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.     

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 enhanced infectivity of Metarhizium flavoviride in oil formulations to desert locusts at low humidities

Formulation in non-evaporative diluents such as oils is required to exploit the advantages of controlled droplet application methods for biopesticides based on conidial suspensions of entomopathogenic fungi. In laboratory assays on desert locusts (Schistocerca gregaria), formulations of Metarhizium flauoviride conidia in cotton seed oil showed superior performance to water-based suspensions and this was especially pronounced at low humidities (35% r.h.). The LD₅₀s for oil and water suspensions at 5 days were 8900 and > 10⁶ conidia/insect respectively. The dose-mortality regression line for oil was more than four times steeper and median lethal times were typically 76% of those for aqueous suspensions. Formulation in oil improves the efficacy of this fungus and may extend the application of species with lipophilic conidia into less humid agricultural environments.     

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.     

Identification of Metarhizium strains highly efficacious against Aedes,Anopheles and Culex larvae

Entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, have been shown to be efficacious in killing mosquito larvae of different mosquito species. The current study compared the pathogenicity and efficacy of two formulations of three fungal strains against different instars of three mosquito species with the aim of identifying the most virulent strain for use under field conditions. Three strains of Metarhizium, ARESF 4556, ARSEF 3297 and V275, were assayed against early (L_2?3) and late (L_3–4) instar larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Two formulations of the fungi were tested, dry conidia and aqueous suspensions (i.e. ‘wet’ conidia). Effects of all combinations of conidia, mosquito species, instar, fungal strain and concentration on mosquito mortality were analysed using Cox regression and Kaplan–Meier analyses. Strain ARSEF 4556 was more virulent than ARSEF 3297 and V275, with LT₅₀ values ranging from 0.3 to 1.1 days, with Anopheles and Culex being more susceptible than Aedes. Early and late instars were equally susceptible independent of species. Although the formulation did influence mortality rates, both ‘wet’ and ‘dry’ conidia applications were highly effective in killing mosquito larvae. Viable spores were more efficacious than heat killed spores. The latter did cause mortality but only at high concentrations. Metarhizium sp. has proved to be effective in reducing survivability of all larval stages of Aedes, Anopheles and Culex under laboratory conditions. Aedes larvae were generally more tolerant than Anopheles and Culex irrespective of fungal strain.     

Recovery of germinating fungal conidia from the nasal cavity after environmental exposure

Background The expression of fungal allergens is increased by the germination of conidia. We assessed the state of germination of fungal conidia recovered by nasal lavage after environmental exposure. Methods Nasal lavage was performed on twenty adults at three stages: the start of the experiment, after 1 h indoors, and after 1 h outdoors. One half of the lavage liquid was immediately treated to prevent in-vitro germination and stained with periodic acid Schiff (PAS) to enable identification of germinated and ungerminated conidia. The untreated half of the lavage liquid was cultured on nutrient agar plates to enumerate and identify viable fungi. Results PAS staining showed that both ungerminated and germinated conidia, and hyphal fragments, were present in the nasal cavity. The most prevalent fungi recovered were Aspergillus, Alternaria, Cladosporium, Epicoccum, Penicillium, and Yeast species. The number of viable fungi recovered after 1 h indoors was significantly less than after 1 h outdoors (P < 0.01). Conclusions Viable fungi and germinating conidia, in addition to ungerminated conidia and hyphal fragments, were present in the nasal cavity after both indoor and outdoor exposure. This provides novel insight into the pathogenicity of exposure to fungal aeroallergens.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

House fly (Musca domestica) (Diptera: Muscidae) mortality after exposure to commercial fungal formulations in a sugar bait†

House flies are pests of medical and veterinary importance. They are known to be susceptible to fungal conidia presented in sugar baits. The purpose of this study was to evaluate the potential for commercial formulations of entomopathogenic fungi to be used as bait for fly control. Differences in sporulation by product were found. Current formulations may influence fly feeding on baits, but because formulations may protect conidia. Additional research into more attractive formulations is warranted.     

In vitro fungitoxicity of the essential oil of Syzygium aromaticum

The in vitro antifungal activity of clove oil was studied against four test fungi namely Alternaria alternata, Fusarium chlamydosporum, Helminthosporum oryzae and Rhizoctonia bataticola by the agar well diffusion method. These test fungi were found to be highly sensitive to clove oil at a concentration of 100 μl/well. The inhibition zone diameter was found to be in the range of 55–65 mm. The toxicity of clove oil on the germination and growth of A. alternata was further examined in liquid medium. Concentration- and time-dependent toxicity was recorded from 0.05 to 20% (v/v) concentration. The minimum fungistatic concentration was found to be 0.05%. Above this concentration, lysis of conidia and inhibition of mycelial growth were detected. Microscopic analysis showed 20–40% lysis of conidia after 72 h of incubation at 5% concentration. However at higher clove oil concentration (10%), up to 20% of conidia were lysed within 24 h of incubation. Similar concentration- and time-dependent toxicity was observed at different concentrations and time intervals. The findings indicated that clove oil possesses fungicidal activity against phytopathogenic fungi. Further study is required to determine whether it could have value in the management of plant infectious diseases. This revised version was published online in July 2006 with corrections to the Cover Date.     

两种杀虫真菌制剂与低用量吡虫啉对温室粉虱的协同防效评价

在昆明市郊蔬菜基地4间大棚(5m×100m·间^-1)栽培的生菜上,用球孢白僵菌(Beauveriabassiana)和玫烟色拟青霉(Paecilomyces fumasonoseus)的纯孢子乳悬剂(有效含孢量10^10·ml^-1)及其与1％和3％(w／v)的吡虫啉10％WP的混配剂对温室粉虱(Trialeurodes vaporariorum)进行了协同药效试验,吡虫啉添加量仅相当于推荐用量的6．4％～9．5％和19．1％～28．6％,试验含6个菌剂处理、2个低用量吡虫啉处理和1个清水对照,各有3个小区(7m×5m·小区^-1)重复,随机区组排列,稀释1000倍的菌液(含孢量10^7·ml^-1)和吡虫啉液于生菜移栽后第11d首次喷雾,15d后喷第二次,首次用菌前调查初始粉虱密度,用菌后每隔5d调查活虫数和死虫数,从8月20日起历时30d,结果表明,连续2次用菌有效地控制了粉虱对当季大棚生菜的危害,各菌剂处理的最终相对防效和虫口减退率均达到95％以上,极显著地高于低用量吡虫啉处理,其中,玫烟色拟青霉制剂对粉虱的控制效果一般优于球孢白僵菌制剂,且与吡虫啉的添加量呈正相关,但是,各菌剂处理间控虫效果的差异主要发生在首次用菌后第5d和10d,此后差异逐渐缩小,至第二次用菌后第10d各菌剂处理间无显著差异,本试验结果表明,两种真菌的孢子乳悬剂均可用于温室粉虱的防治,添加微量吡虫啉可增强菌剂的防效。     

Ecology of nematophagous fungi: Effect of the soil nutrients N,P and K,and seven major metals on distribution

The effects of the major soil nutrients and seven common metals on the distribution of nematophagous fungi were evaluated by comparing the concentration of the elements in soils with and without nematophagous fungi. Mineral nutrients, which were all positively correlated with nematode density, were the most important elements determining the presence of such fungi. Endoparasites producing adhesive conidia were independent of all the elements tested, while those species forming ingested conidia were isolated from soils high in nutrients, indicating a strong dependence on nematode density. Knob forming predators which rely on their ability to spontaneously produce traps to obtain nutrients were isolated from soils with low concentrations of mineral nutrients, while species with constricting rings were isolated from richer soils containing a greater density of nematodes. Net-forming species were largely independent of soil fertility, although generally they were isolated from soils with limited nutrientsd, especially K. Like other fungi, nematophagous fungi are generally not found in soils containing elevated concentrations of heavy metals. The results indicate that even concentrations of heavy metals which naturally occur in Irish soils can restrict the distribution of this fungal group. However the endoparasiteMeria coniospora is tolerant to all metals and to Cu in particular.     

Rapid assessment of antimould efficacies of pressure-treated southern pine

A membrane-screening method was developed in conjunction with flow cytometric (FC) analysis for determining the efficacies of antimould pressure-treatment formulations for mould species of cosmetic significance on southern pine. Fusarium subglutinans, Aspergillus flavus, Penicillium chrysogenum, and Paecilomyces spp. were the predominant moulds colonizing surfaces of the variously treated pine stored in sealed plastic bags over 3- to 6-month periods. Nylon membranes placed directly on pressure-treated pine and membranes saturated with the various formulations were inoculated with the conidia of selected moulds. FC analysis of conidia stained with propidium iodide (PI) before and after exposure to the pressure-treatment formulations permitted a rapid assessment of the inocula and selection of those pressure-treatment formulations with probable inhibitory activity versus probable nonactive preparations. Recoveries of the fungi from the membranes over 9–14 days were in general agreement with the emergence of colonizing fungi on the similarly preserved uninoculated pine stored in sealed plastic bags for 6 months. This combination of procedures provided for a relatively rapid assessment of preservative formulations designed to provide enhanced efficacy against surface mould growth on lumber during storage and retail display. Received 21 December 2001/ Accepted in revised form 20 May 2002     

Fungal colonization of automobile air conditioning systems

Air samples and swab samples of the air conditioning vents were collected from 29 automobiles in the metropolitan region of Atlanta, GA, and cultured for fungi. Among the fungi observed, species of Acremonium, Aspergillus, Alternaria, Aureobasidium, Cladosporium, and Penicillium were in the highest densities. Transparent adhesive tape imprints, SEM observations, and enrichment culture of components of five systems demonstrated fungal hyphae on the metal surfaces and within the matrix of various insulation materials. The evaporator removed from one automobile because of a series of complaints of noxious odors was densely colonized by Penicillium viridicatum. The amplification of known allergenic and odor-producing fungi occurred within the automobile air conditioning systems. Received 27 May 1997/ Accepted in revised form 16 July 1997     

Combining vegetable oil and sub-lethal concentrations of Imidacloprid with Beauveria bassiana and Metarhizium anisopliae against adult guava weevil Conotrachelus psidii (Coleoptera: Curculionidae)

Of the insect pests that attack guava fruits, the guava weevil, Conotrachelus psidii Marshall, 1922 (Coleoptera: Curculionidae), is one of the most important in Brazil. In search of alternatives to chemical pesticides, this study was performed to select fungal isolates of Beauveria bassiana and Metarhizium anisopliae as potential candidates for the control of adult C. psidii. Tests were carried out using three products applied with the entomopathogenic fungi: Tween 80, sunflower oil and Imidacloprid (IMI). A sub-lethal concentration of IMI was determined (100 ppm). The results demonstrated that LPP 19 and LPP 114 were the most effective isolates when used in combination with all of the products. The least virulent isolate, ESALQ 818, when applied in Tween 80 caused only 26.6% mortality, however, this isolate showed significantly improved efficiency when applied together with either sunflower oil or IMI, causing 57.3 and 88.6% mortality, respectively. The efficiency of all the isolates tested here improved when applied together with IMI, with LT₅₀ values of 5.3–10.3 days when compared to LT₅₀ values in Tween alone of 9.5–17 days. The isolate that produced the highest number of conidia on the cadavers of adult C. psidii was LPP138, independent of the product used; however, conidial production was slightly reduced when fungi were applied together with IMI. These results are promising for developing new formulations of the isolates to be tested in the field.     

Whey for mass production of Beauveria bassiana and Metarhizium anisopliae

Spore production of Beauveria bassiana and Metarhizium anisopliae was studied in a novel whey-based culture media. Spore yield and viability were determined for two B. bassiana (GHA-726 and CA-603) and two M. anisopliae (CA-1 and IMI 330189) isolates following production in three whey-based systems: solid, liquid, and a diphasic production system. Our study indicated that whey permeate can be used effectively for production of spores of entomopathogenic fungi. However, spore yield and viability were significantly influenced by fungal isolate, whey concentration, and the type of production process used. Under the conditions defined in the present study, spore yields ranging from 1.3 × 10⁹–10 × 10¹¹ spores l⁻¹ of whey medium could be obtained depending on the strain and production process used. Our study revealed that spores produced by all strains in whey-based solid and liquid media showed between 73–99 % viability; germination rates were comparable with those obtained using the standard SDA medium. In the two-stage production process, the viabilities of conidia produced by GHA-726, CA-603, and CA-1 were 35–86, 32–98, and 6–29 %, respectively; viability was correlated with whey concentration and isolates. Whey permeate can be used as a growth substrate for mass production of biocontrol fungi. We hypothesize that spore yield and viability could be improved by careful selection of whey content in the medium, incorporation of critical additives and optimization of culture conditions.     

Mineral oil-, glycerol-, and Vaseline-coated plates as matrix-assisted laser desorption/ionization sample supports for high-throughput peptide analysis

A novel protocol for rapid and high-quality sample preparation prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed by coating bare stainless steel plates with one of three adhesives: mineral oil, glycerol, or Vaseline. The advantages of these three adhesive coats are that they take little time to both prepare and wipe away, hold the matrices to prevent them from flying from the support, reduce the background matrix, and affect neither the resolution of the peptide peaks nor the accuracy of their determined molecular masses. Consequently, the signal intensity, detection limit, and tolerance of the analytes to contaminants on the three adhesive-coated plates are improved. In the two strategies of on-plate desalting and concentration of the peptide mixture, all three adhesives reduced the loss of peptides, especially in the case of larger molecular mass peptides. The microscope and stereomicroscope images of the deposited droplets showed that after dropping onto the adhesive coats, the droplets formed a reduced spot size, were more homogeneous, and showed sticky crystallization. Therefore, this is an easy-to-use, reproducible, highly sensitive, tolerant (to salts), and high-throughput method of peptide sample preparation for MALDI-TOF MS analysis.     

Inhibitory effect of essential oil from <Emphasis Type="Italic">Nepeta rtanjensis</Emphasis> on fungal spore germination

The ability of Nepeta rtanjensis essential oil to inhibit conidia germination of fungi was evaluated in vitro. Tested fungi included in research were Cladosporium cladosporioides, Trichoderma viride and two Alternaria species originally isolated from N. rtanjensis. The conidia of Cladosporium cladosporioides were most susceptible to the N. rtanjensis essential oil treatment, and the oil concentration of 0.1 μg ml⁻¹ caused the maximum of conidia germination inhibition. The highest concentration used in experiment that caused the maximum of conidia germination inhibition was 0.6 μg ml⁻¹ for Alternaria isolated from leaf surface of N. rtanjensis. The germ tube elongation of Alternaria isolates significantly decreased in response of different concentrations of oil used in experiment.     

Lethal effects of high-intensity violet 405-nm light on Saccharomyces cerevisiae,Candida albicans,and on dormant and germinating spores of Aspergillus niger

This study assessed the effects of high-intensity violet light on selected yeast and mould fungi. Cell suspensions of Saccharomyces cerevisiae, Candida albicans, and dormant and germinating spores (conidia) of the mould Aspergillus niger were exposed to high-intensity narrow band violet light with peak output at 405 nm generated from a light-emitting diode (LED) array. All three fungal species were inactivated by the 405-nm light without a requirement for addition of exogenous photosensitiser chemicals. Of the fungal species tested, S. cerevisiae was most sensitive and dormant conidia of A. niger were most resistant to 405-nm light exposure. Five-log₁₀ colony forming units per millilitre (CFU ml^?1) reductions of the tested species required exposure doses of 288 J cm^?2 for S. cerevisiae, 576 J cm^?2 for C. albicans, and a much higher value of 2.3 kJ cm^?2 for dormant conidia of A. niger. During germination, A. niger conidia became more sensitive to 405-nm light exposure and sensitivity increased as germination progressed over an 8 h test period. Light exposure under aerobic and anaerobic conditions, together with results obtained using ascorbic acid as a scavenger of reactive oxygen species, revealed that 405-nm light inactivation in fungi involved an oxygen-dependent mechanism, as previously described in bacteria. The inactivation results achieved with yeast cells and fungal spores together with operational advantages associated with the use of a visible (nonultraviolet (UV)) light source highlight the potential of 405-nm light for fungal decontamination applications.     

The effect of non-pathogenic phylloplane fungi on life-history traits of urticae (Acari: Tetranychidae)

The direct effect of three common non-pathogenic phylloplane fungi on the life-history traits of Tetranychus urticae Koch was investigated on intact leaves of the garden bean Phaseolus vulgaris L. (var. Tendergreen Improved), under conditions of low and high water-deficit stress. The survival rate of T. urticae was always reduced by an increase in water-deficit stress, whereas the effect of the fungi depended in part on the watering regime. On two of the three fungi tested, Alternaria alternata (Fr.) Keissler and Epicoccum nigrum Link, the mites showed a significant increase in net reproduction compared to those reared under control (i.e. no fungi added) conditions, independent of water-deficit stress, resulting in a higher intrinsic rate of increase. The third fungus tested, Cladosporium cladosporioides (Fres.) de Vries, had a similar response with respect to net reproduction when reared under low water-deficit stress but not when reared under a high water-deficit stress. There was, however, no evidence of an interactive effect with water-deficit stress and presence or absence of fungus affecting the net reproduction of the mites, for any of the three fungal species tested. An interactive effect between fungal species and water-deficit stress was observed for the intrinsic rate of increase of the mites. It is suspected that the fungi are acting as a source of an otherwise limiting resource and the differences observed between the fungal treatments is due to differences in consumption, based on the different sizes of their conidia.     

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.