, or 35°C) for 21 days. In a separate experiment, five root wounding types (cuts, punctures, abrasions, end breaks, and macerating bruises) were compared. All wounded roots were subsequently soaked in a 103 spores/ml suspension and incubated at 100% RH and 23°C for 21 days. This study found 29 and 23°C to be the optimal temperature for black rot disease development and sporulation, respectively. No pathogen growth was observed at 13 and 35°C. Increased inoculum density significantly (P < 0.0001) increased disease incidence, but increasing RH had an effect only on sporulation area. All wound types resulted in increased disease incidence and sporulation as early as 7 days postinoculation. Our results highlight the importance of characterizing factors that affect disease development for achieving successful disease management strategies. Findings from this study will be used to improve disease management for sweetpotato black rot by suggesting tighter regulation of curing and storage conditions and better postharvest handling of sweetpotato roots to avoid unnecessary wounding.