Medium components and culture conditions affect the thermotolerance of aerial conidia of fungal biocontrol agent Beauveria bassiana

AIMS: To produce more thermotolerable conidia of Beauveria bassiana, a well-known fungal biocontrol agent, by optimizing the medium components and culture conditions. METHODS AND RESULTS: The conidia produced on media including 0.5-6% glucose, sucrose or starch as carbon source and 50-300-microg ml(-1) Cu2+, Zn2+, Mn2+ or Fe3+ as additive to Sabouraud dextrose medium at 15-30 degrees C, pH 4-8 or KCl-adjusted water availabilities were exposed to 30-min wet heat stress at 48 degrees C. The medium components for conidial production with greatly enhanced thermotolerance included 4% glucose as optimum or 1% starch as alternative for the carbon source and < or =50-microg ml(-1) Mn2+ for the metal additive. The culture conditions were optimized as 25 degrees C and pH 5-6. Conidial thermotolerance decreased remarkably when sucrose and Fe3+ or Cu2+ were used in the cultures, but altered slightly when 50-200-microg ml(-1) Zn2+ were included. CONCLUSIONS: The tolerance of B. bassiana conidia to the thermal stress was significantly affected by the medium composition and culture conditions under which the conidia were produced. SIGNIFICANCE AND IMPACT OF THE STUDY: Proper treatment of small grains as mass production substrates for more glucose release and supplement of glucose or 50-microg ml(-1) Mn2+ are possible means to enhancing conidial thermotolerance and field persistence for improved insect control.     

蝉拟青霉LB菌株的生物学特性

本文研究碳源、氮源、温度、湿度、pH值和光照等对蝉拟青霉LB菌株生长、产孢和孢子萌发的影响.结果表明,适合该菌株菌落生长和产孢的最佳碳源是可溶性淀粉和蔗糖,最佳氮源为蛋白胨;菌丝生长和孢子萌发的最适温度范围是25℃～27℃,产生分生孢子的最适温度是25℃;分生孢子萌发所需湿度范围是RH 90%～100%,当RH低于90%时很难萌发;在pH值4～10的范围内该菌能生长和产孢,菌丝生长最适pH为6,产生分生孢子和孢子萌发最适pH范围为6-7;光照处理对该菌产孢有一定的影响;分生孢子的致死条件为55℃ 10min.生物学特性显示,蝉拟青霉LB菌株是一株对营养要求不高、对环境适应能力较强的昆虫病原真菌.     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

Production of a Thermostable beta-d-Galactosidase by Alternaria alternata Grown in Whey

In the course of exploring new microbial sources of extracellular beta-d-galactosidase (EC. 3.2.1.23), Alternaria alternata was found to excrete elevated quantities of a thermostable form of the enzyme when cultivated in whey growth medium. Optimum cultural conditions for maximum enzyme production were a whey lactose concentration of 6%, supplementation of the medium with 0.050 M (NH(4))(2)SO(4), an inoculum size of 10 conidia per ml, and a cultivation time at 28 to 30 degrees C of 5 days. The fungus utilized whey lactose for the production of the enzyme most efficiently, and the observed maximum yield, 280 nanokatals of hydrolyzed o-nitrophenyl-beta-d-galactopyranoside per g of whey lactose, was comparable to maximum yields reported for certain commercial fungi. The optimum pH and temperature of the enzymatic reaction were 4.5 to 5.5 and 60 to 70 degrees C, respectively, and the enzyme lost half of its activity when heated at 65 degrees C for 84 min. These properties make the enzyme particularly suitable for processing acid and less-acid (pH 5 to 6) dairy products and by-products.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Formation and Germination of Septoria tritici Secondary Conidia as Affected by Environmental Factors

The effects of medium, isolate, temperature, light and pH on the formation and germination of Septoria tritici secondary conidia were tested. Of the six media tested, the malt–yeast extract agar was the best and generated 1.82 × 10⁹ conidia/plate. The ten isolates tested showed different ability of conidia production. Darkness significantly reduced conidial formation and enhanced the transition of intermediates. The conidial germination and germ tube growth was strongly inhibited at 30°C. The suitable pH for conidial budding in malt–yeast broth (MYB) was between 5 and 9. At pH 2, 10 and 11, almost no new conidia were formed. The number of conidia reached 1.27 × 10⁸ conidia/ml after 7 days in MYB, significantly more than that in potato dextrose broth, wheat leaf extract and H₂O.     

肝素酶产生菌的筛选及发酵条件

从土壤中筛选到一株活性较高的肝素酶产生菌株Corynebacterium sp.。培养及产酶最佳条件表明,最适培养基组成(g/L)：胰蛋白胨20,氯化钠1,磷酸氢二钾25,硫酸镁05,麦芽糖20,pH65。最适生长温度27℃,最佳产酶温度31℃。在500mL三角瓶中装40mL培养基,在30℃,200r/min摇床上培养24 h,每升发酵液可产酶1700u。     

鞘胺醇杆菌肝素酶的产生

肝素类分子是一类结构异常复杂的高度硫酸化的糖胺聚糖,是临床上的一种主要的抗凝剂。除了抗凝血及其相关的抗栓活性以外,肝素还具有多种其他生物学功能,如抗平滑肌细胞及肾小球系膜细胞的增殖^[1]、抗炎症^[2]和阻止肿瘤生长及转移的作用^[3]等。因而肝素可用于防治球囊扩充血管成型术后的血管再狭窄、肾小球系膜细胞增生性肾炎、病理性炎症及肿瘤。但由于完整肝素的抗凝血活性,会引起出血及血小板减少综合症等负作用,限制了肝素在这些方面的临床应用。研究表明,肝素的抗凝血活性依赖于一种独特的肝素五糖序列,约占完整肝素链的1／3,除了五糖序列以外,还需要附近的至少13糖残基,因而不含五糖序列或含五糖序列小于18糖的肝素其抗凝活性大大降低^[4]。而六糖以上的肝素片段就具有抗平滑肌细胞增生、抗炎症及抗肿瘤的活性,并且这些活性与抗凝活性无关。因此,可利用特异性的肝素酶降解肝素长链,产生一系列不同结构及大小的肝素片段,并从中筛选出具有不同生物活性的片段。     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of temperature and relative humidity on sporulation of Metarhizium anisopliae var. acridum in mycosed cadavers of Schistocerca gregaria.

The effects of relative humidity (RH) and temperature on the sporulation of Metarhizium anisopliae var. acridum on mycosed cadavers of desert locust, Schistocerca gregaria, were assessed in the laboratory. Quantitative assessments of conidial production over 10 days under constant conditions showed that sporulation was optimized at RH > 96% and at temperatures between 20 and 30 degrees C. Under both these conditions >10(9) conidia/cadaver were produced. At 25 degrees C, conidial yield was maximized under conditions in which cadavers remained in contact with damp substrate. Relatively little sporulation occurred at 15 degrees C (< 3 x 10(7) conidia/cadaver) and 40 degrees C (< 4 x 10(6) conidia/cadaver) and no sporulation occurred at 10 or 45 degrees C. Following incubation, conidial yield was closely related to the water content of locust cadavers. In separate tests, locust cadavers were incubated for 10 days under diurnally fluctuating temperature and RH that comprised favorable (25 degrees C/100% RH) alternating with unfavorable (40 degrees C/80% RH) conditions for sporulation. In this case, fewer conidia were produced compared with cadavers that were incubated under the favorable conditions for an equal period cumulatively but were not periodically exposed to unfavorable conditions. However, this reduced sporulation observed with the fluctuating condition was not observed when cadavers were similarly incubated under favorable/unfavorable conditions of temperature but were not periodically exposed to the low RH condition. This result implies that sporulation is a dynamic process, dependent not only on periodic exposure to favorable RH but also on the interrelation of this with low RH. Associated tests and the monitoring of changes in cadaver weights imply that the mechanism driving the reduced sporulation under fluctuating RH is the net water balance of cadavers, i.e. the cumulative ability of the fungus/cadaver to adsorb water necessary for sporulation at high RH is restricted by water loss associated with intermittent exposure to a low RH. The duration of daily exposure to high humidity appears to be a crucial constraint to the recycling ability of M. anisopliae var. acridum.     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia.

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.     

The effects of some storage conditions on viability of Lecanicillium lecanii conidia to whitefly (Homoptera: Trialeurodes vaporariorum)

A study on the survival of Lecanicillium lecanii conidia in storage at room temperature was carried out. Firstly, drying methods of conidia powder were compared. Vacuum-freeze drying (VFD) was more suitable for drying conidia as compared to vacuum drying (VD) at room temperature. Vacuum-freeze drying for 24-h resulted in a water content of 5.4%, and a viability, determined as germination of conidia in 2% glucose solution after16 h, was 90.3% and the infection in greenhouse whitefly, Trialeurodes vaporariorum was about 94.7% at a dose of 1×10⁸ conidia/mL. Secondly, the factors influencing viability of conidia stored at room temperature were evaluated in the laboratory. Temperature was the most critical factor influencing conidial storage stability, among the tested factors affecting survival of conidia stored at room temperature for 6 months. Both conidial germination and infection of hosts decreased with storage temperature increasing from 15 to 35°C, and at 35°C the survival of stored conidia for 6 months was near zero. The moisture content of the conidial powder was another major factor influencing viability of stored conidia at room temperature. Conidial powder dried to about 5% moisture content showed higher viability than non-dried conidial powder. For the carriers, clay and charcoal were more suitable for storage of L. lecanii conidia at room temperature. At a room temperature of 25°C, L. lecanii conidia which were dried to 5% water content and mixed with clay or charcoal could retain about 50% survival after 6 months' storage.     

Acid phosphatases of Sporothrix schenckii

Sporothrix schenckii cells were grown on a medium containing yeast extract, neopeptone and glucose at 20 degrees C to obtain a mixture of mycelia and conidia, and at 35 degrees C to obtain yeast-like cells. The organism was maintained in the mycelial form, and its transformation to yeast at the higher temperature proceeded via conidia and 'intermediate cells' that then gave rise to yeast by a blastic mechanism. Cell-free extracts were analysed by PAGE at pH 8.0 and acid phosphatases (EC 3.1.3.2) were revealed by a sensitive detection reagent at pH 5.0. Mycelial, conidial and yeast extracts all had some acid phosphatase activity (M-I, C-I and Y-I) at the origin, although the proportion was highest for the yeast extracts. All of the bands that penetrated the gels had different electrophoretic mobilities. Mycelial and conidial extracts each had one other isoenzyme (M-II and C-II), while the yeast extracts had a total of five electrophoretically distinct acid phosphatases. Isoenzyme Y-II was further resolved into five closely related bands (Y-IIa to Y-IIe), the relative intensities of which varied with the phosphate nutrition of the yeast cells and the history of the extracts. The acid phosphatase isoenzymes were inhibited to various extents by sodium fluoride, L(+)-tartrate and phosphate, and showed interactions with citrate as opposed to acetate as the background buffer at pH 5.0.     

The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h(-1) and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.     

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.     

Optimization of alkaline protease production by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis> ES1 in <Emphasis Type="Italic">Mirabilis jalapa</Emphasis> tuber powder using statistical experimental design

Medium composition and culture conditions for the bleaching stable alkaline protease production by Aspergillus clavatus ES1 were optimized. Two statistical methods were used. Plackett-Burman design was applied to find the key ingredients and conditions for the best yield. Response surface methodology (RSM) including full factorial design was used to determine the optimal concentrations and conditions. Results indicated that Mirabilis jalapa tubers powder (MJTP), culture temperature, and initial medium pH had significant effects on the production. Under the proposed optimized conditions, the protease experimental yield (770.66 U/ml) closely matched the yield predicted by the statistical model (749.94 U/ml) with R (2)=0.98. The optimum operating conditions obtained from the RSM were MJTP concentration of 10 g/l, pH 8.0, and temperature of 30 degrees C, Sardinella heads and viscera flour (SHVF) and other salts were used at low level. The medium optimization contributed an about 14.0-fold higher yield than that of the unoptimized medium (starch 5 g/l, yeast extract 2 g/l, temperature 30 degrees C, and pH 6.0; 56 U/ml). More interestingly, the optimization was carried out with the by-product sources, which may result in cost-effective production of alkaline protease by the strain.     

Effects of stabilizers on shelf-life of Epicoccum nigrum formulations and their relationship with biocontrol of postharvest brown rot by Monilinia of peaches

AIM: To find a formulation of Epicoccum nigrum conidia that maintains a high viability over time and which proves efficient to biocontrol peach rot caused by Monilinia spp. METHODS AND RESULTS: We tested the effect of stabilizers and desiccants on the shelf-life of Epicoccum nigrum conidia. Conidial samples were dried for 40 min at 40 degrees C in a fluidized bed-dryer to obtain moisture contents <15%. The toxicity of additives was tested by assaying production of conidia in fermentations and germinability of the produced conidia: 50% PEG300, 10%-5% KCl (stabilizers) and 95.24% Cl(2)Ca (desiccant) significantly (P = 0.05) reduced conidial germination. To enhance shelf-life of dried conidia, nontoxic stabilizers were added at the following different stages of the production-drying process: (i) to substrate contained in bags before production, (ii) to conidial centrifuge pellets obtained after production, before filtering and drying, (iii) to conidial centrifuge pellets obtained after production, before adding talc and drying, and (iv) to conidial centrifuge pellets obtained after production, before adding silica powder and drying. Conidial germinability was tested at 0, 180 and 365 days after storage at room temperature. Shelf-life of formulations retaining the highest viability were conidia produced with 1% KCl or 50% PEG 8000, conidia dried with 2.5% methylcellulose, and conidia dried with 1% KCl + silica powder. All these formulations improved the shelf-life of E. nigrum conidia and significantly reduced brown rot on peaches. CONCLUSIONS: Our results show that additives improve the shelf-life of E. nigrum and assist controlling brown rot on peaches. SIGNIFICANCE AND IMPACT OF THE STUDY: New improved formulations of a biocontrol agent have been obtained which will improve the control of Monilinia on peach.     

Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus

The June beetle, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), has become a widespread and destructive insect pest of lawns, sport turf, pastures, and horticultural crops in Belgium. The virulence of 34 entomopathogenic fungal isolates from the genera Metarhizium, Beauveria, and Paecilomyces to third-instar H. philanthus was tested in bioassays by dipping larvae in 10(7)conidia/ml suspensions. Two isolates of Metarhizium anisopliae (CLO 53 and CLO 54) caused maximally 90% mortality 10 weeks post-inoculation while other isolates only caused mortalities between 10 and 62%. The virulence of M. anisopliae CLO 53 was further tested by exposing H. philanthus larvae to conidial serial concentrations of 10(4)-10(9)conidia/g sandy soil for up to 11 weeks at 15, 20 or 25 degrees C. Mortality was dependant on the fungal concentration, exposure time, and temperature. Eleven weeks after inoculation, the LC50 values for this isolate ranged from 1.3 to 4.0 x 10(6), 1.0 to 3.2 x 10(5), and 2.5 x 10(4) to 10(5)conidia/g soil at 15, 20, and 25 degrees C, respectively. The LT50 values for this isolate ranged from 3.5 to 21.7, 2.4 to 18.7, and 2.9 to 16.1 weeks at concentrations of 10(9) and 10(4)conidia/g soil at 15, 20, and 25 degrees C, respectively. In glasshouse pot experiment with perennial ryegrass (Lolium perenne L.), the isolate CLO 53 caused mortalities of 50 and 88% of H. philanthus larvae 10 weeks after application of 10(4) and 10(6)conidia/cm(2) soil surface, respectively. The present results suggest that the Belgian isolate CLO 53 has excellent potential for biological control of H. philanthus.