Caractérisation de souches cultivées d'un nouveau champignon comestible: Stropharia rugoso-annulata. II. Anatomie,développement mycélien et fructification

To establish a performing production strategy for the edible mushroom Stropharia rugoso-annulata, investigations on anatomy and physiological characteristics of the basidiomycete were carried out. Scanning electron microscopy analysis of Stropharia strains revealed no significant morphological differences. Spore germination was only obtained in the presence of growing mycelium. On the basis of the influence of temperature on the growth rate of Stropharia strains, three groups were defined without relation to phenotypic classification. Moreover, every strain exhibited a higher growth rate at 25 °C, and residual growth was observed above 35 °C The pH of the medium did not affect mycelium development. Regardless of the initial pH value, after 2 weeks of mycelium growth, the pH was established at a value close to 3.5. However, no acidic compounds were detected in this medium. A preliminary cultivation trial indicated that carpophore formation is strongly affected by spawn run time.     

Dry mycelium preparations of entomopathogenic fungi,Metarhizium anisopliae and Beauveria bassiana

Several different harvesting procedures were used to obtain dry mycelium preparations of the entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. The effects of these procedures on the survival of the fungal preparations and on their conidiation after short periods of storage at room temperature and at 4°C were examined. Harvesting procedures consisted of filtering the mycelium produced in airlift containers from the culture medium, washing with deionized water, spraying with a sugar solution, and incubating for 18 hr at 4°C before drying. Conidial production of treated mycelia stored 1.5 and 4.5 months at 4°C was not significantly different for and procedure. For dry mycelium of M. anisopliae stored 1.5 months at 4°C and then at room temperature for 3 months, maltose- and sucrose-treated preparations produced more conidia than preparations sprayed with dextrose solution, with water only, or not sprayed. B. bassiana preparations dried soon after mat formation were superior to those incubated at 4°C, and maltose-and dextrose-treated mycelia were superior to other treatments when stored at room temperature.     

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.     

Isolation and growth characteristics of a nystatin- resistant mutant of a thermotolerant yeast Hansenula polymorpha

A nystatin-resistant mutant (NR-21) of a thermotolerant yeast, Hansenula polymorpha CK-1, was isolated by mutagenesis with ethyl methanesulfonate, followed by selection for resistance to nystatin (50 units/ml). The mutant was defective in ergosterol biosynthesis. Specific growth rates (h⁻¹ of the mutant were reduced to 0.35 at 40°C and 0.16 at 50°C as compared with the wild type (0.53 at 40°C and 0.28 at 50°C). The mutant grown with ergosterol-phosphatidylcholine emulsion at 50°C incorporated ergosterol and its specific growth rate was increased to 0.41, which was comparable to that of the wild type grown under the same conditions.     

Preservation of Agaricus subrufescens strains at low temperature by using cultures on sorghum grains

BackgroundOne of the main problems for the preservation of genetics resources of Agaricus subrufescens is to maintain the viability of the strains because the mycelium is very sensitive to cooling and therefore it ages rapidly.AimsEvaluate the viability of A. subrufescens strains stored as cultures on sorghum grain (spawn) at different temperatures.MethodsEighteen strains of A. subrufescens and three strains of Agaricus bisporus were studied. Spawn's viability was evaluated under the following conditions: (1) control at 25 °C (C), (2) cooling to 4 °C (R) and (3) freezing in liquid nitrogen at ?196 °C (LN). Samples were recovered from week 4 every 2 weeks until week 12 and week 24 in C and R, whereas in LN samples were recovered at 4, 12 and 24 weeks. Viability was evaluated in 50 seeds, by strain and condition, recovering the mycelium in Petri dishes with potato dextrose agar medium (PDA). Mycelium growth was also evaluated on PDA after 14 days of recovery.ResultsMost strains showed 100% viability and they were recovered usually in 1 day. In LN the viability ranged between 84 and 100% depending on the strain, but in some cases recovery took more than 10 days. Mycelial growth decreased gradually over time and although the results show significant differences between treatments C and R, the decline is associated with ageing of the mycelium rather than the treatment itself.ConclusionsCulture on sorghum grain and storage at low temperature is an interesting way to preserve genetic resources of A. subrufescens.     

Germination and Survival of Fusarium graminearum Macroconidia as Affected by Environmental Factors

The effects of temperature (4–20°C), relative humidity (RH, 0–100%), pH (3–7), availability of nutrients (0–5 g/l sucrose) and artificial light (0–494 μmol/m²/s) on macroconidial germination of Fusarium graminearum were studied. Germ tubes emerged between 2 and 6 h after inoculation at 100% RH and 20°C. Incubation in light (205 ± 14 μmol/m/s) retarded the germination for approximately 0.5 h in comparison with incubation in darkness. The times required for 50% of the macroconidia to germinate were 3.5 h at 20°C, 5.4 h at 14°C and 26.3 h at 4°C. No germination was observed after an incubation period of 18 h at 20°C in darkness at RH less than 80%. At RH greater than 80%, germination increased with humidity. Germination was observed when macroconidia were incubated in glucose (5 g/l) or sucrose (concentration range from 2.5 × 10^?4 to 5 g/l) whereas no germination was observed when macroconidia were incubated in sterile deionized water up to 22 h. Macroconidia germinated quantitatively within 18 h at pH 3–7. Repeated freezing (?15°C) and thawing (20°C) water agar plates with either germinated or non‐germinated macroconidia for up to five times did not prevent fungal growth after thawing. However, the fungal growth rate of mycelium was negatively related to the number of freezing events the non‐germinated macroconidia experienced. The fungal growth rate of mycelium was not significantly affected by the number of freezing events the germinated spores experienced. Incubation of macroconidia at low humidity (0–53% RH) suppressed germination and decreased the viability of the spores.     

Endogenous arabitol and mannitol improve shelf life of encapsulated <Emphasis Type="Italic">Metarhizium brunneum</Emphasis>

Successful commercialization of microbial biocontrol agents, such as Metarhizium spp., is often constrained by poor drying survival and shelf life. Here, we hypothesized that culture age would influence endogenous arabitol, erythritol, mannitol and trehalose contents in M. brunneum mycelium and that elevated levels of these compounds would improve drying survival and shelf life of encapsulated mycelium coupled with enhanced fungal virulence against T. molitor larvae. We found that culture age significantly influenced endogenous arabitol and mannitol contents in mycelium with highest concentrations of 0.6?±?0.2 and 2.1?±?0.2 µg/mg after 72 h, respectively. Drying survival of encapsulated mycelium was independent of culture age and polyol content with 41.1?±?4.4 to 55.0?±?6.2%. Best shelf life was determined for biomass harvested after 72 h at all investigated storage temperatures with maximum values of 59.5?±?3.3% at 5 °C followed by 54.5?±?1.6% at 18 °C and 19.4?±?1.3% at 25 °C after 6 months. Finally, high fungal virulence against T. molitor larvae of 83.3?±?7.6 to 98.0?±?1.8% was maintained during storage of encapsulated mycelium for 12 months with larval mortalities being independent of culture age and polyol content. In conclusion, our findings indicate beneficial effects of endogenous polyols in improving shelf life of encapsulated mycelium and this may spur the successful development of microbial biocontrol agents in the future.     

Copper uptake inAspergillus niger during batch growth and in nongrowing mycelial suspensions

Copper accumulation by the filamentous fungusAspergillus niger from a glucose mineral salts medium containing copper in the concentration range 16 to 157 μM was maximal in the lag phase of growth. In the subsequent linear growth phase, the mycelial copper contents were dramatically reduced on a per gram dry weight basis. The fungal mycelium exhibited pelleted morphology and exponential growth was not apparent. The medium pH was reduced during growth in flask cultures, but this was not responsible for the reduction in copper uptake as indicated by the similar effect in cultures grown in a stirred-tank fermenter with electronic maintenance of pH at 5.5. Voltammetric analysis of medium which had supported growth of the fungus showed that copper added at a final concentration of 40 μM was complexed. Energy-dependent copper uptake from 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5 containing 40 μM copper could not be demonstrated in nongrowing mycelium. Incubation at 4°C reduced copper uptake while the presence of 10 mM glucose or preincubation of the mycelium in 1 mM sodium azide had no effect on copper uptake.     

Effect of relative humidity,temperature and fungicide on germination of conidia of Cercospora canescens caused the Cercospora leaf spot disease in mungbean

The aim of the present investigation was to determine the impact of relative humidity (RH) and temperature on conidial germination, nuclear position and effect of important fungicides on growth and conidial germination of Cercospora canescens. Germination of conidia was observed at RH range 92–100% at 5–35°C. Significant interaction between temperature and RH indicated that higher humidity and high temperature promoted quick germination both in the presence and absence of free moisture. Although in absence of free moisture at 92–95% RH higher temperatures 25–35°C promoted quick evaporation of moisture and no conidial germination. Number of germtube was increased significantly at the optimum temperature 25–30°C and higher humidity (98–100%). But higher temperature 25–35°C with lower RH did not support the conidial germination. This finding is very important for disease forecasting using meteorological data. The spray of Carbendazim as contact fungicide may not be useful since it is not effective against the conidia of C. canescens. Triadimefon did not inhibit the conidia germination but completely inhibited mycelium development at 50 μg/ml. Propriconazole inhibited both conidia germination and mycelial development. Therefore, Propiconazole may be taken as protective as well as curative spray. In non-systemic fungicide, Copper oxychloride gave anticipated result by inhibiting both conidial germination and mycelium development. Therefore, copper oxychloride can be used as protectant fungicides for Cercospora leaf spot caused by C. canescens.     

Host Range,Temperature Response,Survival, and Overwintering of Alternaria cirsinoxia

Alternaria cirsinoxia was evaluated for its host range, the influence of temperature on mycelial growth, and survival and overwintering on Canada thistle (Cirsium arvense) in Saskatchewan. With the exception of leafy spurge, the host range of A. cirsinoxia was limited to species within the Asteraceae. Canada thistle, safflower, and sunflower were most susceptible to A. cirsinoxia, the latter two being crop species of lesser importance in Saskatchewan. Mycelium of A. cirsinoxia grew best at a constant temperature of 25°C and in temperature cycles which alternated around a mean of 20–25°C. Mycelium did not grow when exposed to constant temperatures of 0, 40, or 45°C for 7 days. However, at 0°C, mycelium survived and was able to resume growth, whereas at 40 or 45°C, mycelium was killed. In the field, A. cirsinoxia produced viable conidia on senescent, basal Canada thistle leaves for at least 3–4 months after inoculation in 1998 and 1999. Sporulation tended to be higher in 1998 than in 1999, possibly favored by the warmer, drier, and sunnier conditions prevailing during July to mid-September in 1998. A. cirsinoxia also overwintered and produced viable conidia on infected Canada thistle leaves in the field, and at constant 4°C, when sampled from November 1998 until April 1999. Sporulation of leaves overwintering in the field was lowest in April 1999, probably due to inoculum degradation as a result of surface flooding in the plots. Clusters and chains of chlamydospores were abundant on overwintering leaf and stem debris of Canada thistle in field plots inoculated 10 months previously. A. cirsinoxia subsequently sporulated on this infected debris. Based on these host-range tests, the risks to major nontarget crop species in Saskatchewan should be minimal after the inundative application of A. cirsinoxia as a bioherbicide for Canada thistle. However, this pathogen appears able to persist and remain potentially infectious in the field for a prolonged period of time after inoculation. Hence, longevity and spread of A. cirsinoxia should be evaluated further to minimize the potential risks to susceptible minor crop species.     

Salicylate Degradation by the Fungal Plant Pathogen Sclerotinia sclerotiorum

The fungal plant pathogen Sclerotinia sclerotiorum was studied to determine its ability to degrade salicylate, an important defense-signaling molecule in plants. S. sclerotiorum D-E7 was grown at 25 °C in an undefined medium (50 ml) containing minerals, 0.1 % soytone, 50 mM MES buffer (pH 6.5), 25 mM glucose, and 1 mM salicylate. Glucose, oxalate, and salicylate concentrations were monitored by HPLC. S. sclerotiorum D-E7 was found to be active in salicylate degradation. However, salicylate alone was not growth supportive and, at higher levels (10 mM), inhibited glucose-dependent growth. Biomass formation (130 mg [dry wt] of mycelium per 50 ml of undefined medium), oxalate concentrations (~10 mM), and culture acidification (final culture pH approximated 5) were essentially the same in cultures grown with or without salicylate (1 mM). Time-course analyses revealed that salicylate degradation and glucose consumption were complete after 7 days of incubation and was concomitant with growth. Trace amounts of catechol, a known intermediate of salicylate metabolism, were detected during salicylate degradation. Overall, these results indicated that S. sclerotiorum has the ability to degrade salicylate and that the presence of low levels of salicylate did not affect growth or oxalate production by S. sclerotiorum.     

Lihuaxuella thermophila gen. nov., sp. nov., isolated from a geothermal soil sample in Tengchong, Yunnan, south-west China

A novel filamentous bacterium, designated YIM 77831^T, was isolated from a geothermal soil sample collected at Rehai National Park, Tengchong, Yunnan province, south-west China. Growth occurred from 28 to 65 °C (optimum 50 °C), pH 6.0–8.0 (optimum pH 7.0). The strain formed branched substrate mycelia, endospores were produced on the substrate mycelium and aerial mycelium was not produced on any of the growth media tested. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain YIM 77831^T was affiliated with the family Thermoactinomycetaceae. The stain YIM 77831^T contained meso-diaminopimelic acid in the cell wall. Whole-cell hydrolysates contained glucose, galactose, mannose, ribose and rhamnose. The polar lipids were phosphatidylmethylethanolamine, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid and four unknown phospholipids. The only menaquinone was MK-7. Major fatty acids were iso-C_15:0, anteiso-C_15:0 and anteiso-C_17:0. The G+C content was 55.6 mol%. On the basis of the morphological and chemotaxonomic characteristics as well as genotypic data, strain YIM 77831^T represents a novel genus and species, Lihuaxuella thermophila gen. nov., sp. nov., in the family Thermoactinomycetaceae. The type strain is YIM 77831^T (CCTCC AA 2011024^T = JCM 18059^T).     

Prosopis chilensis is a plant highly tolerant to heat shock

At temperatures between 25 and 35°C, 100% of Prosopis chilensis seeds germinated within 24 h. At higher temperatures, the germination rate was reduced; at 50°C, seeds did not germinate. After germination at 25°C, the optimal temperature for seedling growth was 35°C and the seedlings did not grow at a temperature of 50°C. However, when germination was at 35°C, the optimal temperature for seedling growth was 40°C and some seedlings grew at 50°C, suggesting that thermotolerance was induced during seed germination at 35°C. Further thermotolerance can be induced in seedlings germinated at 35°C, by exposing them to 40°C for 2h. Under these conditions, seedlings exhibited increased growth rate at 45 and 50°C. Fluorography of SDS-polyacrylamide gel electrophoresis of the proteins synthesized and accumulated during 2 h at temperatures of 35, 40, 45 and 50°C in the presence of [³⁵S]methionine revealed the expression of 11 proteins not detectable at 35°C. Most of the proteins present at 35°C also increased in expression. The temperature for maximal expression of these proteins was 45°C.     

Modelling the effects of temperature and wetness on the polycyclic phase of Stemphylium vesicarium,the pathogen causing purple spot on asparagus (Asparagus officinalis L.)

The polycyclic phase of Stemphylium vesicarium is the key factor for the forecast and integrated control of purple spot on asparagus. The annual dynamics of airborne conidia were determined under field conditions by conidia traps. From 2013 to 2015, conidia became airborne at the earliest at mid‐July, but the number trapped was considerably enhanced only after mid‐August, early September. The cumulative percentage of trapped conidia was best described using a logistic function depending on the daily temperature sum (base 0°C) accumulated only on days with >0.2 mm of rainfall (R² = .81). The germination of conidia was modelled by a generalized beta‐modified Chapman Richards function, and the germ tube length was modelled by a generalized beta‐power function. Conidia germinated in a wide temperature range, with an optimum at 23.3°C, whereas germ tube length had a narrow nearly optimum temperature range around 28.7°C, which indicates that infection by conidia is more restricted by germ tube growth than by germination. The effect of temperature on the number of lesions produced by two strains on green asparagus spears had the narrowest optimum range (optimum at 21.9°C) of all parts of the polycyclic phase. In plant tissue, the spread of the fungus depends on the mycelium growth. The mycelium growth of the four strains, which was modelled with data from a petri dish experiment, had an optimum temperature at 24.7°C.     

Temperature-induced changes of malondialdehyde, heat-shock proteins in relation to chlorophyll fluorescence and photosynthesis in Conocarpus lancifolius (Engl.)

The effect of variable temperatures (10–50 °C) on photosynthesis and chlorophyll fluorescence in Conocarpus lancifolius was evaluated. Additionally, the ability of the species to synthesize heat-shock proteins (HSPs) to protect against high temperatures, and malondialdehyde (MDA) as a by-product of lipid peroxidation was investigated. Plants at 10 °C showed virtually no measurable growth, leaf discoloration and a few brown lesions, while high temperatures (40 and 50 °C) promoted growth and lateral branch development. Chlorophyll content index, photochemical efficiency (F _v/F _m) of PS II, electron transport rate and photosynthetic rate declined with decreasing temperature but increased significantly at higher temperatures. Heat-shock protein (HSP 70 kDa) was produced at temperatures 30–50 °C and an additional 90 kDa protein was also produced at 50 °C. Increase in the efficiency of excitation energy captured by the open PS II reaction centers (F _v/F _m) increased linearly (P ≤ 0.05) with the accumulation of HSP 70 at higher temperatures. However, at low temperatures the concentration of MDA increased significantly, indicating lipid peroxidation due to oxidative stress. The production and accumulation of HSP 70 and 90 kDa coupled with increased electron transport rate and photochemical efficiency can be used to assess survival, growth capacity and to some extent the tolerance of C. lancifolius to elevated temperatures.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Do spawn storage conditions influence the colonization capacity of a wheat-straw-based substrate by Agaricus subrufescens?

Storage conditions of the spawn of edible fungi are of major importance to facilitate the production of mushrooms. Here, standard storage conditions at 10 °C or 15 °C were used and the potential of colonization of standard European compost by the tropical species Agaricus subrufescens was assessed during the spawn running phase. Two lignocellulolytic activities, laccase and CMC-cellulase, were enhanced after storage compared to control as well as substrate transformation, as described by the aromaticity ratio and a humification ratio calculated from NMR data. This result indicates that mycelium growth probably occurred during storage at 10 or 15 °C, leading to a larger amount of biomass in the inoculum. Moreover, the microbial functional diversity of the substrate was favored, showing that the electivity of the substrate was maintained. Thus, these findings indicate that recommendations for the mushroom producers can be established for A. subrufescens cultivation under European standard conditions.     

Modelling germination and mycelium growth rates of Monostichella coryli under constant temperature conditions

Monostichella coryli is the causal agent of the hazelnut anthracnose. The increase in the incidence in hazelnut orchards observed during recent years in Central Italy is increasing the need for information and studies on its biology and ecology, and how environmental factors (e.g., temperature) influence its development. For this purpose, seven isolates from different provenances of M. coryli were studied under controlled laboratory conditions with the goal of laying the foundations of a mathematical model describing key aspects of the pathogen's life cycle. The mycelium growth rate was measured at 6 different constant temperatures (5, 10, 15, 20, 25, 30 °C) and fixed relative humidity (100% RH), while the germination rate of the conidia was evaluated at 8 different constant temperatures (5, 10, 15, 20, 23, 25, 27, 30 °C). The dataset was subsequently analyzed estimating the parameters of non-linearfunctions which consider the relationship between the mycelium growth rate and the germination of the spores with environmental temperature. The set of parameters provided as the result of this study constitutes a key step forward in the biological knowledge of the species and the basis for future formulations of mathematical models to be used as decision support systems in an integrated pest management framework.