Effect of nutrition on growth and virulence of the entomopathogenic fungus Beauveria bassiana

Three isolates of the entomopathogen Beauveria bassiana along with one strain of Metarhizium anisopliae were cultured on seven media with different carbon/nitrogen (C/N) ratios. The effect of nutrition on virulence of the isolates was evaluated via measurement of colony growth, spore yield, germination speed, conidial C/N ratio and Pr1 (a serine protease) activity. 'Osmotic stress' medium produced the lowest colony growth with low numbers of conidia in all isolates. However, these conidia showed a high germination rate and virulence. However, conidial Pr1 activity was low in some isolates. In most but not in all cases conidia from 1% yeast extract, 2% peptone and low (10 : 1) C/N medium had higher Pr1 activity compared with conidia from other media. However, in some instances we could not conclude that there was a relationship among germination rate, conidial Pr1 activity and virulence. C/N ratio of conidia was statistically different among various media and fungal isolates. Conidia with lower C/N ratio generally produced lower LT(50) (lowest median lethal time) values (more virulent). Insect-passaged conidia from different media had lower C/N ratio compared with similar conidia from artificial cultures. Therefore, they should be more virulent than in vitro produced conidia. As germination rate, conidial Pr1 activity and C/N ratio are independent of host, it seems that host-related determinants such as insect cuticle and physiology and environmental conditions may influence host susceptibility and therefore fungal isolate virulence towards host insects.     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

Effects of successive subculturing on stability, virulence, conidial yield, germination and shelf-life of entomopathogenic fungi

Aims: To determine the stability and conidial yield of two strains of the entomopathogenic fungus Metarhizium anisopliae and one strain of M. brunneum, being developed for the control of insect pests. Methods and Results: The conidial yields and the shelf‐life of the conidia of two commercially viable strains of M. anisopliae V275 (=F52) and ARSEF 4556 and one strain of M. brunneum (ARSEF 3297) were determined after harvesting conidia from in vitro subcultures on Sabouraud dextrose agar (SDA) and broken basmati rice. The strains were stable and showed no decline in virulence against Tenebrio molitor, even when subcultured successively 12 times on SDA. Conidia‐bound Pr1 protease activity decreased in conidia harvested from SDA and mycosed cadavers after the 1st subculture, but increased in conidia produced on rice. The C:N ratio of conidia from mycosed cadavers was lower than that of conidia from rice or SDA. Irrespective of the number of subcultures, strain ARSEF 4556 produced significantly higher conidial yields than ARSEF 3297 and V275. The 12th subculture of V275 and ARSEF 3297 produced the lowest conidial yield. Shelf‐life studies showed that conidia of strain ARSEF 4556 had a higher conidial viability than V275 and ARSEF 3297 after 4 months, stored at 4°C. Conclusions: The current study shows that determining strain stability and conidial yield through successive subculturing is an essential component for selecting the best strain for commercial purposes. Significance and Impact of the Study: This is the first study to compare quality control parameters in the production of conidia on rice, and it shows that the level of Pr1 is comparatively high for inoculum produced on rice.     

Parallel stimulation of mitochondrial H+ (Mg2+-ATPase and rate of germ tube outgrowth from conidia of Neurospora crassa

Abstract Mitochondrial H⁺ (Mg²⁺)-ATPase (EC 3.6.1.3) activity of Neurospora crassa has been studied during conidial germination in the presence and absence of 2,4-dinitrophenol and dicoumarol. The activity was optimal at pH 8.5–9.0 and was inhibited (80–90%) by oligomycin, N , N '-dicyclo-hexylcarbodiimide and azide.
The enzyme activity already present in mitochondria isolated from dry- and wet-harvested conidia did not change significantly during the 2.5 h pregermination at 25°C (20% outgrowth), but increased sharply up to 7.5 h corresponding to the end of germination period and remained steady until the end of log phase (15 h).
In uncoupler-treated conidia, the stimulation of germ tube outgrowth at 2.5 h was found to parallel the activation (40%) of mitochondrial ATPase.     

Regulation and glutamic acid decarboxylase during Neurospora crassa conidial germination.

Glutamic acid decarboxylase (GAD) from Neurospora crassa was assayed in dormant and germinating conidia that had been permeabilized by toluene and methanol. N. crassa conidia contained 10 times the GAD activity found in vegetativemycelia. During conidial germination, GAD activity rapidly decreased to low levels before germ tubes appeared. GAD activity in germinating conidia closely followed the decreasing rate of glutamic acid metabolism. Inhibiting protein synthesis partially blocked the decrease in GAD activity, but eliminating exogenous carbon sources did not alter the initial rate of decrease in this enzyme. However, when conidia were incubated for more than 3 h in distilled water, GAD activity began to increase and eventually reached levels comparable to those in dormant conidia. Either GAD was reversibly inactivated or this enzyme could be synthesized from endogenous storage compounds when conidia were incubated in distilled water. These results are consistent with the hypothesis that GAD is a developmentally regulated enzyme that is responsible for catalyzing the first step in the metabolism of the large pool of free glutamic acid during conidial germination.     

Successive subculturing alters spore-bound Pr1 activity,germination and virulence of the entomopathogenic fungus,Beauveria bassiana

One of the hurdles in the development of entomopathogenic fungi such as Beauveria bassiana is loss of virulence when successively maintained in vitro. This may result in products of inferior quality in mass production programs. Also, there are many contradicting data and unclear points in this case. Three isolates of B. bassiana were subcultured successively 15 times. Spore-bound Pr1 activity, germination rate, and virulence of conidia against mealworm (Tenebrio molitor L.) larvae were studied. Results showed that isolates normally retained their virulence during 10 subculturings. However, they clearly offered decreased virulence (elevated LT₅₀ values and lower percent mortality). The activity of Pr1 bound to conidia declined as subculturing continued; the lowest spore-bound activity and germination potential of conidia was recorded for the 15th subculture. Virulence data were in agreement with Pr1 activity and germination rate as there was a positive correlation between germination rate and spore-bound Pr1 activity with fungal virulence. This explains that at least a part of attenuation in fungal virulence can be explored in enzymatic activity, especially in the important cuticle-degrading protease, Pr1.     

Effect of sunscreens,irradiance and resting periods on the germination ofMetarhizium flavoviride conidia

Chemical sunscreens were incorporated into oil formulations of conidia of two isolates of the entomopathogenic fungusMetarhizium spp. and the formulations exposed to simulated solar radiation. After 2 h exposure several sunscreens gave protection as demonstrated by conidial germination after 24 h incubation on gelatin plates at 26°C but only the formulations with Eusolex 8021 showed higher germination than the unprotected control after 48 h incubation. During 5h of exposure, Eusolex 8021 failed to offer significant protection as demonstrated by conidial germination after 48 h incubation. Conidial damage was proportional to the duration of radiation received. Allowing periods of darkness between exposures did not result in decreased loss of viability. Storing conidia, after exposure to simulated radiation, for 24 h prior to germination reduced their viability.     

白僵菌对茶小绿叶蝉田间防效的关键影响因素研究

白僵菌孢子萌发率和萌发速度是影响其田间防效的关键因素。通过对球孢白僵菌YP9(CCTCC No:M209238)孢子萌发的关键影响因素进行研究,得到以下田间应用条件:营养物质为2%麸皮浸出液(自然pH);孢子悬浮液喷洒浓度为5×10~7孢子/mL;在气温为23℃~28℃时喷洒。经昆明和无锡两试验地田间防效验证,该白僵菌在上述条件下对茶小绿叶蝉的田间防效高达65%,与茶农常用化学农药——天王星防效相当。     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Evaluation of Beauveria bassiana (Hyphomycetes) isolates as potential agents for control of Dendroctonus valens

Abstract The red turpentine beetle (RTB), Dendroctonus valens LeConte, as a destructive invasive pest, has become one of the most economically important forest pest in China. Effective control measures are desperately needed. Entomopathogenic fungi, such as Beauveria bassiana, have shown great potential for the management of some bark beetle species. In this study, 12 isolates of B. bassiana from bark beetle were examined for biological characteristics and virulence, to assess their potential as biocontrol agents for RTB. There were significant differences (at P= 0.05) in colony growth rate, conidial yield, conidial germination, tolerance to UV light and extracellular proteases activity among the tested B. bassiana isolates. Isolates, including Bb1801, Bb1906, Bb789 and Bb773, exhibited the best characteristics, because they have faster hyphal growth rate, higher spore production and faster spore germination, higher UV tolerance and protease (Pr1) production. The results of a pathogenicity test of B. bassiana on RTB larvae showed that most isolates of B. bassiana have demonstrated high efficacy and the highest virulent isolate was Bb1801, which killed 100% of the treated insects and had a median lethal time (LT₅₀) of 4.60 days at a concentration of 1×10⁷ conidia/mL. Therefore, isolate Bb1801 has a great potential for sustainable control of RTB in the forest. The correlation between biological characteristics and virulence of the fungal isolates is discussed and the possibility of combination of entomopathogenic fungi with semiochemicals, as one of the promising strategy for RTB control, is considered.     

Factors affecting sporulation and germination of Pandora nouryi (Entomophthorales: Entomophthoraecae), a pathogen of Myzus persicae (Homoptera: Aphididae)

Pandora nouryi discharged large numbers of primary conidia between 8 and 25°C from cadavers on the surface of water-agar. At 8°C conidial discharge lasted for 120 h, but most conidia were produced within 48 h when temperature was >15°C. Saturated humidity alone was not enough to allow for sporulation to occur freely and where RH?<?95%, no conidia were discharged. Light did not affect the pattern of conidial production nor the total number of conidia. Germination percentages of conidia on the surface of water-agar were 40 and 66% at 8 and 30°C, respectively, and were significantly lower than that at 15–25°C where germination was >95%. Conidia on leaves germinated well when RH?>?74%, while no germination occurred when RH?<?100% on cover slips. All eight insecticides tested entirely inhibited conidial germination at recommended doses (R), in particular, both the organophosphorus pesticides Lorsben (chlorpyrifos) and the organochlorine pesticides Thiodan (endosulfan) completely inhibited conidial germination even at 0.2R dose.     

Characterization of Beauveria bassiana and Metarhizium anisopliae isolates for management of tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Selected morphological and physiological characteristics of four Beauveria bassiana (Balsamo) Vuillemin isolates and one Metarhizium anisopliae (Metschnikoff) Sorokin isolate, which are highly pathogenic to Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), were determined. There were significant differences in conidial size, viability, spore production, speed of germination, relative hyphal growth, and temperature sensitivity. Spore viability after incubation for 24h at 20 degrees C ranged from 91.4 to 98.6% for the five isolates tested. Spore production on quarter-strength Sabouraud dextrose agar plus 0.25% (w/v) yeast extract after 10 days incubation at 20 degrees C ranged from 1.6x10(6) to 15.5x10(6)conidia/cm(2). One B. bassiana isolate (ARSEF 1394) produced significantly more conidia than the others. Spore germination was temperature-dependant for both B. bassiana and M. anisopliae. The time required for 50% germination (TG(50)) ranged from 25.0 to 30.9, 14.0 to 16.6, and 14.8 to 18.0h at 15, 22, and 28 degrees C, respectively. Only the M. anisopliae isolate (ARSEF 3540) had significant spore germination at 35 degrees C with a TG(50) of 11.8h. A destructive sampling method was used to measure the relative hyphal growth rate among isolates. Exposure to high temperature (40-50 degrees C) for 10min had a negative effect on conidial viability. The importance of these characteristics in selecting fungal isolates for management of L. lineolaris is discussed.     

Effect of temperature and sources of carbon and nitrogen on the conidial germination and appresoria formation in Colletotrichum capsici

The effect of temperatures and exogenous supply of different carbon and nitrogen sources on the conidial germination and appresoria formation inC. capsici has been studied. 25 °C was observed to be the best temperature for conidial germination. At 18 °C, conidia germinated only in hanging drops and did not germinate on 2 % agar. Amongst carbon sources, 1-rhamnose supported maximum conidial germination and appresoria formation. Potassium nitrate supported very good conidial germination and appresoria formation. Ammonium nitrate, ammonium sulphate and ammonium chloride were found to be inhibitory. A few aminoacids stimulated conidial germination but dl-alanine, dl-methionine, tyrosine and l-glutamine were found to be inhibitory.     

Control of the synthesis, activity, and turnover of enzymes of sulfur metabolism in Neurospora crassa

The synthesis of aryl sulfatase, choline-O-sulfate permease, and two distinct sulfate permeases are repressed by methionine, but the activity of these enzymes is not subject to feedback inhibition. The permease species, but not aryl sulfatase, are also regulated by dynamic turnover, displaying a functional half-life of approximately 2 hr. The rate of turnover of these permeases is not influenced by the presence of the end product, methionine. Development of sulfate permease activity occurs only by de novo synthesis which requires both a lifting of methionine repression and a functional cys-3 product. The turnover system for sulfate permease is not present in dormant conidia but appears to be synthesized relatively rapidly during germination. Preexisting conidial sulfate permease is lost by turnover during germination and outgrowth into the mycelial phase, during which both permease species are synthesized anew, although the high affinity system contributes most of the total activity in growing mycelia.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.     

In vitro conidial germination in Arthrinium aureum and Arthrinium phaeospermum

This paper describes the microscopic details of conidial germination and the influence of pH, sodium chloride concentration, 3% glucose, 3% saccharose and ultraviolet irradiation on the conidial germination in Arthrinium species. Under laboratory conditions, germination started after an incubation period of 90 minutes in 2% malt extract broth at 25 degrees C. In vitro, the conidia of Arthrinium species have a very low percentage of germination (A. phaeospermum: 7.9%; A. aureum: 15.8%). Conidia of this genus have a characteristic equatorial slit. Conidia may break spontaneously at this slit, releasing their cytoplasmic contents. Arthrinium phaeospermum attains its optimum germination percentage when its conidia are suspended in a sterile saline solution (pH 3.5) in a water bath at 20 degrees C for 15 minutes before being inoculated on 2% malt extract agar. Conidial suspensions of A. aureum may be held in the same conditions, but for 30 minutes.     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

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.