Mortality in Three African Tephritid Fruit Fly Puparia and Adults Caused by the Entomopathogenic Fungi, Metarhizium anisopliae and Beauveria bassiana

The pathogenicity of 13 isolates of Metarhizium anisopliae and two isolates of Beauveria bassiana to Ceratitis capitata and Ceratitis var. rosa fasciventris exposed as late third instar larvae in sand was evaluated in the laboratory. All isolates caused a significant reduction in adult emergence and a corresponding large mortality on puparia of both species. All isolates also induced large deferred mortality in emerging adults following treatment as late third instar larvae. On C. capitata , seven isolates ( M. anisopliae ICIPE 18, 20, 32, 60 and 69 and B. bassiana ICIPE 44 and 82) caused significantly higher mortality on puparia than other isolates. With the exception of ICIPE 32, the other four isolates of M. anisopliae above were the most pathogenic against C. r. fasciventris . Dose-response study carried out with these isolates of M. anisopliae on the two species of flies above plus another species, Ceratitis cosyra showed that the dose-mortality regression lines of ICIPE 18 and 20 were steeper with lower LC 50 values when compared with ICIPE 60 and 69 on the three species. When these two isolates were evaluated with regard to their pathogenicity to different pupal age, adult emergence was found to increase with increasing pupal age with a corresponding decrease in mortality in puparia and emerging adults in the three species of fruit flies. M. anisopliae ICIPE 18 and 20 were equally pathogenic to all pupal ages tested in C. capitata and C. cosyra but ICIPE 18 was more pathogenic to older puparia of C. r. fasciventris than ICIPE 20. Our results suggest that soil inoculation with M. anisopliae under mango trees might form an important component of integrated pest management strategies in areas where these three species of fruit fly coexist.     

Effect of temperature on virulence of Beauveria bassiana and Metarhizium anisopliae isolates to Tetranychus evansi

The virulence of three isolates of Beauveria bassiana (Bals.) Vuill. and 23 isolates of Metarhizium anisopliae (Metschnik.) Sorok. (Ascomycota: Hypocreales) against the tomato spider mite, Tetranychus evansi Baker and Pritchard (Acari: Tetranychidae), was assessed in the laboratory. The effect of temperature on germination, radial growth and virulence of selected isolates (two isolates of B. bassiana and nine of M. anisopliae) on T. evansi was also investigated in the laboratory. All the fungal isolates tested were pathogenic to the adult females of T. evansi, and there were significant differences in mortality between fungal isolates. The lethal time to 50% mortality (LT(50)) values ranged from 4.2 to 8.1 days and the LT(90) values from 5.6 to 15.1 days. Temperature had significant effects on germination, radial growth and virulence of the various isolates. The best fungal germination was observed at 25 and 30 degrees C, while for the fungal radial growth it was 30 degrees C. All the isolates germinated and grew at all temperatures, but germination and radial growth varied with isolate and temperature. The selected isolates were all virulent to T. evansi, but virulence varied also with isolate and temperature.     

Effect of soil temperature and moisture on survival and infectivity of Metarhizium anisopliae to four tephritid fruit fly puparia

The infectivity of 4 isolates of Metarhizium anisopliae to puparia of Ceratitis capitata treated as late third-instar larvae in unsterilized soil was investigated in the laboratory under controlled temperature and moisture. At 20-30 degrees C, mortality in puparia was highest at water potential of -0.1 and -0.01 mega Pascal (MPa) and lowest at water potential of -0.0055 and -0.0035 MPa in all the isolates. In wetter soil however, isolates ICIPE 20 and 60 caused significantly higher mortality than ICIPE 18 and 69. The survival of conidia in drier soil (-0.1 MPa) was not adversely affected at all temperatures. However, in wet soil (-0.0035 MPa) there was drastic reduction in colony counts in ICIPE 18 and 69 at 25 and 30 degrees C but conidial density in ICIPE 20 and 60 remained at the initial level at 14 days after inoculation at all temperatures. When ICIPE 20 was evaluated against three other fruit fly species (Ceratitis cosyra, Ceratitis rosa, and Ceratitis fasciventris), significant reduction in adult emergence and higher pupal mortality occurred in C. cosyra and C. fasciventris than in C. rosa at a combination of 15 and 20 degrees C and -0.1 and -0.0035 MPa. However, at higher temperature and the same moisture level, the isolates were equally pathogenic across the 3 species. It is probable that in addition to pathogen cycling and multiplication from dead infected insects in the soil, a balance between microbial degradation and replenishment of inoculum of virulent isolates occur through fluctuations in, and intricate interactions between temperature and moisture levels. This study is indicative of the potential of using isolate ICIPE 20 for soil inoculation against pupariating third-instar larva of fruit flies, thus providing a novel alternative to chemical soil application.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO4, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20-35 degrees C in both fungal species.     

Pathogenicity of Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo) Vuillemin,to three adult fruit fly species: Ceratitis capitata (Weidemann), C. rosa var. fasciventris Karsch and C. cosyra (Walker) (Diptera :Tephritidae)

The pathogenicity of two isolates of Beauveria bassiana and 12 of Metarhizium anisopliae towards adult fruit flies, Ceratitis capitata and Ceratitis rosa var. fasciventris was tested in the laboratory. Fruit flies were exposed to dry conidia evenly spread on velvet material covering the inner side of a cylindrical plastic tube. All isolates tested were pathogenic to both species of fruit flies. Mortality ranged from 7 to 100% in C. capitata and from 11.4 to 100% in C. rosa var. fasciventris at 4 days post-inoculation. Six isolates, M. anisopliae ICIPE 18, 20, 32, 40, 41 and 62, were highly pathogenic to both C. capitata and C. rosa var. fasciventris. The LT90 values of the most pathogenic isolates ranged between 3-4 days in both insects. Because of the difficulties in rearing C. cosyra, only the isolates that were highly pathogenic to both C. rosa var. fasciventris and C. capitata were tested against adult C. cosyra. They caused mortality of between 72-78% at 4 days post-inoculation. The LT90 values in all the isolates did not exceed 4 days. One of the most pathogenic isolates, M. anisopliae ICIPE 20, was evaluated against C. capitata and C. rosa var. fasciventris in cage experiments using three autoinoculators (maize cob, cheesecloth and Petri dish) in an autoinoculative device consisting of plastic mineral bottle. Mortality of between 70-93% was observed in flies of both species that were captured from the cages and held under laboratory conditions. These results indicate the possibility of fruit fly suppression with entomopathogenic fungi using an autoinoculative device.     

Viability and virulence of blastospores of Metarhizium anisopliae (Metch.) Sorokin after storage in various liquids at different temperatures

Blastospores of three strains of Metarhizium anisopliae were stored in 18 liquids at 4°C, 20°C and 35°C for 18 weeks, 12 weeks or 9 days respectively. Viability was quantified by determination of their germination. In bioassays the virulence of stored blastospores was studied using adults and third instars of Locusta migratoria migratorioides (R. & F.) and compared to those of freshly produced blastospores and conidia. Generally, there was great variability in the viability of blastospores, depending on the fungal strain and the liquids used. Blastospores survived best at 4°C in 10% hydroxyethyl starch; for example, germination of M. anisopliae strain 97 still amounted to more than 80% after storage for 18 weeks. Other suitable liquids were deionized water, 25% Ringer's solution and 1% sodium alginate. The viability of blastospores stored at 20°C was considerably shorter than at 4°C. During storage for 12 weeks at 20°C the best protective liquids for M. anisopliae strain 97 were 25% Ringer's solution (43% germination), deionized water (23%) and 10% hydroxyethyl starch (23%). At 35°C, 45% of M. anisopliae strain 97 blastospores still germinated after storage for 7 days in 25% glycerol. The bioassays revealed that the virulence of blastospores after storage was comparable to that of fresh ones and even better than that of fresh conidia. In general, the LT₅₀ was about 4–6 days at an alternating day/night temperature of 28/20°C.     

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.     

Effects of Entomopathogenic fungi and Bacillus thuringiensis‐based biopesticides on Spoladea recurvalis (Lepidoptera: Crambidae)

Spoladea recurvalis (Fabricius) is one of the most devastating pests of amaranths causing severe yield losses of 60%–100% to the crop. Unfortunately use of chemical pesticides is the most common control strategy that vegetable farmers rely on to control the pest. However, it is not effective and harmful to environmental and human health. Aiming to provide more environmentally friendly alternatives, this study evaluated the effects of various entomopathogenic fungal isolates and commercial based Bacillus thuringiensis Subsp. kurstaki product Halt^®, on the pest. Twenty‐four entomopathogenic fungal (EPF) isolates from three genera (14 Metarhizium anisopliae, 9 Beauveria bassiana and 1 lsaria fumosorosea) were screened in the laboratory to assess their pathogenicity against second instar larvae of S. recurvalis. Only M. anisopliae ICIPE 30 reached a moderate threshold, causing 58.3% larval mortality. All the 11 isolates (8 M. anisopliae, 2 B. bassiana and 1 l. fumosorosea) tested against adult S. recurvalis were pathogenic, with M. anisopliae ICIPE 30 and B. bassiana ICIPE 725 causing the highest mortality of 92% and 83%, respectively. Metarhizium anisopliae ICIPE 30 had the shortest LT₅₀ value of 4.8 days. Bacillus thuringiensis Subsp. kurstaki product Halt^® caused <50% mortality on S. recurvalis larvae. A consecutive application of M. anisopliae ICIPE 30 and Bt did not cause a significant increase in larval mortality compared to separate applications of both products. Results of this study suggest that M. anisopliae ICIPE 30 was the most potent candidate and could be used in an autodissemination approach for management of adult S. recurvalis.     

Production of blastospores by three strains of metarhizium anisopliae (metch.) sorokin in submerged culture

Studies on blastospore production in different liquid media were conducted with three strains of Metarhizium anisopliae var. anisopliae (M. a.) derived from various countries (M. a. 43: Austria, M. a. 57: Brazil, M. a. 97: Philippines). Variation of six fermentation parameters (cornsteep products, carbohydrates, pH values, temperature, Tween 80, and polyethyleneglycol (PEG) 200) disclosed that the three strains of M. anisopliae differed in their growth pattern and physiology. In standard medium and in all tests, M. a. 57 produced the highest number of blastospores invariably amounting to > 10⁸ per ml, while mycelial pellets were never formed. The preferred carbohydrates were glucose and fructose. Blastospore production of M. a. 43 was increased by growth at 30°C, at pH 6.5 or by addition of 5% PEG 200. However, it was impaired by different concentrations of Tween 80 or higher concentrations of PEG 200 (10–15%). M. a. 97 produced most blastospores at 30°C, and the strain preferred basic (pH 8.0) as well as acid (pH 4.5) media. Blastospore production was increased by the addition of 5% PEG 200 or 0.4–1.2% Tween 80. Moreover, PEG 200 suppressed pellet formation effectively. Altogether, our results showed that for optimal blastospore production of Metarhizium anisopliae, suitable strain‐specific parameters have to be evaluated.     

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.     

Variability in response to UV-B among species and strains of Metarhizium isolated from sites at latitudes from 61 degrees N to 54 degrees S.

The effects of irradiances of 920 and 1200 mW m(-2) (biologically effective weighted irradiance) were examined in 2 Metarhizium album strains, 26 M. anisopliae strains, 1 M. flavoviride strain, and 1 M. taii strain isolated from sites located at latitudes from 61 degrees N to 54 degrees S. Conidia were exposed to UV-B from 1 to 6 h and subsequently examined for relative percentage culturability. Total dosage received at the end of the exposure periods ranged from 3.3 to 19.9 kJ m(-2) for the lower irradiance and from 4.3 to 25.9 kJ m(-2) for the higher irradiance. Both the irradiance values and the doses are environmentally realistic and can be observed even in temperate regions. The relationships between latitude of origin and UV-B tolerance were compared for the two levels of irradiance for the data from 1 and 2 h exposure. Exposure to both irradiances drastically reduced the relative percentage culturability of all strains. Tolerance to UV-B varied widely among strains and high variation was observed for both irradiances after all periods of exposure. After 1 h of exposure, a difference between the two irradiance levels was detectable, and this difference was magnified at longer irradiations. A significant quadratic relationship of decreasing UV-B tolerance with increasing latitude was observed after exposure of 1 and 2 h. The shape of the relationship did not differ for the two levels of irradiance. Also, we studied the effect of 1200 mW m(-2) irradiance on conidial germination time in 1 M. album strain, 7 M. anisopliae strains, and 1 M. taii strain. Exposure to UV-B delayed the germination of surviving conidia of all strains. In general, the delay in germination was directly proportional to the dose.     

Enzyme activities associated with oxidative stress in Metarhizium anisopliae during germination, mycelial growth, and conidiation and in response to near-UV irradiation

Metarhizium anisopliae isolates have a wide insect host range, but an impediment to their commercial use as a biocontrol agent of above-ground insects is the high susceptibility of spores to the near-UV present in solar irradiation. To understand stress responses in M. anisopliae, we initiated studies of enzymes that protect against oxidative stress in two strains selected because their spores differed in sensitivity to UV-B. Spores of the more near-UV resistant strain in M. anisopliae 324 displayed different isozyme profiles for catalase-peroxidase, glutathione reductase, and superoxide dismutase when compared with the less resistant strain 2575. A transient loss in activity of catalase-peroxidase and glutathione reductase was observed during germination of the spores, whereas the intensity of isozymes displaying superoxide dismutase did not change as the mycelium developed. Isozyme composition for catalase-peroxidases and glutathione reductase in germlings changed with growth phase. UV-B exposure from lamps reduced the activity of isozymes displaying catalase-peroxidase and glutathione reductase activities in 2575 more than in 324. The major effect of solar UV-A plus UV-B also was a reduction in catalase-peroxidases isozyme level, a finding confirmed by measurement of catalase specific activity. Impaired growth of M. anisopliae after near-UV exposure may be related to reduced abilities to handle oxidative stress.     

Differential expression of genes involved in entomopathogenicity of the fungi Metarhizium anisopliae var. anisopliae and M. anisopliae var. acridum (Clavicipitaceae)

Expression analysis of the genes involved in germination, conidiogenisis and pathogenesis of Metarhizium anisopliae during its saprophytic and pathogenic life stages can help plan strategies to increase its efficacy as a biological control agent. We quantified relative expression levels of the nitrogen response regulator gene (nrr1) and a G-protein regulator of genes involved in conidiogenesis (cag8), using an RT-qPCR assay. Comparisons were made between M. anisopliae var. anisopliae and M. anisopliae var. acridum during germination and conidiogenesis and at different stages of pathogenesis. The cag8 gene was repressed during germination and induced during conidial development and the pathogenic phase, and the nrr1 gene was induced during germination, conidiogenesis and the pathogenic phase. Both genes were more expressed in M. anisopliae var. anisopliae, demonstrating that different varieties of M. anisopliae differ in activation of genes linked to virulence for certain environments and hosts. This suggests that differences among these varieties in the ability to adapt could be attributed not only to specific genomic regions and genes, but also to differential gene expression in this fungus, modulating its ability to respond to environmental stimuli.     

Germination, Radial Growth, and Sporulation of Beauveria bassiana and Metarhizium anisopliae Isolates and Their Virulence to Chilo partellus (Lepidoptera: Pyralidae) at Different Temperatures

The effect of temperature regimes on conidia germination, radial growth and virulence of Beauveriabassiana and Metarhizium anisopliae against the spotted stalk borer Chilo partellus was investigated with Ethiopian isolates. Conidia germination, radial growth and sporulation of all isolates were retarded at 15 and 35°C. A suitable temperature range for the isolates was between 20 to 30°C. Conidia germination was less tolerant of low temperature (15°C) than radial growth. Radial growth and sporulation reacted differently to temperature. At both 25 and 30°C, all isolates induced 100% mortality to C. partellus larvae in six days. The LT₅₀ decreased for the isolates with increasing temperature.     

Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

The effect of temperature and relative humidity on the formation of Metarhizium anisopliae chlamydospores in tick eggs

The influence of ambient conditions on the development of Metarhizium anisopliae chlamydospores in tick eggs is reported for the first time. The infection of tick eggs by M. anisopliae involves common events, such as adhesion, conidial germination, appressoria formation, invasion, and development within the eggs. However, the final stage of fungal development differs according to the environmental conditions. At high humidity (close to 100%) and moderate temperature (25°C) the fungus emerged from the eggs and formed conidiophores and conidia externally on the dead eggs. Elevating the temperature to 30°C or reducing humidity to 55-75% induced the production of chlamydospores inside the eggs, without conidiogenesis. When eggs with mature chlamydospores were returned to the appropriate conditions (25°C and 100% RH), conidiogenesis was recovered. Formation of chlamydospores, observed by means of histology and TEM, began with the thickening and septation of hyphae. As the chlamydospore wall thickened a new external undulated wall layer appeared. The mature chlamydospore in eggs has an oval shape (5.3 ± 0.9 microm long, 2.5 ± 0.2 microm wide); its wall comprises three distinct layers. The ability of M. anisopliae to produce chlamydospores under harsh conditions is advantageous and should be considered in application.     

The Influence of Water Activity and Temperature on Germination,Growth and Sporulation of <Emphasis Type="Italic">Stachybotrys chartarum</Emphasis> Strains

The objectives were to determine the influence of water activity (a_w, 0.997–0.92) and temperature (10–37°C) and their interactions on conidial germination, mycelial growth and sporulation of two strains of Stachybotrys chartarum in vitro on a potato dextrose medium. Studies were carried out by modifying the medium with glycerol and either spread plating with conidia to evaluate germination and germ tube extension or centrally inoculating treatment media for measuring mycelial growth rates and harvesting whole colonies for determining sporulation. Overall, germination of conidia was significantly influenced by a_w and temperature and was fastest at 0.997–0.98 a_w between 15 and 30°C with complete germination within 24 h. Germ tube extension was found to be most rapid at similar a_w levels and 25–30°C. Mycelial growth rates of both strains were optimal at 0.997 a_w between 25 and 30°C, with very little growth at 37°C. Sporulation was optimum at 30°C at 0.997 a_w. However, under drier conditions, this was optimum at 25°C. This shows that there are differences in the ranges of a_w x temperature for germination and growth and for sporulation. This may help in understanding the role of this fungal species in damp buildings and conditions under which immune-compromised patients may be at risk when exposed to such contaminants in the indoor air environment.     

Effect of temperature on growth, germination, germ-tube extension and survival of Paecilomyces lilacinus strain 251

Paecilomyces lilacinus and in particular the commercial strain 251 has been intensively tested for biological control of plant parasitic nematodes. Since this species has been mentioned in a number of reports concerning infection of humans, the human health risk for Paecilomyces lilacinus strain 251 was investigated. The effects of time, temperature and growth medium on radial colony growth and germination were determined. Additionally, exposure to 36°C and its effect on germ-tube extension and on survival of conidia was evaluated. Radial growth was significantly affected by temperature, growth medium and their interaction. Optimum temperatures were between 24 and 30°C, but no growth was found at 36°C. Germination rate was significantly influenced by time, medium, temperature and their interactions. The optimum temperature range for germination was between 28 and 30°C. Formulated conidia were capable of germinating at 36°C. However, studies on germ-tube extension conducted at 36°C showed a delay in development for 28-49 h and no further germ-tube extension was found after exposure for 80-95 h. Slopes of survival curves were significantly influenced by the type of conidia tested. In general, conidia did not survive exposure to 36°C for 168 h. These experiments indicate the temperature conditions where the strain is likely to be active and provide supporting data for full environmental and health risk assessments of biocontrol fungi.     

The Susceptibility of Boophilus annulatus (Ixodidae) Ticks to Entomopathogenic Fungi

The pathogenicity of five species of entomopathogenic fungi (Deuteromycetes, species: Beauveria bassiana, Metarhizium anisopliae, Metarhizium flavoviride, Paecilomyces fumosoroseus and Verticillium lecanii ) to the various developmental stages of Boophilus annulatus ticks was compared under laboratory conditions. M. anisopliae and B. bassiana strains were most virulent to engorged females and caused 85-100% mortality within 7-10 days post-inoculation (PI). The highest mortality of engorged females caused by other fungi reached only 25-60%. All tested fungi prevented or reduced the egg laying capability of the ticks several days before their death. Females surviving after treatment with the most virulent M. anisopliae strain (Ma-7) reached only 7-8% of their egg laying capacity as compared with the control. Other fungi caused a reduction of the weight of laid eggs by 35.4-80.8% as compared with untreated females. Only M. anisopliae and B. bassiana strains caused 70-98% mortality of the treated eggs. Unfed larvae of Boophilus annulatus were sensitive to M. anisopliae and M. flavoviride strains. The Ma-7 strain was most virulent to unfed larvae, with a mortality rate of 80.4% at a concentration of 1 ×107 spores ml -1 and 100% mortality at a concentration of 1 ×108 spores ml -1 .     

Variability in conidial thermotolerance of Metarhizium anisopliae isolates from different geographic origins

Notable variability in thermotolerance was found among conidia of 16 isolates of the insect-pathogenic fungi Metarhizium anisopliae var. anisopliae and one M. anisopliae var. acridum isolated from latitudes 61 degrees N to 54 degrees S. Conidial suspensions were exposed to 40 or 45 degrees C for 2, 4, 8, and 12 h. Most of the isolates tolerated 40 degrees C very well, with relative germination (germination relative to unheated controls) above 90% after 12 h of exposure. Exceptions were three isolates originating from high latitude, viz., ARSEF 2038 (38 degrees N, South Korea), 4295 (54.4 degrees S, Australia), and 5626 (61.2 degrees N, Finland) that had approximately 80% germination. High variability, however, was observed among isolates at 45 degrees C; viz., after 2 h exposure, relative germination was above 80% for six isolates, between 50 and 70% for three isolates, and between 0 and 30% for eight isolates. After 8 and 12 h at 45 degrees C, only two M. anisopliae isolates pathogenic to grasshoppers, viz., ARSEF 324 (latitude 19 degrees S, Australia) and 3609 (15 degrees N, Thailand), had high relative germination (91.6 and 79.4%, respectively, for 8 h exposures; and 90 and 47.1%, respectively, for 12 h). These isolates also were the most tolerant to UV-B radiation [J. Invertebr. Pathol. 78 (2001) 98-108]. The median lethal dose (LD50) for isolate ARSEF 324 was 49.4 and 47.9 degrees C, for 2 and 4 h of exposures, respectively. Exposure of conidia to wet-heat greatly delayed germination of some isolates. In general, isolates from higher latitudes demonstrated greater heat susceptibility than isolates from nearer the equator. Dry conidia tolerated 50 degrees C better than 45 degrees C in aqueous suspension.