Influence of two formulation types and moisture levels on the storage stability and insecticidal activity of Beauveria bassiana

The formulation of mycopesticides may require a physical separation of conidia from the substrate and subsequent drying. In the present study, Beauveria bassiana conidia produced by solid-state fermentation were harvested either through a dry or washing protocol. Washed conidia were used to design a water-dispersible granule (WG) formulation, whereas sieved conidia were mixed with an emulsifiable oil to achieve an oil-based formulation (OD). Potential harmful effects caused by the formulation type on the storage stability and insecticidal activity against Hypothenemus hampei were assessed. As expected, the time for initial conidial germination to drop 50% (GT₅₀) in all treatments was deeply influenced by storage temperatures, which varied from over 180 days at 4 °C to less than 90 days at 35°C. In all four tested temperatures, GT₅₀s for unformulated dry conidia were significantly higher than for those formulated as WG, and the latter was similar to conidia formulated as OD in the two highest temperatures. Residual water content in the OD formulation (1,600 vs. 340?ppm) had a negative influence on conidial survival under storage, whereas WG granules immediately dried after the washing protocol showed conidial germination similar to granules exposed to a slower dehydration regime. Mortality of H. hampei adults exposed to different concentrations of B. bassiana formulated as WG was slightly lower (10–15%) than either the OD or the unformulated conidia. In brief, we have demonstrated that formulation type and their moisture level can affect the storage stability and insecticidal activity of B. bassiana conidia toward the coffee berry borer. Of particular importance, we have shown that drying oils prior to formulation could improve the storage of mycopesticides, an approach that may find industrial applications.     

Effects of inoculation method on efficacy of wettable powder and oil dispersion formulations of Beauveria bassiana against Colorado potato beetle larvae under low-humidity conditions

The effects of inoculation method on efficacy of two formulations of Beauveria bassiana strain GHA against Colorado potato beetle larvae were investigated. Under dry greenhouse conditions, ca. 58% mortality was observed among second-instar larvae exposed directly to sprays of B. bassiana conidia, whereas mortality among larvae exposed to similarly treated foliage (either leaf dorsal or ventral surfaces) was <10%. Mortality was ca. 64% among larvae exposed to both direct sprays and foliar spray deposits. Equivalent rates of mortality were observed among larvae treated with a clay-based wettable powder versus an emulsifiable oil-based formulation of B. bassiana conidia; however, this was observed despite application of an approximately 40% greater dose of WP-formulated conidia, indicating greater efficacy of the emulsifiable oil formulation. These results suggest that, under dry conditions, potato beetle larvae do not readily acquire an effective dose of conidia from treated foliage and that development of improved application technologies to more effectively target the larvae may ultimately prove more beneficial than development of formulations with greater foliar persistence.     

两种杀虫真菌制剂对茶小绿叶蝉的田间防效评价

于2002年盛夏在浙江遂昌一高山茶园对茶小绿叶蝉(Empoasca spp．)进行了真菌杀虫剂的田间药效试验。所用菌剂为球孢白僵菌(Beauveria bassiana)和玫烟色拟青霉(Paecilomyces fumosoroseus)的纯孢子悬乳剂及其与3％吡虫啉10％可湿剂的混配剂．各菌剂稀释500倍喷雾2次,间隔12d．结果表明。两种真菌的混配剂明显优于纯菌剂,其中球孢白僵菌混配剂的最高防效达83．4％,而玫烟色拟青霉的最高防效为71．3％．根据25d期间历次调查结果计算平均防效,球孢白僵菌混配剂达66．8％,玫烟色拟青霉混配剂为62．1％,含微量吡虫啉的矿物油为50．3％,球孢白僵菌和玫烟色拟青霉的纯菌剂分别为49．5％和19．0％,结合试验期间气候及田间种群结构特征,讨论了各处理问差异的来源及提高真菌杀虫剂控制茶小绿叶蝉效果的可能途径。     

Influence of substrate and relative humidity on the efficacy of three entomopathogenic fungi for the hide beetle,Dermestes maculatus (Coleoptera,Dermestidae)

Dermestes maculatus De Geer (Coleoptera: Dermestidae) is a carrion feeder that is also a pest of poultry houses, museums, silkworm culture, and many stored foods. The Hypocreales, Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff), and Isaria fumosorosea Wize, were tested for efficacy against D. maculatus larvae on concrete, plastic, leather, and wood surfaces. Only wood had a significant negative effect on efficacy, and B. bassiana was the most efficacious species. The conidia of all three species lost viability quickly on wood with various responses on the other surfaces. When beetle larvae were exposed to deposited B. bassiana and incubated at 43, 56, 75, or 82% relative humidity, mortality was greatest at the lowest humidity suggesting enhancement of fungal infection by desiccation stress. The results indicate that entomopathogenic Hypocreales, especially B. bassiana, may be useful for control of hide beetles when applied at a low dose to surfaces that do not impact the viability of conidia.     

Effects of combining the fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhiziumanisopliae</Emphasis> on the mortality of the tick <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> (ixodidae) in relation to seasonal changes

The effect of formulation, fungal concentration, type and seasonal changes on the mortality of the tick Amblyomma variegatum was investigated. A previous study demonstrated high pathogenicity of strains of the fungi Beauveria bassiana and Metarhizium anisopliae against the ticks Rhipicephalus appendiculatus and Amblyomma variegatum (Kaaya et al. J Invertebr Pathol 1996; 67: 15–20). The present study was undertaken to explore possible additive or synergistic effects of the two fungi on A. variegatum. The effects of oil and water formulations at different concentrations of each fungus and combination of the two on the mortality of A. variegatum in the laboratory and in the field during the wet and dry seasons were determined and compared. The oil formulation performed better in all assays, with highest tick mortality of 92% occurring during the wet season at conidia concentration of 1 × 10¹⁰ conidia/ml of the mixed fungi compared to 49% for the water formulation at similar conidia concentration. However, at the same conidial concentration during the dry season, mortalities in the field were relatively low with the mixture of the fungi recording 24% and 17% tick mortality for the oil and water formulation respectively. The effect of infecting the ticks with a cocktail of the two fungi was inconclusive under more controlled conditions in the laboratory but field results under both wet and dry seasons indicated significant differences between the separate and mixed fungi infections. The results demonstrate a potential of cocktail formulations in the control of ticks and possibly of other arthropod pests.     

Enhanced ovicidal activity of an oil formulation of the fungus Metarhizium anisopliae on the mosquito Aedes aegypti

Abstract.  The effect of humidity on the activity of Metarhizium anisopliae IP 46 (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) formulated in sunflower oil against Aedes aegypti (L.) (Diptera: Culicidae) eggs was examined. After exposure of eggs at 75% relative humidity (RH) for ≤ 25 days, ovicidal activity was not increased by oil-in-water formulated conidia, hyphal bodies or pure-oil formulated conidia, compared with conidia or hyphal bodies prepared in water only. At optimal > 98% RH, eclosion was ≤ 13.7% after treatment with oil-in-water formulated propagules in ≤ 10% oil, and it was completely inhibited when conidia were applied in pure oil. At 86–100% RH, new conidia were found on eggs treated with oil-formulated conidia and incubated down to 91% RH. Ovicidal activity was still detected at 93% RH and was augmented with increasing humidity and time of exposure of eggs. Eclosion of larvae was distinctly reduced by IP 46 pure-oil formulated conidia after a minimal initial exposure of 3 days at > 98% RH, followed by: (a) a 12-day exposure at 75% RH before submersion in water; (b) a minimal 5-day exposure at > 98% RH and direct subsequent transfer of treated eggs to water, or (c) a minimal daily 20-h exposure at > 98% RH alternating with 4 h at 75% RH for 10 days. We demonstrate that oil-based formulations of conidia of M. anisopliae enhance ovicidal activity at high humidities and conclude that these formulations have potential in the integrated control of Ae. aegypti .     

Using particle size analysis to determine the hydrophobicity and suspension of fungal conidia with particular relevance to formulation of biopesticide

Fungal formulations are vital for effective biopesticide development. Good formulations help to optimise field efficacy, while poor formulations result in product failure. This study aimed to produce a hydrophobicity test that would be appropriate for fungal conidia produced to a commercial quality and determine relative hydrophobicity of fungi from four different genera by using laser diffraction. A particle size analyser was used to determine the hydrophobicity of: three Metarhizium acridum samples, M. anisopliae, Beauveria bassiana, Trichoderma stromaticum, T. harzianum, T. viride and Alternaria eichhorniae conidia by suspending the conidia in three different liquids: Shellsol T (a mineral oil), water and 0.05 % Tween 80. Hydrophobicity was determined by the size of the particles formed in each of the liquids. All the Metarhizium samples were the most hydrophobic followed by B. bassiana and A. eichhorniae. The Trichoderma samples were the least hydrophobic. As a comparison, a phase exclusion assay and a salt-mediated aggregation and sedimentation (SAS) test were performed. It was not possible to get a reliable reading for the B. bassiana, A. eichhorniae and T. viride samples using the phase exclusion assay. The addition of salt in the SAS test did not affect the rate of sedimentation. It was hypothesised that conidia size affected the results of the SAS test that made A. eichhorniae the most hydrophobic conidia. Particle size analysis (PSA) was a more accurate test for comparing fungi from difference genera compared to the SAS test and phase exclusion assay. PSA was also used to test three emulsions and demonstrated that different formulations had an effect on particle size.     

The influence of relative humidity on Beauveria bassiana infectivity and replication in the chinch bug,Blissus leucopterus

Beauveria bassiana conidia were bioassayed for pathogenicity against adult chinch bugs, Blissus leucopterus leucopterus, at varying percent relative humidities (RH). The conidia were found to be invasive and pathogenic at all humidities tested. Normal fungal replication and conidiogenesis, however, occurred only on the hosts incubated at the 75% or higher RH levels.     

The potential of Beauveria bassiana inoculum formulated into a polymeric matrix for a microbial control of spruce bark beetle

Two local strains of Beauveria bassiana originally isolated from naturally infected spruce bark beetles in Slovakia were tested for their virulence to Ips typographus (IT) and for their compatibility with a polymeric matrix composed of low-molecular polyethylene. Conidia could be homogenously immobilized in the low-molecular polyethylene matrix with no adverse effect on their viability and infectivity. At constant temperature (25°C), viability of immobilized conidial decreased only by 1–2% after 7 or 14 days when compared with non-formulated conidia. In field conditions, viability of conidia formulated in the matrix was even significantly higher than non-formulated conidia 35 days after their application in traps. Conidia incorporated into the polymeric matrix were infective to IT adults in laboratory bioassays. Mean values of LC₅₀ for native conidia (0.72–2.05?×?10⁶ conidia?ml^?1) and conidia immobilized in the polymeric matrix (0.64–1.03?×?10⁵ conidia?mm^?2) demonstrated high virulence. The efficacy of the local strains was significantly higher than that of B. bassiana strains from mycoinsecticides (Boverol^®, Botanigard^® ES and Naturalis-L^®). Results showed potential of this polymeric material for its use in microbial control of IT when mixed with conidia of B. bassiana.     

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.     

Influence of humidity on the infection of western flower thrips, <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> (Thysanoptera: Thripidae), by <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

The insecticidal activity of Beauveria bassiana GHA derived from a commercial mycoinsecticide BotaniGard ES against Frankliniella occidentalis was determined in a bioassay by dipping the female adults into a conidial suspension. The 90% lethal concentration of B. bassiana GHA was estimated to be 9.7 × 10⁶ conidia/ml. The lethal times for achieving 90% mortality of thrips inoculated with a 1/500-diluted solution of BotaniGard ES and a 10^7.5 (3.16 × 10⁷) conidia/ml suspension of B. bassiana GHA were estimated to be five and six days, respectively. When the treated thrips were exposed to a high relative humidity (RH) of over 99% for various periods and then transferred to 60% RH, the requisite lengths of the high-humidity period to achieve 90% mortality of the thrips at six days after inoculation were estimated to be 46 and 47 h in BotaniGard ES and B. bassiana GHA, respectively. Fungal multiplication in the thrips was detected between 48 to 60 h after inoculation by measuring Beauveria-specific DNA in the host following inoculation with a B. bassiana GHA suspension of 10^7.5 conidia/ml using a real-time quantitative PCR. The mycelial growth in the host hemocoel was not influenced by the low-humidity condition.     

The mechanism of the mycoinsecticide diluent on the efficacy of the oil formulation of insecticidal fungus

Adverse conditions, including low humidity, UV irradiation, and high temperature, appreciably affect the efficacy of mycoinsecticides. Oil formulation increased the virulence of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against locusts and grasshoppers by reducing the dependence on saturated water. A mycoinsecticide diluent (a water-in-oil emulsion) has been widely used to dilute the oil formulation of M. anisopliae in China. The aim of our study was to elucidate the mechanism by which the mycoinsecticide diluent improves the virulence of M. anisopliae. We investigated the effects of the mycoinsecticide diluent on the virulence, invasion speed, and viability of the conidia under various adverse conditions. The results demonstrated that the mycoinsecticide diluent significantly improved the virulence of conidia at low humidity (68, 75, and 84%). In particular, at an RH of 68%, the LT₅₀ for locusts treated with the emulsion was 5.4 days and was 31.6% lower than the value for locusts treated with an oil formulation. In addition, the concentration of the hyphal bodies found in the haemolymph of locusts treated with emulsion was about 27-fold higher than that in locusts treated with oil formulation four days after inoculation. This result was further confirmed by determining the concentration of M. anisopliae var. acridum DNA in locust haemolymph using quantitative PCR. The percentage germination of conidia in the emulsion was also significantly higher than that in oil at 68% RH. There was no significant difference in percentage germination between conidia treated with the emulsion and oil when exposed to irradiation with ultraviolet-B (UV-B) or high temperature. These results demonstrate that the mycoinsecticide diluent enhances the virulence of M. anisopliae formulated in oil at low humidity by providing adequate water for germination without interfering with the UV tolerance and thermotolerance of the conidia.     

Laboratory and field evaluation of Beauveria bassiana bait against two cockroach species (Dictyoptera: Blattellidae,Blattidae) in Jinan City,East China

A Beauveria bassiana bait formulation was evaluated for its efficacy in the laboratory and field against German cockroaches, Blattella germanica (L.), and American cockroaches, Periplaneta americana (L.). The LT₅₀ values were 46 and 119?h, respectively, for B. germanica and P. americana. The cockroach species were lured and killed effectively when linear distance was about 5?m between B. bassiana bait and fresh mouse food. In the field, more than 90% relative decline in density was found 14?d after treatment, which lasted for 90?d. In conclusion, laboratory and field studies demonstrated the high efficacy of a B. bassiana bait formulation against common cockroach species.     

Target-oriented dissemination of Beauveria bassiana conidia by the predators,Harmonia axyridis (Coleoptera: Coccinellidae) and Chrysoperla carnea (Neuroptera: Chrysopidae) for biocontrol of Myzus persicae

Dissemination of microbial biocontrol agents via predators may have advantages for safe spore dispersal to targeted pests with the added benefit of predation. A laboratory study was conducted to test the target-oriented dissemination of conidia of Beauveria bassiana using larvae of both the multicoloured Asian lady beetle (Harmonia axyridis) and common green lacewing (Chrysoperla carnea) for control of aphids. Maximum dry conidial attachment occurred within approximately 7 min after exposure. After release of the treated predators on leaves of Chinese cabbage, within 12 hours lacewing larvae dispersed 89% of the attached conidia while Asian lady beetles dispersed 93%. Both predators dispersed conidia up to 2.4 m from the release site. Leaf disk bioassays were conducted to compare two application methods; the dissemination of conidia of B. bassiana by predators and the direct application of conidial suspensions. Mortality in sprayed aphids was 91±2.1% compared to 88±2.1 and 84±4.2%, respectively, when conidia were disseminated by lacewings and lady beetles. Predation was not affected in treated lacewing larvae whereas there was a 20% reduction in predation by lady beetle larvae. It appears that B. bassiana can be effectively delivered using certain insect predators.     

Comparison of two methods for estimation of spray deposits after application of microbial pesticides

The effectiveness of two methods for the determination of deposition of mycopesticides on hemlock trees by spray application was compared. One method employs water and oil sensitive paper cards; the other uses a combination of two adhesive tapes (scotch tape) with different adhesive properties. Two fungi, Beauveria bassiana and Lecanicillium muscarium, and three fungal formulations based on whey, oil, and whey together with oil were used to evaluate the efficacy of the two methods. The new method has shown the certain advantages in comparison with the traditional method which is based on the sensitive paper cards. Generally, number of droplets counted was greater in the case of adhesive tape utilization. These observations were noted for the two entomopathogenic fungi. However, different results were noted for the three different types of fungal formulations. The presence of conidia was observed in all three formulations using adhesive tape. The number of conidia localized on hemlock twigs was affected by fungal formulation. The adhesive tape method allowed counting more droplets than the sensitive cards. Other advantages of the adhesive tape method include the ability to observe the outer appearance and internal structure of the droplets and to count fungal propagules directly on the leaf surfaces.     

Pathogenicity, formulation and storage of insect pathogenic hyphomycetous fungi tested against Diabrotica speciosa

Studies were conducted tosearch for fungal strains with potentialpathogenicity against Diabrotica speciosa(Germar) (Coleoptera: Chrysomelidae).Among sixteen fungal isolates screenedthe most virulent was a Beauveria bassiana(Balsamo) Vuillemin isolate (FHD13) thatcaused 70% mortality of D. speciosathird instar larvae. The LC₅₀ value ofB. bassiana isolate FHD13 was3.48 × 10¹⁰ conidia/ml.Different temperatures (4, 17 and 26 °C)and vegetable oils (corn, sunflower and canola)used for storage did not significantly affectviability of conidia. A pathogenicity trialagainst D. speciosa larvae performed withthe corn oil formulation (1 × 10⁸ conidia/mlof oil) caused 65% of mortality.     

Entomogenous Fungi as Promising Biopesticides for Tick Control

When ticks were sealed in nylon tetrapacks and infected with the entomogenous fungi, Beauveria bassiana and Metarizium anisopliae and maintained in potted grass in the field, the fungal oil formulations (10⁹ conidia per ml) induced 100% mortality in larvae of Rhipicephalus appendiculatus and Amblyomma variegatum, whereas mortalities in nymphs varied between 80–100% and in adults 80–90%. The aqueous formulations (10⁹ conidia per ml) induced mortalities of 40–50% and reductions in egg hatchability of 68% (B. bassiana) and 48% (M. anisopliae) when sprayed on Boophilus decoloratus engorging on cattle. The strains of B. bassiana and M. anisopliae isolated from naturally infected ticks were also found to induce high mortalities in both R. appendiculatus and A.variegatum in tetrapacks placed in potted grass. Both aqueous and oil-based formulations were found to be effective, although the latter induced higher mortalities. These fungal strains in aqueous formulation (10⁸ conidia per ml) suppressed on-host populations of adult R. appendiculatus by 80% (B. bassiana) and 92% (M. anisopliae) when sprayed on tick-infested grass once per month for a period of 6 months. The feasibility of using entomogenous fungi for tick control in the field is discussed.     

Selection of surfactant compounds to enhance the dispersion of Beauveria bassiana

This study aimed to select surfactant compounds to improve the dispersion of emulsifiable conidial suspensions of Beauveria bassiana for field application while maintaining high conidia viability. Surfactant compatibility was initially evaluated by colony growth in culture media amended with increasing doses of surfactants. In a second experiment, the conidia germination rate was evaluated after temporary exposure to the compounds under laboratory conditions. The effect of the hydrophilic–lipophilic balance (HLB) of the surfactants on conidia dispersion and viability was assessed using mixtures of the most compatible products and concentration initially selected. Unitol L/20 and Ultranex NP/100 did not show deleterious effect on colony growth on culture media, while Surfom 3409 strongly inhibited conidial germination. Unitol L/20, Ultranex NP/100 and Ultratan D did not interfere in the germination of conidia during the first 10 minutes of exposure. After 120 minutes, germination was greatly reduced at a 0.5% concentration, with the exception of Ultranex NP/100. The higher conidial dispersion and germination were obtained with HLB values between 8 and 12 and lower remaining clusters at HLB 10. Moreover, there was no significant effect of HLB values on insect mortality. Unitol L/20 and Ultranex NP/100 were the most compatible surfactants in concentrations lower than 0.5%. The HLB value selected for dispersion of B. bassiana on water based formulations was 10.     

Effect of formulated Metarhizium anisopliae on eggs and eclosing nymphs of Triatoma infestans

Little is known about the ovicidal effects of fungi that attack nymphs and adults of triatomine vectors. A combined formulation of Metarhizium anisopliae IP 46 conidia prepared with diatomaceous earth (DE) and vegetable oil was tested against eggs of Triatoma infestans. Eggs were highly susceptible to fungal infection at relative humidity close to saturation [>98% relative humidity (RH)] but not at 75% RH regardless of the formulation applied. Susceptibility of eggs decreased with longer post‐ovipositional embryonation periods before treatments. The eventual eclosion of nymphs was best suppressed by application of conidia prepared with DE + oil and at a >98% RH incubation. Moreover, nymphs were less affected by the fungus when exposed for only a 24‐h period after eclosion to a treated surface than individuals that were in constant contact with the conidia. These findings contribute to a better understanding of the potential of M. anisopliae as an agent against all developmental stages of T. infestans.     

白僵菌孢子悬乳剂田间控蚜效果与常温货架寿命测定

将高纯度球孢白僵菌分生孢子粉兑入一种含悬浮稳定剂及紫外保护剂的矿物油中,配成含孢量为100亿/ml的孢子悬乳剂(纯菌剂),再添加3%的吡虫啉10%可湿剂而得菌药混配剂,于2003年5月在浙江余姚进行了防治蔬菜蚜虫的田间药效试验和农户示范。在两块夏熟白菜地中,纯菌剂和菌药混配剂均用1000倍稀释液(107个孢子/ml)常规喷雾2次,并设清水对照和低用量吡虫啉对照。结果表明,混配剂在两块菜地的相对防效分别达94.5%和86.2%,纯菌剂分别达86.4%和73.1%,而低用量吡虫啉处理的最高防效仅为50%左右。在入夏豇豆、甘蓝、白菜、萝卜及梨树上用菌药混配剂1000倍稀释液对多种蚜虫进行的农户示范试验中,一次性喷雾后5~10d内对菜蚜防效为48%~68%,尤对梨二叉蚜的防效高达99%。在对含有和不含添加剂的两配方孢子悬乳剂进行的常温避光18个月贮存中,活孢率在头7个月均稳定在95%左右且未受配方影响,第10个月活孢率仍有75%左右。而后,含添加剂配方的活孢率下降加速,至16个月时基本失活;而不含添加剂配方的活孢率在第16、17及18个月时还有57%、51%和42%。