Spore Germination and Mycelial Growth of Streptomycetes at Different Humidity Levels

This study is the first to show the ability of streptomycetes to develop at a very low humidity level. All of the streptomycetes studied produced growth at low humidity (a_w 0.86 and 0.67). This capacity was most markedly pronounced in Streptomyces odorifer, whose spores were capable of germinating, and mycelial germs increased in length, at the air humidity a_w 0.50. The formation of lateral branches (mycelium branching) at this humidity was noted only in single S. odorifer germs and only after 72 h of incubation. Study of streptomycete growth on an agarized medium with different osmotic pressures, created by various glycerol concentrations in the medium, showed that, at a_w 0.67, the spores of all the streptomycetes studied germinate, producing mycelial germs but not microcolonies. The ecological significance of mycelial prokaryotes in soil microbial communities that develop and function under conditions of extremely low humidity is discussed.     

Water activity and temperature effects on germination and growth of Eurotium amstelodami, E. chevalieri and E. herbariorum isolates from bakery products

This study investigated the effect of temperature (5-30 degrees C), water activity (0.775-0.90 aw) and their interactions on the temporal rates of germination and mycelial growth of three species of Eurotium on flour wheat sucrose medium. Germination was quite rapid at aw >0.85, with an almost linear increase with time for all isolates. However, under more extreme water stress, germination was slower. The aw minima for germination were usually lower than those for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h) prior to germination and on the germination rates (h-1) were predicted using the Gompertz model modified by Zwietering. Eurotium spp. had shown short lag times at 0.90 aw over a wide range of temperatures. At marginal temperatures, lag phases were significantly longer, especially at >15 degrees C. The temperature x aw profiles for mycelial growth varied between species in terms of rates (mm d(-1)). Predictions of the effect of important environmental factors, such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth, are important in the development of hurdle technology approaches to predict fungal spoilage in food products.     

Effect of water activity and temperature on germination and growth of Penicillium digitatum,P. italicum and Geotrichum candidum

AIMS: This study compares the effect of temperature (4-37 degrees C) and water activity (aw: 0.99-0.87) and their interactions on the germination rates, lag times prior to germination and mycelial growth 'in vitro' of Penicillium digitatum, P. italicum and Geotrichum candidum, the main postharvest pathogens affecting citrus fruits. METHODS AND RESULTS: Germination and growth were markedly influenced by temperature and aw. Generally, lag times were longer and germination and growth rates were slower when conditions of temperature and aw were far from optimum. All the studied species were able to germinate over a range of 4-30 degrees C at 0.995 aw, although in non-optimal conditions P. digitatum only reached 40-60% of germinated conidia. At low temperatures, P. italicum germinated and grew faster than P. digitatum and G. candidum, particularly at 0.95 aw. Penicillium italicum was also able to germinate and grow in the driest studied conditions (0.87 aw), while G. candidum did not germinate under 0.95 aw. CONCLUSIONS: Knowledge of the ecological requirements of these fungi is important in order to understand their behaviour in natural situations and to predict fungal spoilage on citrus fruits.     

Ecological determinants for germination and growth of some Aspergillus and Penicillium spp. from maize grain

This study compared the effect of temperature (5-45 degrees C), water availability (water activity, aw; 0.995-0.75) and their interactions on the temporal rates of germination and mycelial growth of three mycotoxigenic strains of Aspergillus ochraceus and one isolate each of A. flavus, A. niger, Penicillium aurantiogriseum and P. hordei in vitro on a maize extract medium. Germination was very rapid at > 0.90 aw with an almost linear increase with time for all species. However, at < 0.90 aw, the germination rates of A. flavus and P. hordei were slower. The aw minima for germination were usually lower than for growth and varied with temperature. The effect of aw x temperature interactions on the lag phases (h), prior to germination, and on the germination rates (h(-1)), were predicted for the first time for these fungi using the Gompertz model modified by Zwietering. This showed that A. flavus, A. niger and the two Penicillium spp. had very short lag times between 0.995-0.95 aw over a wide temperature range. At marginal temperatures, these were significantly higher, especially at < 10 degrees C for Aspergillus spp. and > 30 degrees C for Penicillium spp. There were also statistically significant differences between lag phases and germination rates for three different isolates of A. ochraceus. The Aspergillus spp. also germinated faster than the Penicillium spp. The temperature x aw profiles for mycelial growth varied considerably between species, both in terms of rates (mm d(-1)) and tolerances. Predictions of the effects of important environmental factors such as temperature, aw and their interactions on lag times to germination, germination rates and mycelial growth are important in the development of hurdle technology approaches to predicting fungal spoilage in agricultural and food products.     

Dynamics of spore germination and mycelial growth of streptomycetes under low humidity conditions

At extremely low values of moisture pressure (?96.4 MPa; a_w 0.50), the spores of xerotolerant streptomycetes (Streptomyces odorifer and S. rubiginosohelvolus) germinated, their germ lengths increased, and lateral branching of the mycelium was observed after 5 days of incubation in a thin layer of agarized nutrient medium. At ?22.6 MPa (a_w 0.86), the mycelium begins to branch after a 2-day incubation; over a 5-day incubation at ?2.8 MPa (a_w 0.98), it goes through a reproduction cycle, which culminates in spore formation. The mathematical model approach enabled us to elucidate the behavioral patterns of Streptomyces spores in a thin layer of agarized nutrient medium at low humidity levels. The dynamics of spore germination is governed by the exponential law, which allows calculation of the average duration of the period a before spore germination, as well as the time needed for 50% of viable spores to germinate.     

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G.

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G

The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated. The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose. The minimum pH (at the optimum aw) for growth and toxin production of C. botulinum type G was found to be 5.6. Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C. These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6. It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.     

Response surface methodology study of the combined effects of temperature, pH, and aw on the growth rate of Trichoderma asperellum

AIMS: To evaluate the influence of environmental parameters (water activity aw, temperature, and pH) on the radial growth rate of Trichoderma asperellum (strains PR10, PR11, PR12, and 659-7), an antagonist of Phytophthora megakarya, the causal agent of cocoa black pod disease. METHODS AND RESULTS: The radial growth of four strains of T. asperellum was monitored for 30 days on modified PDA medium. Six levels of aw (0.995, 0.980, 0.960, 0.930, 0.910, and 0.880) were combined with three values of pH (4.5, 6.5, and 8.5) and three incubation temperatures (20, 25, and 30 degrees C). Whatever the strain, mycelial growth rate was optimal at aw between 0.995 and 0.980, independently of the temperature and pH. Each strain appeared to be very sensitive to aw reduction. In addition, all four strains were able to grow at all temperatures and pH values (4.5-8.5) tested, highest growth rate being observed at 30 degrees C and at pH 4.5-6.5. The use of response surface methodology to model the combined effects of aw, temperature, and pH on the radial growth rate of the T. asperellum strains confirmed the observed results. In our model, growth of the T. asperellum strains showed a greater dependence on aw than on temperature or pH under in vitro conditions. CONCLUSION: aw is a crucial environmental factor. Low aw can prevent growth of T. asperellum strains under some conditions. The observed and predicted radial growth rate of strain PR11 showed its greater capacity to support low aw (0.93) as compared with other tested strains at 20 degrees C. This is in agreement with its better protective level when applied in medium-scale trials on cocoa plantations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study should contribute towards improving the biocontrol efficacy of T. asperellum strains used against P. megakarya. Integrated into a broader study of the impact of environmental factors on the biocontrol agent-pathogen system, this work should help to build a more rational control strategy, possibly involving the use of a compatible adjuvant protecting T. asperellum against desiccation.     

Modulation of water activity on fungicide effect on Aspergillus niger growth in Sabouraud dextrose agar medium*

AIMS: To examine whether water activity (aw) in combination with low concentration of fungicides can be used to effectively control Aspergillus niger van Tieghem growth in cultural medium, the Sabouraud dextrose agar (SDA). The data would be used as baseline information for reducing A. niger contamination in insect artificial diets. METHODS AND RESULTS: Aspergillus niger was isolated from an insect artificial diet. Four concentration levels (i.e. 0, 1, 10 and 20 micromol) of two fungicides (i.e. amphotericin B and itraconazole) were tested against A. niger under four aw levels (i.e. 0.994, 0.961, 0.921 and 0.859) adjusted by including 0, 12.5, 25 and 38% of glycerol in the medium mixture. Aspergillus niger growth was significantly reduced at low fungicide concentration (1 micromol), and at reduced aw. The spore germination was prevented with either higher fungicide concentration (>10 micromol), or low aw in the medium (aw < 0.921). The two ecological determinants (fungicides and aw) showed a significant impact on A. niger survival in the medium (P < 0.0001). Itraconazole is more effective than amphotericin B in controlling A. niger contamination in the agar medium. CONCLUSION: Adjustment of aw (with 12.5% of glycerol) in combination with 1 mumol of itraconazole can effectively prevent A. niger growth in the SDA cultural medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus niger contaminations have frequently affected the quality of insects produced from mass rearing facilities. Low aw in combination with low fungicide concentration has the potential to become one of the most cost-effective management strategies to prevent A. niger contamination in insect artificial diets. The effect of fungicides and low aw in artificial diets on insect biology needs to be further examined.     

Endospore formation by Streptomyces avermitilis in submerged culture

The capability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycles on synthetic medium CP1 with glucose under phosphate limitation, and (2) in autolysing cell suspensions of high density obtained by tenfold concentration of a stationary-phase culture grown in a synthetic medium resuspended in phosphate buffer (pH 7.2) with 0.2% CaCl2. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to pertain to the main species-defining features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in a surface culture.     

Differentiation and protease production in Micromonospora echinospora (ATCC 15837)

Micromonospora echinospora differentiates in both submerged and surface cultures producing abundant dark spores after a period of vegetative mycelial growth. In submerged batch cultures, under either carbon or nitrogen limiting conditions, protease activity was found to coincide with sporulation indicating a relationship between proteolytic activity and differentiation in this organism. Further evidence for this link was provided from surface grown cultures wherein sporulation was inhibited by the serine protease inhibitors TLCK and TPCK. The association between proteolysis and differentiation apparent in this organism correlates with evidence of a similar phenomenon observed in the streptomycetes, suggesting that this may be a common response associated with differentiation in filamentous actinomycetes.     

Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.     

碳源对玫烟色虫草菌丝生长、芽生孢子产生及其耐热性的影响

提高虫生真菌孢子应对热胁迫的能力是生防菌应用研究的关键,为研究菌丝培养阶段碳源对玫烟色虫草Cordyceps fumosorosea IF-1106耐热性的影响,选择了麦芽糖、可溶性淀粉、蔗糖、葡萄糖、果糖、海藻糖为碳源的培养基对玫烟色虫草IF-1106进行液体培养,评估了不同碳源条件下菌丝的生长、产孢及所产芽生孢子的耐热性。结果表明,在菌株培养阶段,培养基中碳源的种类及浓度对菌丝产量、产孢量及所产芽生孢子的耐热性有显著影响,其中蔗糖为碳源时,所产芽生孢子的耐热性强,45 ℃热胁迫条件下LT₅₀为1.65 h;蔗糖浓度为40 g/L时,可产生大量耐热芽生孢子,液体培养3 d后产孢量可达3.43×10⁷个孢子/mL。为探索不同培养条件下所产芽生孢子耐热性不同的原因,提取了孢子内的海藻糖并采用离子色谱法对其进行了定量分析,发现耐热性高的芽生孢子胞内海藻糖含量普遍较低,可见海藻糖是与芽生孢子耐热性密切相关的内源物质。综上所述,选择适宜的培养基是调控孢子耐热性的有效途径,本研究为生产高耐热的玫烟色虫草生防制剂提供了有益的指导。     

Microcycle Conidiation and Spore-Carrying Capacity of Colletotrichum gloeosporioides on Solid Media

The effect of spore inoculum density, medium concentration, and temperature on slime-spot formation, spore yield, and mycelium production by Colletotrichum gloeosporioides on agar media were studied with a simple microplate assay. A steady-state spore yield (spore-carrying capacity) independent of inoculum density was reached only on media that supported good fungal growth and sporulation. The spore-carrying capacity was reached earlier, the denser the inoculum. On standard mycological media a high inoculum density (2.5 × 10⁶ spores per ml) resulted in a slimy mass of conidia forming a slime spot, a phenomenon associated with greatly reduced mycelium formation and indicative of microcycle conidiation. In contrast, for a similar inoculum density, enhanced mycelial growth preceded sporulation and overrode slime-spot formation on highly concentrated media; a very low medium concentration resulted in much less mycelium, but spore production was also decreased. Exposure to suboptimal growth temperatures of 36 to 48°C for up to 8 days did not induce microcycle conidiation from inocula that did not form a slime spot at 28°C.     

Screening of compounds stimulating spore formation and mycelial growth of pock-forming plasmid-carrying strains in Streptomyces azureus

Summary Sporulation-stimulating compounds were screened by using mutant PK100a of Streptomyces azureus ATCC 14921, in which spore formation was markedly inhibited by the pock-forming plasmid pSA1.1. On agar media, cysteine, bacitracin, glutathione and -NAD induced the formation of coloured spores or spore mass of strain PK100a. These compounds also stimulated the spore formation of the wild-type and its plasmid-cured (good spore-forming) strains and the growth of aerial mycelia of these three strains. This screening method appears to be an effective method for the screening of substances that stimulate spore formation and mycelial growth of streptomycetes or that overcome the inhibitory effect of pock-forming plasmids. Correspondence to: S. Ogata     

Effect of Additives on the Production, Viability and Virulence of Blastospores of Metarhizium anisopliae

Studies on the blastospore production of Metarhizium anisopliae var. anisopliae were conducted in Adamek's medium used as a standard, enriched with lecithin, collagen, lactic acid or polyethylene glycol 200 (PEG 200) to increase spore yield and suppress mycelial pellet formation. The addition of 5% lecithin resulted in a significant 10-fold increase in spore yield up to 1.9 108 blastospores/ml compared with 1.9 107 spores/ml in the standard medium. Collagen (3%) increased the number of blastospores 3.7-fold, and lactic acid (1.5%) two-fold. A reduction of mycelial pellet formation in favour of spore production was noted with each additive. The viability of blastospores at 40IC from media with lecithin, collagen and lactic acid suspended in 25% Ringer's solution was comparable to that of spores produced in the standard medium. Striking differences were noticed in the viability of spores produced with 5% PEG 200 in standard medium. The half-life of blastospores produced in standard medium suspended in sunflower oil was 33.6 h and that of 5% PEG 200 spores only 25.2 h. In bioassays, the virulence of spores produced in standard medium to which 3% lecithin, 3% collagen, 1.5% lactic acid or 5% PEG 200 had been added was tested against third-instar nymphs of Locusta migratoria migratorioides (R. & F.). The median lethal time and the mortality of L. migratoria achieved with blastospores produced with 3% lecithin (5.7 days, 99%) was comparable to that of blastospores from standard medium (5.1 days, 98%). The virulence of blastospores from all other media with additives was significantly reduced.     

Influence of humidity on production of pelleted fungal inoculum

One of the practical problems in scaling-up the production of fungal inocula for environmental applications is how to provide essential humidity for fungal growth. Pelleted solid substrate was used as a fungal biomass carrier. It was coated with alginate or agar hydrogels that contained mycelial fragments of the white-rot fungi Trametes versicolor or Irpex lacteus. To follow fungal growth and formation of mycelial coat over pelleted substrate, the fluorescein-diacetate hydrolysing activity (FDA) assay and visual inspection were used. Both fungi were able to overgrow the pelleted substrate in 5–6 days, at a relative humidity (RH) of 86.3% or higher. The enrichment of alginate hydrogel with nutrients or coating of pelleted substrate with more hydrophilic agar hydrogel enabled I. lacteus to overgrow the pellets at a lower RH of 83.6%. Fungal inocula produced at lower RH possessed lower final moisture contents and had greater mechanical strength. Conditioning of T. versicolor mycelial fragments, by a 3-h incubation in fresh growth medium, enhanced fungal growth over the pelleted substrate. A mathematical model was used to simulate and to explain moisture distribution in a hydrogel-coated pellet and the formation of mycelial coat, for various conditions of fungal inocula production.     

Unlocking Streptomyces spp. for Use as Sustainable Industrial Production Platforms by Morphological Engineering

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.     

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.