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.     

Potential of a Dried Rice/Mycelium Formulation of Entomopathogenic Fungi to Suppress Subterranean Pests in Small Fruits

The potential of a dried rice/mycelium formulation of three species of entomopathogenic fungi ( Metarhizium anisopliae, Beauveria bassiana and Paecilomyces farinosus ) was assessed according to five criteria: storage life, field efficacy against two subterranean pests of cranberry, persistence and efficacy against first-instar black vine weevil larvae in potted strawberry and field persistence in a cranberry bog. The number of conidia sporulated from the formulation did not decline between 4 and 20 days. In small plot field trials, the numbers of black vine weevil, Otiorhynchus sulcatus (F.), were significantly lower in plots treated with 1000 gm - 2 of formulated M. anisopliae than in untreated plots. In similar trials, the numbers of adult cranberry girdler, Chrysoteuchia topiaria (Zeller), inside emergence cages were significantly lower on plots treated with the formulated M. anisopliae than on untreated plots. The formulation suppressed first-instar black vine weevil larvae in potted strawberry regardless of the species of entomopathogenic fungus or the post-inoculation interval. M. anisopliae was periodically isolated for up to 637 days from plots in cranberry bogs treated with the formulation. In general, the dried rice/mycelium formulation may be a useful fungal mycopesticide for the management of subterranean pests of small fruits, especially if applied at high rates to areas of serious pest infestation.     

Development of a Spray-drying Technique for Submerged Spores of Entomopathogenic Fungi

The stability of blastospores or submerged conidia of entomopathogenic fungi is one of the key problems associated with the practical use of fungal biopreparations. A spray-drying technique was developed which allows the drying of blastospores and/or submerged conidia, here called submerged spores, without a significant loss of viability and with only a slight delay in eY cacy. The method was tested successfully using the fungal species Metarhizium anisopliae , M. flavoviride, Beauveria bassiana and Paecilomyces fumosoroseus . For all experiments, a laboratory spray dryer was used. The optimum inlet and outlet temperatures were found to be 64 2oC and 48 2oC respectively. The best protective agent for submerged spores was skimmed milk powder at concentrations of 10 or 20%. The addition of sugars, especially 2.5% sugar-beet syrup, slightly improved the viability after spray-drying. Submerged spores of eight isolates of M. anisopliae, M. flavoviride, B. bassiana and P. fumosoroseus were suspended in 20% skimmed milk powder and 2.5% sugar-beet syrup, and spray-dried. Germination rates of about 90% were achieved, which were comparable to those of freshly produced submerged spores. Finally, the virulences of spray-dried submerged spores of M. anisopliae (Ma 97) and M. flavoviride (Mfl 5) were tested in bioassays using third- and fourth-instar nymphs of the African locust, Locusta migratoria . In contrast to M. flavoviride , there was no significant diVerence in the median lethal time between spray-dried and fresh submerged spores of M. anisopliae . The investigations demonstrated that spray-drying is possible to preserve sensitive submerged spores of entomopathogenic fungi.     

Influence of Pesticide Use on the Natural Occurrence of Entomopathogenic Fungi in Arable Soils in the UK: Field and Laboratory Comparisons

The spectrum and abundance of entomopathogenic fungi in agricultural soil receiving different pesticide applications were evaluated . Seven small field plots within a barley crop were selected . Each plot had received a different pesticide treatment at slightly higher than the field rate each year for the previous 12-19 years . The field plots received either benomyl (fungicide) , triadimefon (fungicide) , aldicarb (insecticide) , chlorfenvinphos (insecticide) , glyphosate (herbicide) , all five of these pesticides or no pesticides at all (control) . Soil sampled from each plot was baited with Galleria mellonella larvae at either 18 or 26 C . Five species of entomopathogenic fungi infected these larvae . Beauveria bassiana was the dominant species , and the only species for which infection levels were high enough to be analyzed statistically . Significantly fewer G. mellonella larvae became infected with B. bassiana in soil treated with benomyl than in other treatments . This deleterious effect was confirmed in in vitro experiments where benomyl inhibited fungal growth significantly . Chlorfenvinphos also reduced the proportion of G. mellonella becoming infected with B. bassiana slightly (although not statistically significantly) . This effect was much greater in in vitro experiments in which chlorfenvinphos inhibited fungal growth significantly . B. bassiana- induced mortality was significantly greater in G. mellonella larvae placed in soil treated with triadimefon than in other treatments . In vitro studies , however , demonstrated that triadimefon inhibited rather than stimulated growth . A similar anomaly between field and laboratory observations was seen in the case of aldicarb . There was no significant difference in the number of G. mellonella larvae found infected with B. bassiana in soil treated with aldicarb than in the control soil . However , in the in vitro studies , aldicarb stimulated fungal growth at all concentrations except 10 times the field rate . In general , significantly fewer larvae became infected with B. bassiana at 26 C than at 18 C in all treatments . Pesticides may have a direct impact on the natural occurrence , infectivity and population dynamics of entomopathogenic fungi . They also affect other macro - and microorganisms in the soil which may interact with the ento mopathogenic fungi . In combination with climate and soil variables , these effects are difficult to separate . Although any suppression of these fungi may be detrimental to their capacity as natural enemies , this study has demonstrated that pesticides used under field conditions are unlikely either to kill all the entomopathogenic fungi present in the treated area or to limit their recolonization . It seems likely , therefore , that pesticides have the potential to be used in conjunction with mycoinsecticides in integrated pest management systems .     

Susceptibility of Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) to Entomopathogenic Fungi from Ethiopia

The efficacy of 13 isolates of entomopathogenic fungi belonging to Beauveria , Metarhizium or Paecilomyces spp. was assessed against Sitophilus zeamais (Coleoptera: Curculionidae) and Prostephanus truncatus (Coleoptera: Bostrichidae) using a total immersion bioassay technique in the laboratory. Fungi were applied at concentrations of 1 ×10 7 and 1 ×10 8 conidia mL -1 for P. truncatus and S. zeamais , respectively. All isolates tested were virulent to P. truncatus (98-100% mortality, and median survival time (MST) ranged from 2.85-4.05 days). Metarhizium anisopliae and B. bassiana were also virulent to S. zeamais (92-100% mortality, MST ranged from 3.58-6.28 days). The isolate of Paecilomyces sp. was found to be the least virulent against S. zeamais , causing only 26.32 ±4.29% mortality with MST of 10.38 ±0.29 days. P. truncatus proved more susceptible to the entomopathogenic fungi tested than S. zeamais . One M. anisopliae (PPRC-EE) and three B. bassiana isolates (PPRC-HH, PPRC-9609 and PPRC-9614) were selected for further study and dose-mortality relationships were assessed on S. zeamais . The tested concentrations ranged from 1 ×10 4 -1 ×10 7 conidia mL -1 . M. anisopliae (PPRC-EE) showed the lowest LC 50 (3.39 ×10 5 conidia mL -1 ) followed by B. bassiana PPRC-HH (2.04 ×10 6 conidia mL -1 ). PPRC-9609 and PPRC-9614 showed slight differences in LC 50 but not at LC 90 . The results revealed the higher potency of M. anisopliae as compared with the B. bassiana isolates tested. The study suggests that the use of entomopathogenic fungi may hold promise as an alternative method to control pests of stored-products in Ethiopia.     

Infection of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Scolytidae) by Brazilian Isolates of the Entomopathogenic Fungi Beauveria bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes)

The virulence of four fungal isolates (three Beauveria bassiana and one Metarhizium anisopliae ) against adult female coffee berry borers (CBB) was investigated. The most virulent isolate from initial bioassays, B. bassiana LPP1, with a LT 50 of 3.4 days, was further investigated by application to berries prior to infestation and to berries already infested with CBB. At the highest concentration applied to berries (1 ×10 7 conidia mL -1 ), CBB mortality was 83% (berries inoculated prior to infestation) and 62% (berries inoculated after infestation).     

Entomopathogenic Fungi Associated with Natural Populations of the Moroccan Locust Dociostaurus maroccanus (Thunberg) (Orthoptera: Gomphocerinae) and Other Acridoidea in Spain

The entomopathogenic fungi Beauveria bassiana Vuill . and Metarhizium anisopliae (Metschnikoff) have been found in natural populations of the Moroccan locust Dociostaurus maroccanus (Thunberg) and other species of acridoids that cohabit the same locust breeding areas in southern Spain . Infection levels of B. bassiana on insects collected in the field and maintained under laboratory conditions were relatively high (1 . 6 - 20 . 5%) . The prevalence of the disease extended from spring to summer in the three consecutive years monitored . A local isolate of this fungus demonstrated high virulence (LD = 440 conidia / insect) against the 50 locust D. maroccanus in the laboratory bioassay . The relatively wide host range of B. bassiana and its adaptation to the dry and hot conditions dominating the locust breeding area suggest that this isolate could be considered in the development of a biological control programme against D. maroccanus.     

Screening for Virulent Isolates of Entomopathogenic Fungi Against the Desert Locust, Schistocerca gregaria Forskal

The pathogenicity of 159 isolates of Metarhizium and Beauveria spp. was screened using Schistocerca gregaria adults. The median lethal time MLT , the average survival time and its standard deviation SD were used to describe the rate of kill from the cumulative observed response curve over time, following the application of conidia in bioassays. All screens included a standard strain of Metarhizium sp. from a single spore of IMI 330189ss , which gave an average MLT value of 4.4 days SD 0.4 in 46 assays. Approximately 50 isolates, all belonging to the genus Metarhizium, showed virulence that was comparable with this strain. Two previously unreported preliminary tests were carried out. The first test assessed the response to the standard isolate by an established laboratory stock of desert locusts compared with a recently introduced locust stock from Niger. There were insignificant differences in MLT and LD50 on day 5 between the different locust strains. The second test assessed the effects of inoculation at different times after the final ecdysis. This similarly had little effect, although applications before day 10 appeared to minimize the risk of control mortality. A computer program was developed to store bioassay results in a standardized format and to produce quick estimates of these statistics.     

Evaluation of Beauveria bassiana and Metarhizium anisopliae for Controlling Chilo partellus (Lepidoptera: Crambidae) in Maize

Maize (variety Katumani) was planted in a greenhouse and plants were infested with 20 Chilo partellus second instar larvae 3 and 4 weeks after plant emergence. One isolate of Beauveria bassiana (BB-01) and four isolates of Metarhizium anisopliae (PPRC-4, PPRC-19, PPRC-61 and EE-01) were sprayed onto the leaf whorl at 2×10⁸ conidia/mL 24 h after infestation. Leaf damage by the larvae was greatly reduced by the treatments. The mean daily temperature and relative humidity in the greenhouse ranged from 10 to 35°C and 30 to 90%, respectively. The growth of infested unsprayed plants was less than that of fungi treated plants. Stem tunneling (1-5%), deadheart (0-33%), number of attacked nodes (0.3-2.5) and holes (0.2-3.3) were also reduced in plants sprayed with conidial suspensions. Isolates PPRC-4, PPRC-19 and PPRC-61 seemed to be the best candidates for further development.     

Proteases Released by Entomopathogenic Fungi Impair Phagocytic Activity, Attachment and Spreading of Plasmatocytes Isolated from Haemolymph of the Greater Wax Moth Galleria mellonella

Reliable assays for the in vitro quantification of the attachment and spreading of isolated Galleria mellonella plasmatocytes have been established. The effects of extracellular proteases released by the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana on the phagocytic activity, spreading, attachment and cytoskeleton formation of isolated plasmatocytes of G. mellonella were examined to elucidate their putative role in the suppression of cellular immune responses in infected insects. In addition, the influence of different commercially available proteases on isolated plasmatocytes was studied. Among the proteases tested, the metalloprotease thermolysin produced the strongest inhibitory activity on plasmatocytes. The results obtained support the conclusion that invading fungal cells could interfere with the insect immune system via the release of proteases which affect cellular defence reactions. Isolated G. mellonella plasmatocytes incubated with fungal proteases had an impaired ability to ingest yeast cells and exhibited alterations in morphology and cytoskeleton formation. The effects were similar to those observed in plasmatocytes from infected larvae. The role of extracellular fungal proteases in the interactions of entomopathogens with the insect immune system is discussed.     

Susceptibility to Fungi of Cotton Boll Worms Before and After a Natural Epizootic of the Entomopathogenic Fungus Nomuraea rileyi (Hyphomycetes)

Epizootics caused by Beauveria bassiana and Nomuraea rileyi have been observed on boll worms and Spodoptera litura in south Indian fields during winter since the last 15 years. During the N. rileyi-induced natural epizootics, some boll worms were found surviving without infection. Whether they represent pathogen-resistant genotypes was investigated. Two insect populations, collected 3 months prior to and during the epizootic were established. Their sensitivity to both the fungi was compared in laboratory bioassays. No significant difference in sensitivity was observed between the two populations. It was concluded that the boll worm population surviving the epizootic was not genotypically resistant.     

Control of Leaf-footed Bug Leptoglossus zonatus and Shield-backed Bug Pachycoris klugii with Entomopathogenic Fungi

The pathogenicity of three isolates of Beauveria bassiana (Bals.) Vuil. and one isolate of Metarhizium anisopliae (Metsch.) Sorok. (Deuteromycotina: Hyphomycetes) was assessed in the laboratory against adults of the leaf-footed bug Leptoglossus zonatus (Dallas) (Heteroptera: Coreidae) and the shield-backed bug Pachycoris klugii Burmeister (Heteroptera: Scutelleridae), the two most frequent pest species in physic nut ( Jatropha curcas L., Euphorbiaceae) plantations in Nicaragua. In a dipping bioassay, the median lethal concentration (LC) of the most efficient strain, M. anisopliae NB, was determined as 4.34 106 50 conidia/ml for adult P. klugii . In a field trial, a scheduled high-volume spray regime using B. bassiana increased fruit yield by 28%, and was more effective than malathion or an aqueous extract of ground neem seeds. The effectiveness of M. anisopliae was further tested in field cages covering entire trees and containing a predetermined number of insects. Mineral oilbased ultra-low volume controlled droplet applications of M. anisopliae at a rate of 1 1010 conidia/tree were made using hand-held Micron ULVA + sprayers. The corrected mortalities ranged from 65% in P. klugii to 94% in L. zonatus.     

Pathogenicity of the Hyphomycete Fungi Verticillium lecanii and Metarhizium anisopliae to the Western Flower Thrips,Frankliniella occidentalis

The pathogenicity of several isolates of the hyphomycete fungi Verticillium lecanii and Metarhizium anisopliae to Frankliniella occidentalis was investigated. Treatment of adult thrips with M. anisopliae resulted in at least 94% mortality at 7 days post-inoculation. In contrast, V. lecanii isolates only gave mortalities of between 20 and 70%. Detailed studies were made on the most virulent isolate of M. anisopliae (275) to determine its efficacy at different doses and temperatures. At 23 C the LC was ca. 3 105 conidia ml-1 after 5 days and the LTs were 50 50 3 and 4.5 days at 10 7 and 106 conidia ml-1 respectively. Temperature influenced fungal virulence to adult thrips; the LT at 18 and 20 C was ca. 4 days and at 23 or 26 C it was ca. 3 days. 50 Larvae were less susceptible to infection than adult thrips (27% versus 100% mortality), presumably due to the inoculum being shed with the exuvium during ecdysis. Conidia of M. anisopliae isolate 275 germinated rapidly on the surfaces of larvae, pupae and adults, with most germlings producing appressoria within 24 h post-inoculation. Fungal elements were present in significant amounts in the body 3 days after treatment.     

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.     

Susceptibility of Ceratitis capitata Wiedemann (Diptera: Tephritidae) to Entomopathogenic Fungi and Their Extracts

The effectiveness of seven strains of entomopathogenic fungi against Ceratitis capitata adults was evaluated in the laboratory. Adults were susceptible to five of seven aqueous suspensions of conidia. Metarhizium anisopliae and strain CG-260 of Paecilomyces fumosoroseus were the most pathogenic fungi, with 10-day LD₅₀ values of 5.1 and 6.1 × 10³ conidia/fly, respectively, when applied topically. Sublethal effects on fecundity and fertility of the fungal-exposed females were also studied. The most effective fungus in reducing fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at 1 × 10⁶ conidia/fly. M. anisopliae and Aspergillus ochraceus also showed significant reductions of fecundity (40–50% for most of the assayed concentrations). Fertility was moderately affected by the fungi. M. anisopliae at 1 × 10⁶ conidia/fly was the most effective fungus, showing egg eclosion reduction of over 50% compared with the control. In addition, culture broth dichloromethane extracts from the entomopathogenic fungi were tested for insecticide activity against C. capitata, including effects on fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in about 90% mortality at a concentration of 25 mg/g of diet; under these conditions, fecundity and fertility of treated females were reduced by 94 and 53%, respectively, compared with untreated controls.     

Susceptibility of the Hymenopteran Parasitoids Apoanagyrus ( = Epidinocarsis ) lopezi (Encyrtidae) and Phanerotoma sp. (Braconidae) to the Entomopathogenic Fungus Metarhizium anisopliae var. acridum (Deuteromycotina: Hyphomycetes)

Laboratory and so-called extended laboratory bioassays were conducted in Benin, West Africa, to investigate the pathogenicity and virulence of the entomopathogenic fungus Metarhizium anisopliae var. acridum , a biocontrol agent against locusts and grasshoppers, to two hymenopteran parasitoids, Apoanagyrus ( = Epidinocarsis ) lopezi and Phanerotoma sp. Treatments were carried out under simulated field conditions at standard field dose rates of 2.5 and 5.0 ×10 12 conidia ha -1 . Test organisms were continuously (3 weeks) exposed to spray residues in artificial or simulated natural environments. The standard strain IMI 330 189 of the mycopesticide Green Muscle caused a significant reduction of 24% in the longevity ( = average survival time, AST) of A. lopezi , relative to the untreated control. Mycosis was confirmed in 16% of all cadavers. AST was shorter under low relative humidity (RH) conditions, and these conditions seemed to enhance susceptibility to fungal infection. However, this effect was only marginally significant. In contrast, average longevity of untreated A. lopezi was slightly yet significantly shorter at low RH (50-60%) than at high RH (80-90%). In the extended laboratory assay, the same isolate had no significant effect on mortality, parasitoid emergence ( = beneficial capacity) and sex ratio. In a further screening test with isolates IIBC I91 609, IIBC I93 833 and IMI 330 189 (reference), no infection of A. lopezi was confirmed. Similarly, Phanerotoma sp. was not susceptible to IMI 330 189. It is concluded that mycopesticides based on the three strains tested pose a low risk to parasitic hymenopterans under field conditions.