Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi

Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were grown on Sabouraud Dextrose Agar (SDA) modified with KCl to give a range of water activity (a(w)) from 0.938 to 0.998. Growth of all three species was optimal at 0.983 a(w) and growth occurred over the a(w) range tested. Acyclic sugar alcohol (polyol) and trehalose content of conidia was determined by HPLC and found to vary with species and a(w). Conidia of B. bassiana and P. farinosus were found to contain totals of 1.5% and 2.3% polyols respectively at 0.998 a(w), and double these amounts at <0.950 a(w). Conidia of M. anisopliae contained from 5.7% to 6.8% polyols at each a(w) tested. In conidia of all three species the predominant polyol was mannitol. The lower molecular weight polyols, arabitol and erythritol, were found to accumulate at reduced a(w). Small amounts of glycerol were present in conidia of each species; <15% total polyols. Conidia of B. bassiana and M. anisopliae contained about 0.5% trehalose from 0.970 to 0.998 a(w), but only trace amounts below 0.950 a(w). Conidia of P. farinosus contained 2.1% trehalose at 0.998 a(w) and this decreased to <0.1% below 0.950 a(w). Potential to manipulate the endogenous reserves of conidia of these biological control agents to enhance viability and desiccation tolerance is discussed.

Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi

The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon 1-1 for 30 d at 25°C. The water activity (a(w)) of the media ranged from 0.925 to 0.998. Growth of M. anisopliae and P. farinosus was stimulated between 0.975 and 0.995 a(w) on glucose media and that of P. farinosus at 0. 975 a(w) on glycerol media. At < 0.970 a(w), growth of each fungal species was significantly reduced (P < 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1 for B. bassiana, 77.3 and 90.3 mg g-1 for M. anisopliae, and 26.7 and 76.1 mg g-1 for P. farinosus. The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1 when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon 1-1 than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1 on glucose media with 142.7 g added carbon 1-1, depending on species. Conversely, conidia of B. bassiana and P. farinosus contained maximum amounts of trehalose ( ≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon l-1, and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon 1-1; conidia of B. bassiana contained up to 154.0 mg glycerol plus erythritol g-1. hen B. bassiana and P. farinosus were grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliae contained < 17.0 mg trehalose g-1 under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of culture media in viability test of conidia of entomopathogenic fungi

O presente trabalho objetivou investigar se meios de cultura utilizados em teste de viabilidade afetam a germinação de conídios de cinco isolados de Lecanicillium lecanii, cinco de Beauveria bassiana e quatro de Paecilomyces fumosoroseus. Os testes foram realizados em lâminas de microscopia contendo um dos seguintes meios de cultura: Ágar-água (AA), Meio Mínimo (MM), Batata, dextrose e ágar (BDA), Batata, dextrose, ágar e 1% de extrato de levedura (BDAL), Sabouraud, dextrose, ágar e extrato de levedura (SDAL) e Meio Completo (MC). Delimitaram-se três áreas por lâmina e em cada uma aplicou-se 0,05mL de uma suspensão com concentração de 5,5 x 105 conídios ml-1. Para cada isolado foi realizado um bioensaio, com seis tratamentos e cinco repetições. Avaliou-se a germinação 15 horas após incubação, a 26±0,5ºC. Os meios de cultura influíram na capacidade de germinação das três espécies estudadas, ocorrendo variações inter e intraespecíficas. Verificou-se que os meios Completo e BDA proporcionaram as maiores porcentagens de germinação dos isolados de L. lecanii, sendo que e as menores foram obtidas nos meios SDAL e AA. Os meios ricos em nutrientes (BDA, BDAL e Completo) favoreceram a germinação dos isolados de B. bassiana, o que não ocorreu com os meios pobres (AA e MM). Nos meios Completo e BDA foram obtidas as maiores porcentagens de germinação dos isolados de P. fumosoroseus. As menores percentagens, por sua vez, foram obtidas no meio SDAL. Entretanto, alguns isolados apresentaram alta germinação em meios pobres em nutrientes (AA e MM).

Carrying and Effect of Granulated Baits Formulated with Entomopathogenic Fungi among Atta sexdens rubropilosa Colonies (Hymenoptera: Formicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Interactions Between the Entomopathogenic Fungi Beauveria bassiana (Ascomycota: Hypocreales) and the Aphid Parasitoid Diaeretiella rapae (Hymenoptera: Braconidae) on Myzus persicae (Hemiptera: Aphididae)

The interactions between the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) and the aphid parasitoid Diaeretiella rapae McIntoch (Hymenoptera: Braconidae) were evaluated under laboratory conditions. Nymphs of Myzus persicae Sulzer (Hemiptera: Aphididae) were first exposed to parasitoid females for 24 h and then 0, 24, and 48 h afterwards sprayed with a solution of B. bassiana. Likewise, aphids were also sprayed with B. bassiana and then exposed to parasitoids at 0, 24, and 48 h afterwards. Parasitism rate varied from 13 to 66.5%, and were signi_cantly lower in treatments where the two agents were exposed within a 0-24 h time interval compared with the control (without B. bassiana). Parasitoid emergence was negatively affected in treatments with B. bassiana spraying and subsequent exposure to D. rapae. Decreases in longevity of adult females of the D. rapae F1 generation were observed in treatments with B. bassiana spraying. The application of these two biological control agents can be used in combination on the control of M. persicae, wherein this use requires effective time management to avoid antagonistic interactions.

Population trends of the red palm mite, raoiella indica hirst (acari: tenuipalpidae) and associated entomopathogenic fungi in trinidad, antigua, st kitts and nevis and dominica

A survey to determine population trends and entomopathogenic fungi associated with the red palm mite (RPM), Raoiella indica, was conducted in Trinidad, Antigua, St. Kitts and Nevis and Dominica. RPM population density was evaluated by sampling a total of ten coconut palms per site in Antigua, St. Kitts and Nevis, Dominica, and Trinidad (Manzanilla and Icacos). Mites from the four islands were either surface sterilized or left unsterilized before being cultured on Tap Water Agar (TWA). A total of 318 fungal colonies were retrieved. A further 96 mites from Dominica were kept on sterile moist filter paper in a humidity chamber and a further 85 colonies were isolated. Based on morphological observations of all 403 isolates, a sample consisting of 32 colonies (8 %) was sent for identification at CABI-UK. Of the 27 fungi positively identified, 15 isolates belonged to the genera Cladosporium, three to Simplicillium spp., and one to Penicillium. Other fungi genera with limited or no entomopathogenic potential included: Aspergillus, Cochliobolus, Fusarium, Pestalotiopsis and Pithomyces. The results show a potential use of entomopathogenic fungi for population management of the red palm mite in the Caribbean region.

Biological Control of Oilseed Rape Pests with Entomopathogenic Nematodes

Biological control techniques attract increasing attention as one of the sustainable alternatives to pesticide use in integrated pest management programs. In order to develop sustainable pest management methods for arable crops based on entomopathogenic nematodes (EPN), their efficacy and persistence needed to be investigated, and an economically feasible delivery system had to be developed. In this study, first a survey of entomopathogens was conducted, and a system approach was tested, using the oilseed Brassica (OSB) growing system (OSB, spring wheat, and red clover) as a model. The system approach aimed at determining the potential of Steinernema feltiae (Filipjev) for the control of OSB pests, developing OSB rotation schemes that support EPN persistence, and investigating the impact of the selected biotic and abiotic factors on efficacy and persistence of EPN. This study employed abductive logic (which employs constant interplay between the theory and empirical observation), quantitative methods, and a case study on OSB. Laboratory and field experiments were carried out, and two types of pathogen surveys. A horizontal survey included OSB fields across Estonia, Germany, Poland, Sweden and the UK, while a vertical survey included sampling from two sets of differently managed experimental fields during three years. A new approach was introduced for measuring occurrence, where the prevalence and relative intensity of entomopathogens, biotic agents, and unidentified insect antagonists were determined. The effect of dose, timing, and the application method on S. feltiae in the control of pests in OSB, and the potential of a controlled release delivery system (CRS) were evaluated in the field. Studies on the impact of selected biotic and abiotc factors (Brassica plant, bait insects, developmental stages of Meligethes aeneus Fab., Isaria fumosorosea Wize (Ifr), and organic and synthetic fertilizers) on the efficacy of S. feltiae were conducted in the laboratory. Persistence of S. feltiae in the OSB growing system, and the effect of dose, timing, and the application method, was assessed in the field as part of the efficacy experiments. The impact of selected biotic and abiotic factors on S. feltiae persistence was assessed in laboratory experiments. The pathogen survey showed that the occurrence of entomopathogens is low in the OSB growing system, and that a management system causing less disturbance (ICM) to the soil increases the relative intensity of insect parasitic nematodes and other insect antagonists. A longer study period is required to show any possible impact of ICM on the relative intensity of entomopathogenic fungi, or on the prevalence of entomopathogens. Two different measures of the occurrence yielded different results: the relative intensity revealed the difference between the two different crop management methods, while prevalence did not. The highest efficacy of S. feltiae was achieved by using a low dose and targeting all stages of M. aeneus. When only the larval stage was targeted, the application method and dose had no significant effect. The CRS decreased the pest abundance significantly more than the surface application method. S. feltiae persisted in the OSB fields in Finland for several months, but did not survive the winter. The strain survived for 7 months when it was applied in autumn in Germany, but its populations declined rapidly after winter. The examined biotic and abiotic factors had variable impacts on S. feltiae efficacy and persistence. The two measures, prevalence and relative intensity of entomopathogens, gave valuable information for their use in biocontrol programs. The recommended biocontrol strategy for OSB growing in Finland is inundation and seasonal inoculation of EPN. The impact of some biotic and abiotic factors on S. feltiae efficacy and persistence is significant, and can be used to improve the efficacy of EPN. The CRS is a novel alternative for EPN application, and should also be considered for use on other crops. Keywords: Biological control, inundation, inoculation, conservation, formulation, slow release method, crop rotation, Entomopathogenic nematodes, Steinernema feltiae, oilseed rape pests, Meligethes aeneus, Phyllotreta spp., occurrence, prevalence, intensity, efficacy, persistence, field, Isaria fumosorosea, biotic factors, abiotic factors, interaction, impact, insect stages, integrated crop management, standard (conventional) crop management

Stress tolerance and virulence of insect-pathogenic fungi are determined by environmental conditions during conidial formation

The virulence to insects and tolerance to heat and UV-B radiation of conidia of entomopathogenic fungi are greatly influenced by physical, chemical, and nutritional conditions during mycelial growth. This is evidenced, for example, by the stress phenotypes of Metarhizium robertsii produced on various substrates. Conidia from minimal medium (Czapek's medium without sucrose), complex medium, and insect (Lepidoptera and Coleoptera) cadavers had high, moderate, and poor tolerance to UV-B radiation, respectively. Furthermore, conidia from minimal medium germinated faster and had increased heat tolerance and were more virulent to insects than those from complex medium. Low water-activity or alkaline culture conditions also resulted in production of conidia with high tolerance to heat or UV-B radiation. Conidia produced on complex media exhibited lower stress tolerance, whereas those from complex media supplemented with NaCl or KCl (to reduce water activity) were more tolerant to heat and UV-B than those from the unmodified complex medium. Osmotic and nutritive stresses resulted in production of conidia with a robust stress phenotype, but also were associated with low conidial yield. Physical conditions such as growth under illumination, hypoxic conditions, and heat shock before conidial production also induced both higher UV-B and heat tolerance; but conidial production was not decreased. In conclusion, physical and chemical parameters, as well as nutrition source, can induce great variability in conidial tolerance to stress for entomopathogenic fungi. Implications are discussed in relation to the ecology of entomopathogenic fungi in the field, and to their use for biological control. This review will cover recent technologies on improving stress tolerance of entomopathogenic fungi for biological control of insects.

Selective isolation of dematiaceous fungi from the workers of Atta laevigata (Formicidae: Attini)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Potencial bioinsecticida de hongos entomopatógenos de plagas en granos almacenados

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Culture Age, temperature, and pH affect the polyol and trehalose contents of fungal propagules

The growth and conidial physiology of the entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae, and Paecilomyces farinosus were studied under different conditions. The effects of culture age (up to 120 days), temperature (5 to 35°C), and pH (2.9 to 11.1) were determined. Growth was optimal at pH 5 to 8 for each isolate and between 20 and 35°C, depending on the isolate. The predominant polyol in conidia was mannitol, with up to 39, 134, and 61 mg g of conidia-1 for B. bassiana, M. anisopliae, and P. farinosus, respectively. Conidia of M. anisopliae contained relatively small amounts of lower-molecular-weight polyols and trehalose (less than 25 mg g-1 in total) in all treatments. Conidia of B. bassiana and P. farinosus contained up to 30, 32, and 25 mg of glycerol, erythritol, and trehalose, respectively, g-1, depending on the treatment. Conidia of P. farinosus contained unusually high amounts of glycerol and erythritol at pH 2.9. The apparent effect of pH on gene expression is discussed in relation to the induction of a water stress response. To our knowledge, this is the first report of polyols and trehalose in fungal propagules produced over a range of temperature or pH. Some conditions and harvesting times were associated with an apparent inhibition of synthesis or accumulation of polyols and trehalose. This shows that culture age and environmental conditions affect the physiological quality of inoculum and can thereby determine its potential for biocontrol.

Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability

The insect pathogen Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosos can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (a(w)) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0 05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951 - 0.935 a(w) (≡ 95.1 - 93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 a(w) (≡ 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930 - 0.920 a(w)). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.