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Characterization of Beauveria bassiana isolates from Kyrgyzstan
Abstract
We report on the enzootic foci of the insect pathogenic fungus, Beauveria bassiana, found in high meadows in the middle mountain steppes of Kyrgyzstan, at elevations from 1000 m to 2200 m. The growth characteristics of various B. bassiana isolates on different media and as a function of temperature were studied. In addition, the ability of the fungal isolates to produce enzymes with amylase, protease and lipase activities was investigated. Dense biomass production on inexpensive solid media (oatmeal and bean oil meal) produced conidia used for insect bioassays targeting white grub larvae (Phyllophaga fullo, Coleoptera, Scarabaeidae) and nymphal and adult populations of whiteflies (Trialeurodes vaporariorum, Hemiptera, Aleyrodidae). The efficacies of the tested B. bassiana strains for third instar white grub larvae varied, with only two strains showing high entomopathogenic activity. At 25 °C, mortality reached 73% for Bav.5-Gal and 74% for Bav.1-Lep at 55 d post-infection, but was lower, 27% and 29%, respectively, at 12 °C. These two strains produced significantly higher mortality in adult and whitefly nymphs, with 65-75% mortality 6 d post-infection. Based on morphological characters, including production of ellipsoidal conidia, and molecular characters (ITS, partial 18S (SSU rDNA) and EF1-α sequences), the isolates were identified as Beauveria bassiana belonging to Clade E from Asia. Our results add to data on the diversity of ecosystems inhabited by B. bassiana and provide a local resource for pest control efforts.
... The samples were examined in the laboratory of Research on Biological Systems and Geometry LRSBG (Faculty of life Sciences, Mascara University) where they were observed under a binocular magnifying glass. The cadavers were sterilized by immersion in a solution of sodium hypochlorite (1%) for 2 to 3 min and then rinsed successively with sterile distilled water, and finally they were dried with sterile filter paper (Doolotkeldieva et al. 2019). The sterile cadavers were placed in Sabouraud Dextrose Agar (SDA) and incubated for 5 days at 25 ± 2 °C and 75 ± 5% R.H. ...
... Identification of the fungal isolate was based on the different morphological characteristics of its colonies such as, growth, color, shape and texture (Doolotkeldieva et al. 2019). An aliquot of the fungal culture was placed on a glass slide, and then a drop of lactophenol cotton blue stain was added and covered with a cover slip. ...
... The amylolytic activity of B. bassiana isolate was tested by using plates of starch agar medium; the plates were then incubated at 28 °C for 3 days. The halo appeared around the fungal colonies in the case of amylase production (Doolotkeldieva et al. 2019). Protease activity of B. bassiana isolate was demonstrated using Skim milk agar. ...
Background
The cowpea aphid, Aphis craccivora Koch (Hemiptera: Aphidiidae) is a polyphagous aphid species that causes various damage on different crops. The conventional method of controlling this pest is the use synthetic insecticides that threaten both the environmental safety and human health. Moreover, it contributes to the emergence of insecticide-resistant generations. Hence, relying on Entomopathogenic fungi (EPF) remains one of the most safe and effective alternative solutions to control insect pests. For the mentioned reasons, the EPF, Beauveria bassiana was isolated and characterized; besides, its efficiency against adults’ A. craccivora was evaluated both in the laboratory and in the greenhouse.
Results
A new isolate of B. bassiana was isolated from collected cadavers’ insects associated with the population of A. craccivora in a rural area in the Northwest of Algeria. This isolate was identified on the basis of its morphological and molecular characteristics and was referred to as B. bassiana BBAA. The enzymatic activities of this isolate revealed a high production of chitinase, protease and lipase, without any production of amylase. The use of different concentrations of B. bassiana BBAA conidia against A. craccivora led to a high mortality rate, ranging from 64 to 74% mortality on the seventh day post-treatment in vitro and 58 to 70% in greenhouse.
Conclusion
Virulence and enzymatic activities produced by B. bassiana BBAA demonstrated the necessity to exploit entomopathogenic fungi (EPFs) in pest control.
... Accordingly, the highest conidial length was obtained with 7.10 and 3.24 μm for M. robertsii isolate K-63 and B. bassiana isolate APPRC-27, respectively. This agrees with previous work conducted by Doolotkeldieva et al. (2019), who reported the conidial length and width of B. bassiana were 2.27 and 1.85 μm, respectively, with a length to width ratio of 1.23 μm. Bich et al. (2021) also indicated that the conidial diameters of B. bassiana showed a mean value of 1.7 to 2.3 μm. ...
Background Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 ⁷ spores ml ⁻¹ and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day ⁻¹ . The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 ⁸ conidial ml ⁻¹ at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii , are promising bioagents for the control of insect pests.
... Accordingly, the highest conidial length was obtained with 7.10 and 3.24 μm for M. robertsii isolate K-63 and B. bassiana isolate APPRC-27, respectively. This agrees with previous work conducted by Doolotkeldieva et al. (2019), who reported the conidial length and width of B. bassiana were 2.27 and 1.85 μm, respectively, with a length to width ratio of 1.23 μm. Bich et al. (2021) also indicated that the conidial diameters of B. bassiana showed a mean value of 1.7 to 2.3 μm. ...
Background
Entomopathogenic fungi exists naturally in plants as an asymptote and have the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella.
Methods
From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were isolated using the galleria bait method. All fungi isolate were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their virulence was evaluated on second instar larvae of Galleria melonella under laboratory conditions.
Results
In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11.
Conclusions
In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of insect pests.
... For 15 days, daily observations were made to track the development of a fungus colony. The isolated colony was cleaned, identified macroscopically by its distinctive colour and shape, and studied microscopically in accordance with the colour atlases of pathogenic fungi and entomopathogenic fungi (Doolotkeldieva et al., 2019). ...
... For 15 days, daily observations were made to track the development of a fungus colony. The isolated colony was cleaned, identified macroscopically by its distinctive colour and shape, and studied microscopically in accordance with the colour atlases of pathogenic fungi and entomopathogenic fungi (Doolotkeldieva et al., 2019). ...
... For 15 days, daily observations were made to track the development of a fungus colony. The isolated colony was cleaned, identified macroscopically by its distinctive colour and shape, and studied microscopically in accordance with the colour atlases of pathogenic fungi and entomopathogenic fungi (Doolotkeldieva et al., 2019). ...
... Accordingly, the highest conidial length was obtained with 7.10 and 3.24 μm for M. robertsii isolate K-63 and B. bassiana isolate APPRC-27, respectively. This agrees with previous work conducted by Doolotkeldieva et al. (2019), who reported the conidial length and width of B. bassiana were 2.27 and 1.85 μm, respectively, with a length to width ratio of 1.23 μm. Bich et al. (2021) also indicated that the conidial diameters of B. bassiana showed a mean value of 1.7 to 2.3 μm. ...
Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella. Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions. Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x107 spores ml-1 and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day-1. The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x108 conidial ml-1 at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11. Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii , are promising bioagents for the control of pests.
... Accordingly, the highest conidial length was obtained with 7.10 and 3.24 μm for M. robertsii isolate K-63 and B. bassiana isolate APPRC-27, respectively. This agrees with previous work conducted by Doolotkeldieva et al. (2019), who reported the conidial length and width of B. bassiana were 2.27 and 1.85 μm, respectively, with a length to width ratio of 1.23 μm. Bich et al. (2021) also indicated that the conidial diameters of B. bassiana showed a mean value of 1.7 to 2.3 μm. ...
Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella .
Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions.
Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 ⁷ spores ml ⁻¹ and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day ⁻¹ . The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 ⁸ conidial ml ⁻¹ at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11.
Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.
... Accordingly, the highest conidial length was obtained with 7.10 and 3.24 μm for M. robertsii isolate K-63 and B. bassiana isolate APPRC-27, respectively. This agrees with previous work conducted by Doolotkeldieva et al. (2019), who reported the conidial length and width of B. bassiana were 2.27 and 1.85 μm, respectively, with a length to width ratio of 1.23 μm. Bich et al. (2021) also indicated that the conidial diameters of B. bassiana showed a mean value of 1.7 to 2.3 μm. ...
Background: Entomopathogenic fungi exists naturally in plants as an asymptote and has the potential to reduce the population of insect pests through indirect interactions. This study was conducted to detect and characterize the endophytic fungi Beauveria bassiana and Metarhizium robertsii from the rhizosphere soil of tomato plants and their virulence effect on Galleria melonella .
Methods: From the rhizosphere soil of 40 tomato fields, three Beauveria bassiana and seven Metarhizium robertsii isolates were identified using the galleria bait method. All fungi isolates were morphologically characterized by their colony color, shape, and surface texture. Isolates with the highest percentages of germination, conidial yield, and radial growth were selected, and their pathogenicity was evaluated on second instar larvae of Galleria melonella under laboratory conditions.
Results: In this study, Beauveria bassiana showed white colony color and aseptate hyphae, whereas Metarhizium robertsii showed dark green to light green colony color and septate hyphal structures. Maximum spore production and conidial length were obtained by Beauveria bassiana isolate APPRC-27 with 2.67x10 ⁷ spores ml ⁻¹ and 3.24 µm, respectively. Colony radial growth rates ranged from 1.73 to 3.24 mm day ⁻¹ . The results revealed that the highest mortality rate of Galleria melonella (100%) was obtained by Metarhizium robertsii isolates K-61 and K-102 at a concentration of 1x10 ⁸ conidial ml ⁻¹ at 7 days post-inoculation. The lowest mortality rate was registered by Metarhizium robertsii isolate RST-11.
Conclusions: In the present study, isolates that produced the most spores and had the highest germination rates were the most virulent to Galleria mellonella second instar larvae. Therefore, virulent entomopathogenic fungi, Beauveria bassiana and Metarhizium robertsii, are promising bioagents for the control of pests.
... As fermentações de fungos em resíduos agrícolas no estado sólido são consideradas adequadas e podem ser convertidas em produtos lucrativos, a exemplo disso, a produção de fungos entomopatogênicos em meio sólido de baixo custo apresentam bons resultados, uma vez que não necessitam de elevada tecnologia e permitem à produção direta de unidades infectivas, a exemplo disso, a farinha de aveia, farinha de óleo de feijão, além de casca de batata e casca de arroz apresentaram bons resultados para a produção de esporos de B. bassiana em condições ambientes (Doolotkeldieva et al., 2019;Chilakamarry et al., 2021). Em suma nossos resultados sugerem que os meios avaliados apresentaram uma boa produção do fungo Beauveria sp o que pode ser um indicativo para o desenvolvimento de estratégias inovadoras de produção do patógeno, além de um indicativo de reaproveitamento de resíduos da indústria da palma de óleo tendo em vista reduzir os impactos ambientais dos seus subprodutos no meio ambiente e a utilização de produtos químicos na agricultura. ...
A palma de óleo (Elaeis guineensis Jacq.) é uma cultura industrial amplamente utilizada no mundo todo, contudo, é afetada pelo ataque de insetos praga que representam um fator limitante para a sua produção em grande escala, neste sentido, os fungos entomopatogênicos são importantes agentes de controle biológico de pragas, pois podem infectar o inseto em diversos estágios de desenvolvimento. Por isso, alternativas ambientalmente viáveis e de baixo custo são importantes para a produção destes fungos. Deste modo, o presente trabalho tem por objetivo avaliar o desenvolvimento do fungo Beauveria sp. (BP05) em resíduos da palma de óleo, bem como, determinar a maior produção do fungo, por meio das análises da atividade, concentração e viabilidade dos conídios desenvolvidos. Para avaliar o desenvolvimento do fungo utilizou-se, Arroz parboilizado-AP; Torta de dendê molhada-TDM; Torta de dendê lavada-TDL; Torta de palmiste lavada-TPL e torta de palmiste molhada-TPM, onde avaliou - se a atividade, concentração e viabilidade dos conídios do fungo. Sendo os tratamentos T3 (TDM 30% + TPM 30% + AP 40%) e T5 (TDM 25% + TPM 25% + AP 50%) mais produtivos para o desenvolvimento do fungo. Esses resultados podem contribuir para uma maior produção de estruturas reprodutivas de Beauveria sp. (BP05) e o reaproveitamento dos resíduos da palma de óleo, reduzindo os seus impactos ambientais, e os custos de produção em grande escala de fungos entomopatogênicos.
... Entomopathogenic fungi (EF) are highly whidespread geographically and are highly capable of colonizing soil, which has led to their use as biological control agents worldwide. One of these EF is Beauveria bassiana fungus, which is an ascomycete that belongs to the Cordycipitaceae family of the Hypocreales order, capable of infecting more than 700 species of invertebrates through the cuticle via its conidia [3,4]. Furthermore, this order has great incidence as an asymptomatic endophyte in tissues of different plant species (Zea mays, Theobroma cacao, Phoenix dactylifera, Coffea L., Musa paradisiaca L., Pinus radiata, Vicia faba, Papaver somniferum, Gossypium, Manihot esculenta, Phaseolus vulgaris, and Solanum lycopersicum L.). ...
The objective of this work was to evaluate the effect of Beauveria bassiana (Bb 1205) on controlling Fusarium oxysporum f. sp. lycopersici (Fol 17108) in tomato plants in greenhouse conditions. Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymes β-1,3-glucanases and chitinases. Inoculation of Bb 1205 occurred at a concentration of 1 × 108 conidia·mL−1, which was administered onto the leaves, directly into the soil and via injection. Infection with Fol 17108 occurred with 1 × 106 spores·mL−1, which were added directly to the soil. Spectrophotometry was used for measuring agronomic parameters, namely activity of chitinases and β-1,3-glucanases in foliage and roots. When Bb 1205 was added to the soil, the chlorophyll index and aerial part length showed significant differences. In addition, it was determined that root length, fresh weight of foliage, flower, and fruit count increased 82 days after inoculation (dai). Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai (41.8 and 11.6-fold, respectively). Inoculation on the foliage showed a 10-fold increase of β-1,3-glucanases in the roots after 82 dpi. As for leaves, a 3.8-fold increase was found when the stem was inoculated. In the different in vivo applications, Bb 1205 activated its defenses by expressing the chitinase enzymes and β-1,3-glucanase, thus reducing the damage caused by Fol 17108, demonstrating increase plant growth thereafter.
The fungi from the genus Cordyceps have been used in the preparation of remedies in traditional medicine due to the several health benefits attributed to their consumption. In the present day, the production of fungal biomass and metabolites from Cordyceps is highly dependent on manual harvesting, which has a high environmental impact and is impractical for the large-scale production of biomass and valuable metabolites. With an increasing demand for these substances due to their antioxidant, anti-inflammatory, antidiabetic, antimutagenic, antimicrobial, anti-insomnia, antiviral, antimicrobial, and immunomodulatory activities, it is necessary to identify opportunities for their use in everyday products and alternatives for their production on an industrial scale. This chapter highlights how the substances from Cordyceps can be extracted and recovered to be used in nutraceuticals, functional foods, cosmeceuticals, pigments, and drug formulations. Besides that, several options for the implementation of biotechnological processes with Cordyceps are discussed, emphasizing the use of vegetable biomass as a sustainable source of nutrients and the functionalization of foods through fermentation. The prospects for the use of Cordyceps in the industry are overwhelming; however, more research is needed to develop feasible and safe biotechnological processes with these fungi.
The entomopathogenic fungus Beauveria majiangensis strain MJ1015, recently isolated from white grubs on a blueberry farm in Guizhou, China, could be used as a biocontrol agent. As a first step toward determining the effect of different solid culture media, temperature, and pH on colony growth rate and sporulation, we evaluated the optimum solid medium for mycelial growth and conidia production on a commercial scale. Subsequently, we also used single-factor analysis and response surface optimization to optimize the composition of the solid culture medium. On potato dextrose agar (PDA) medium, MJ1015 grew fastest and produced the highest spore yield at 29°C and pH 5. The best solid medium for the growth and sporulation of strain MJ1015 comprised 64.70 g/l of rice, 13.00 g/l of wheat, 0.30 g/l of NaNO 3 , 0.36 g/l of K 2 HPO 4 · 3H 2 O, and 1.00 g/l of CaCO 3 . Rice, NaNO 3 , and K 2 HPO 4 · 3H 2 O were the main influencing factors. The predicted value of cultured spores using the optimal medium was 4.56 x 10 ¹⁰ conidia/l. The validation test results showed that the average growth rate of strain MJ1015 on the optimal medium was 85% and 96% faster than that on Sabouraud dextrose agar with yeast extracts medium (SDAY) and PDA, respectively. Sporulation was 43.90 times and 9.65 times of that produced on SDAY and PDA, respectively. Our findings provide a theoretical basis for the commercial production of B. majiangensis to control white grubs.
We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated
methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing
systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The
new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict
gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line.
The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command
line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput
and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
Significant progress has been made in the biochemical and genetic characterization of the host-pathogen interaction mediated by insect pathogenic fungi, with the most widely studied being the Ascomycetes (Hypocrealean) fungi, Metarhizium robertsii and Beauveria bassiana. However, few studies have examined the consequences and effects of host (insect) microbes, whether compatible or antagonistic, on the development and survival of entomopathogenic fungi. Host microbes can act on the insect cuticular surface, within the gut, in specialized insect microbe hosting structures, and within cells, and they include a wide array of facultative and/or obligate exosymbionts and endosymbionts. The insect microbiome differs across developmental stages and in response to nutrition (e.g., different plant hosts for herbivores) and environmental conditions, including exposure to chemical insecticides. Here, we review recent advances indicating that insect-pathogenic fungi have evolved a spectrum of strategies for exploiting or suppressing host microbes, including the production of antimicrobial compounds that are expressed at discrete stages of the infection process. Conversely, there is increasing evidence that some insects have acquired microbes that may be specialized in the production of antifungal compounds to combat infection by (entomopathogenic) fungi. Consideration of the insect microbiome in fungal insect pathology represents a new frontier that can help explain previously obscure ecological and pathological aspects of the biology of entomopathogenic fungi. Such information may lead to novel approaches to improving the efficacy of these organisms in pest control efforts.
Broad host range insect pathogenic fungi penetrate through the host cuticle, necessitating an ability to confront and overcome surface lipids and other molecules that often include antimicrobial compounds. In this context, induction of lipid assimilatory pathways by exogenous substrates is crucial for successful infection to occur, and lipid growth substrates can have significant effects on the virulence of fungal infectious propagules, e.g. conidia. The production of lipases is a critical part of the cuticle-degrading repertoire and pathways involved in triglyceride metabolism and phospholipid homeostasis have been shown to contribute to host invasion. Mobilization of endogenous lipid stores via the activities of the caleosin and perilipin lipid storage-turnover proteins, have been linked to diverse processes including formation of penetration structures, e.g. germ tubes and appressoria, spore properties and dispersal, and the ability to respond to lipid growth substrates and virulence. Here, we summarize recent advances in our understanding of lipid assimilation and mobilization pathways in the ability of entomogenous fungi to infect and use host substrates. Host surface and internal lipids can alternatively act as antifungal barriers, inducers of pathogenesis-related pathways, and/or as fungal growth substrates. Lipids and lipid assimilation can be considered as forming a co-evolutionary web between in insect host and entomogenous fungi.
Significance
Since the discovery of oosporein more than 70 years ago, there have been conflicting reports on its potential antimicrobial and insecticidal activities. Our results indicate that oosporein is unlikely to function as an insect toxin or to be involved in early to mid-infection processes, including penetration and immune evasion. Instead, oosporein most likely functions after death of the host to thwart bacterial competition on a host cadaver, allowing the fungus to maximally use host nutrients and complete its life cycle. Our data also reveal that oosporein production is regulated by a cascade of transcription factors, with BbSmr1 acting as an upstream negative regulator, targeting the expression of OpS3, which in turn acts as a positive regulator of the oosporein biosynthetic gene cluster.
Research on the insect pathogenic filamentous fungus, Beauveria bassiana has witnessed significant growth in recent years from mainly physiological studies related to its insect biological control potential, to addressing fundamental questions regarding the underlying molecular mechanisms of fungal development and virulence. This has been in part due to a confluence of robust genetic tools and genomic resources for the fungus, and recognition of expanded ecological interactions with which the fungus engages. Beauveria bassiana is a broad host range insect pathogen that has the ability to form intimate symbiotic relationships with plants. Indeed, there is an increasing realization that the latter may be the predominant environmental interaction in which the fungus participates, and that insect parasitism may be an opportunist lifestyle evolved due to the carbon- and nitrogen-rich resources present in insect bodies. Here, we will review progress on the molecular genetics of B. bassiana, which has largely been directed toward identifying genetic pathways involved in stress response and virulence assumed to have practical applications in improving the insect control potential of the fungus. Important strides have also been made in understanding aspects of B. bassiana development. Finally, although increasingly apparent in a number of studies, there is a need for progressing beyond phenotypic mutant characterization to sufficiently investigate the molecular mechanisms underlying B. bassiana's unique and diverse lifestyles as saprophyte, insect pathogen, and plant mutualist.
Entomopathogenic fungi have a great potential as control agents, as they constitute a group with over 750 species that, when dispersed in the environment, provoke fungal infections in insect populations. These fungi begin their infective process when spores are retained on the integument surface, where the formation of the germinative tube initiates, the fungi starting to excrete enzymes such as proteases, chitinases, quitobiases, Upases and lipoxygenases. These enzymes degrade the insect's cuticle and help in the process of penetration by mechanical pressure that is initiated by the apresorium, a specialized structure formed in the germinative tube. Once inside the insect, the fungi develop as hyphal bodies that disseminate through the haemocele and invade diverse muscle tissues, fatty bodies, Malpighian tubes, mitochondria and haemocytes, leading to death of the insect 3 to 14 days after infection. Once the insect dies and many of the nutrients are exhausted, fungi start micelar growth and invade all the organs of the host. Finally, hyphae penetrate the cuticle from the interior of the insect and emerge at the surface, where they initiate spore formation under appropriate environmental conditions.
Air conidia production of Beauveria bassiana, strain CA-603 was studied based on modified diphasic system. The biomass yield obtained in the first phase based on submerged cultivation of fungus was processed using methodology providing different contact with air space. Our study indicated that productivity of the second stage of diphasic system is had inversely proportional dependence on depth of liquid fungal biomass. Increase of biomass depth is significantly decreased production of air conidia. Two methodology of biomass processing extending contact biomass with air space including distribution of fungal material on surface of hygroscopic paper and starch packaging peanuts were investigated. The novel substrates provided optimal contact between the submerged fungal biomass and the air, and overall, conidial production was directly proportional to the total area of air-to-fungal surface. Technologies based on the starch peanuts and hygroscopic paper were the most productive in comparison to the common technology where the submerged culture was transferred to flat containers. The advantages and disadvantages of these different production methods are discussed.
The entomopathogenic fungi-like Beauveria bassiana must penetrate via the integument of an insect to reach the hemocoel.
Since proteins are the molecules responsible for integument strength in insects, the proteins must synthesise the cuticle degrading
proteases which will then enable the proteases to penetrate. It is important to determine the biochemical properties of these proteases
so that fungal virulence can be better understood. In the current study, a recently collected isolate of B. bassiana, namely AM-118, was
inoculated in liquid media containing 0.5% of Andrallus spinidens Fabricus cuticle to obtain specific proteases. The crude samples were
purified via a three step process using ammonium sulfate, Sepharyl G-100, and DEAE-Cellulose Fast Flow. The results revealed two
proteases known as subtilisin-like (Pr1), and trypsin-like (Pr2), with the molecular weights of 105 and 103 kDa. The optimal pH and
temperature values were found to be 8 and 35°C for Pr1 and 8 and 40°C for Pr2, respectively. Inhibitors like AEBSF, EDTA, TPCK, and
phenanthroline significantly affected proteolytic activities. Here, we reported two fungal proteases by high molecular weight from an
Iranian isolate of B. bassiana. These findings will help us to better understand fungal virulence against insects.
Infections mediated by broad host range entomopathogenic fungi represent seminal observations that led to one of the first germ theories of disease and are a classic example of a co-evolutionary arms race between a pathogen and target hosts. These fungi are able to parasitize susceptible hosts via direct penetration of the cuticle with the initial and potentially determining interaction occurring between the fungal spore and the insect epicuticle. Entomogenous fungi have evolved mechanisms for adhesion and recognition of host surface cues that help direct an adaptive response that includes the production of: (a) hydrolytic, assimilatory, and/or detoxifying enzymes including lipase/esterases, catalases, cytochrome P450s, proteases, and chitinases; (b) specialized infectious structures, e.g., appressoria or penetrant tubes; and (c) secondary and other metabolites that facilitate infection. Aside from immune responses, insects have evolved a number of mechanisms to keep pathogens at bay that include: (a) the production of (epi) cuticular antimicrobial lipids, proteins, and metabolites; (b) shedding of the cuticle during development; and (c) behavioral-environmental adaptations such as induced fever, burrowing, and grooming, as well as potentially enlisting the help of other microbes, all intended to stop the pathogen before it can breach the cuticle. Virulence and host-defense can be considered to be under constant reciprocal selective pressure, and the action on the surface likely contributes to phenomena such as strain variation, host range, and the increased virulence often noted once a (low) virulent strain is “passaged” through an insect host. Since the cuticle represents the first point of contact and barrier between the fungus and the insect, the “action on the surface” may represent the defining interactions that ultimately can lead either to successful mycosis by the pathogen or successful defense by the host. Knowledge concerning the molecular mechanisms underlying this interaction can shed light on the ecology and evolution of virulence and can be used for rational design strategies at increasing the effectiveness of entomopathogenic fungi for pest control in field applications.
The aim of this study was to evaluate the effectiveness of the entomopathogenic fungi (EPF), Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) strain PPRI 5339 [BroadBand, an emulsifiable spore concentrate (EC) formulation] and Metarhizium anisopliae (Metsch.) Sorokin (Hypocreales: Clavicipitaceae) strain F52 [Met52, both EC and granular (GR) formulations] against the larvae of Polyphylla fullo (L.) (Coleoptera: Scarabaeidae). Larvicidal bioassays were performed in foam boxes (100 by 75 by 50 cm; length by width by height), containing moist soil medium with some humus and potato tubers as food. Although the B. bassiana product (min. 4 × 10(9) conidia/ml) was applied at 100, 150, and 200 ml/100 l water; M. anisopliae strain F52 was applied at 500, 1,000, and 1,500 g/m(3) of moist soil medium for GR (9 × 10(8) cfu/g) and 75, 100, and 125 ml/100 l water for EC (5.5 × 10(9) conidia/ml) formulation. Both fungi were pathogenic to larvae of the pest; however, young larvae (1st and 2nd instars) were more susceptible to infection than older ones (3rd instar). Mortality rates of young and older larvae varied with conidial concentration of both fungi and elapsed time after application. The B. bassiana product was more effective than both of the formulations of the M. anisopliae product, causing mortalities up to 79.8 and 71.6% in young and older larvae, respectively. The highest mortality rates of young and older larvae caused by the M. anisopliae product were 74.1 and 67.6% for the GR formulation, 70.2 and 61.8% for the EC formulation, respectively. These results may suggest that both fungi have potential to be used for management of P. fullo.
© The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.
The desire for decreased reliance on chemical pesticides continues to fuel interest in alternative means for pest control including the use of naturally occurring microbial insect pathogens. Insects, as vectors of disease causing agents or as agricultural pests, are responsible for millions of deaths and significant economic losses worldwide, placing stresses on productivity (GDP) and human health and welfare. In addition, alterations in climate change are likely to affect insect ranges, expanding their access to previously constrained geographic areas, a potentially worrisome outcome. Metarhizium anisopliae and Beauveria bassiana, two cosmopolitan fungal pathogens of insects found in almost all ecosystems, are the most commonly applied mycoinsecticides for a variety of insect control purposes. The availability of the complete genomes for both organisms coupled to robust technologies for their transformation has led to several advances in engineering these fungi for greater efficacy and/or utility in pest control applications. Here, we will provide an overview of the fungal-insect and fungal-plant interactions that occur and highlight recent advances in the genetic engineering of these fungi. The latter work has resulted in the development of strains displaying (1) increased resistance to abiotic stress, (2) increased cuticular targeting and degradation, (3) increased virulence via expression of insecticidal protein/peptide toxins, (4) the ability to block transmission of disease causing agents, and (5) the ability to target specific insect hosts, decrease host fecundity, and/or alter insect behaviors.
Covering: 2000 up to April 2014This highlight discusses the secondary metabolite potential of the insect pathogens Metarhizium and Beauveria, including a bioinformatics analysis of secondary metabolite genes for which no products are yet identified.
A Brazilian isolate of Beauveria bassiana (CG425) that shows high virulence against the coffee berry borer (CBB) was examined for the production of subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases. Fungal growth was either in nitrate-medium or in CBB cuticle-containing medium under both buffered and unbuffered conditions. In unbuffered medium supplemented with cuticle, the pH of cultures dropped and Pr1 and Pr2 activities were detected in high amounts only at a pH of 5.5 or higher. In buffered cultures, Pr1 and Pr2 activities were higher in medium supplemented with cuticle compared to activities with nitrate-medium. The Pr1 and Pr2 activities detected were mostly in the culture supernatant. These data suggest that Pr1 and Pr2 proteases produced by strain CG425 are induced by components of CBB cuticle, and that the culture pH influences the expression of these proteases, indicating the occurrence of an efficient mechanism of protein secretion in this fungus. The results obtained in this study extend the knowledge about protease production in B. bassiana CG425, opening new avenues for studying the role of secreted proteases in virulence against the coffee berry borer during the infection process.
The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.
This review presents and discusses the merits of the methodologies available for implementing integrated pest management
(IPM) of B. tabaci populations: namely, chemical control with selective insecticides, biological control, crop plant resistance and physical/mechanical
methods. Insecticides, by their poisonous nature, are often harmful to natural enemies and therefore, disruptive to overall
pest management. However, the more modern materials that are effective for B. tabaci control are relatively specific to the target pests, and therefore are less harmful to natural enemies and the environment;
consequently, they are also more suitable for integrative combination with other methods. Natural enemies, by themselves,
usually do not form a suitable solution of B. tabaci- caused problems. However, their occurrence and use greatly reduces the pest’s populations. Since viral plant diseases transmitted
by B. tabaci are not curable, the principal tactics for their management should be based on prevention of transmission by physical-mechanical
methods and/or on utilization of host-plant resistance. The correct implementation of natural enemies will help to reduce
whitefly numbers, which can then be more readily managed using cultural and, only if necessary, chemical countermeasures.
Thus, adopting IPM will alleviate the numerous concerns that accompany the use of chemicals, including those associated with
environmental pollution and the widespread resistance that plagues B. tabaci management.
A substantial number of mycoinsecticides and mycoacaricides have been developed worldwide since the 1960s. Here we present an updated, comprehensive list of these products. At least 12 species or subspecies (varieties) of fungi have been employed as active ingredients of mycoinsecticides and mycoacaricides for inundative and inoculative applications, although some are no longer in use. Products based on Beauveria bassiana (33.9%), Metarhizium anisopliae (33.9%), Isaria fumosorosea (5.8%), and B. brongniartii (4.1%) are the most common among the 171 products presented in this paper. Approximately 75% of all listed products are currently registered, undergoing registration or commercially available (in some cases without registration), whereas 15% are no longer available. We were unable to determine the status of the remaining 10%. Insects in the orders Hemiptera, Coleoptera, Lepidoptera, Thysanoptera, and Orthoptera comprise most of the targets, distributed among at least 48 families. A total of 28 products are claimed to control acarines (mites and ticks) in at least 4 families, although only three products (all based on Hirsutella thompsonii) were exclusively developed as acaricides. Eleven different technical grade active ingredients or formulation types have been identified, with technical concentrates (fungus-colonized substrates) (26.3%), wettable powders (20.5%) and oil dispersions (15.2%) being most common. Approximately 43% of all products were developed by South American companies and institutions. Currently, what may be the largest single microbial control program using fungi involves the use of M. anisopliae for control of spittlebugs (Cercopidae) in South American sugarcane and pastures.
Biopesticides based on living microbes and their bioactive compounds have been researched and promoted as replacements for synthetic pesticides for many years. However, lack of efficacy, inconsistent field performance and high cost have generally relegated them to niche products. Recently, technological advances and major changes in the external environment have positively altered the outlook for biopesticides. Significant increases in market penetration have been made, but biopesticides still only make up a small percentage of pest control products. Progress in the areas of activity spectra, delivery options, persistence of effect and implementation have contributed to the increasing use of biopesticides, but technologies that are truly transformational and result in significant uptake are still lacking.
The virulence of three isolates of Beauveria bassiana (Bals.) Vuill. and 23 isolates of Metarhizium anisopliae (Metschnik.) Sorok. (Ascomycota: Hypocreales) against the tomato spider mite, Tetranychus evansi Baker and Pritchard (Acari: Tetranychidae), was assessed in the laboratory. The effect of temperature on germination, radial growth and virulence of selected isolates (two isolates of B. bassiana and nine of M. anisopliae) on T. evansi was also investigated in the laboratory. All the fungal isolates tested were pathogenic to the adult females of T. evansi, and there were significant differences in mortality between fungal isolates. The lethal time to 50% mortality (LT(50)) values ranged from 4.2 to 8.1 days and the LT(90) values from 5.6 to 15.1 days. Temperature had significant effects on germination, radial growth and virulence of the various isolates. The best fungal germination was observed at 25 and 30 degrees C, while for the fungal radial growth it was 30 degrees C. All the isolates germinated and grew at all temperatures, but germination and radial growth varied with isolate and temperature. The selected isolates were all virulent to T. evansi, but virulence varied also with isolate and temperature.
Molecular biology methods have elucidated pathogenic processes in several fungal biocontrol agents including two of the most commonly applied entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. In this review, we describe how a combination of molecular techniques has: (1) identified and characterized genes involved in infection; (2) manipulated the genes of the pathogen to improve biocontrol performance; and (3) allowed expression of a neurotoxin from the scorpion Androctonus australis. The complete sequencing of four exemplar species of entomopathogenic fungi including B. bassiana and M. anisopliae will be completed in 2010. Coverage of these genomes will help determine the identity, origin, and evolution of traits needed for diverse lifestyles and host switching. Such knowledge combined with the precision and malleability of molecular techniques will allow design of multiple pathogens with different strategies to be used for different ecosystems and avoid the possibility of the host developing resistance.
Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana.
Entomopathogenic fungi are widely produced for use as mycoinsecticides. Therefore, improvement of the shelf life of fungal propagules under good and adverse conditions should be a pre-requisite of their production. In order to improve conidial physiology as well as mycoinsecticide efficiency, culture conditions may be varied. The Doehlert design was used to generate response surfaces with an estimation of the parameters of the quadratic model allowing the study of three different factors at a different number of levels. This experimental design was applied to optimize water activity (aw), pH, and fermentation time for Beauveria bassiana conidial production and accumulation of polyols in solid-state fermentation. Thus, it was possible to identify the region in the experimental range in which the optimum values of these parameters were simultaneously achieved. Maximal conidia production was achieved at pH 5-6 and aw=0.999. Under these conditions, polyol accumulation was 3 mg erythritol/g conidia and 29.6 mg glycerol/g conidia. However, maximal polyol accumulation was achieved at pH 4.5 and aw 0.950; erythritol production increased 33-fold and glycerol production 4.5-fold. Under these conditions conidia production was 1,000 times lower. The possibilities of increasing the quality of the biocontrol agent without neglecting yield are discussed.
The entomopathogenic fungus Beauveria bassiana produces at least three distinct single-cell propagules, aerial conidia, vegetative cells termed blastospores, and submerged conidia, which can be isolated from agar plates, from rich broth liquid cultures, and under nutrient limitation conditions in submerged cultures, respectively. Fluorescently labeled fungal cells were used to quantify the kinetics of adhesion of these cell types to surfaces having various hydrophobic or hydrophilic properties. Aerial conidia adhered poorly to weakly polar surfaces and rapidly to both hydrophobic and hydrophilic surfaces but could be readily washed off the latter surfaces. In contrast, blastospores bound poorly to hydrophobic surfaces, forming small aggregates, bound rapidly to hydrophilic surfaces, and required a longer incubation time to bind to weakly polar surfaces than to hydrophilic surfaces. Submerged conidia displayed the broadest binding specificity, adhering to hydrophobic, weakly polar, and hydrophilic surfaces. The adhesion of the B. bassiana cell types also differed in sensitivity to glycosidase and protease treatments, pH, and addition of various carbohydrate competitors and detergents. The outer cell wall layer of aerial conidia contained sodium dodecyl sulfate-insoluble, trifluoroacetic acid-soluble proteins (presumably hydrophobins) that were not present on either blastospores or submerged conidia. The variations in the cell surface properties leading to the different adhesion qualities of B. bassiana aerial conidia, blastospores, and submerged conidia could lead to rational design decisions for improving the efficacy and possibly the specificity of entomopathogenic fungi for host targets.
Beauveria is a globally distributed genus of soil-borne entomopathogenic hyphomycetes of interest as a model system for the study of entomopathogenesis and the biological control of pest insects. Species recognition in Beauveria is difficult due to a lack of taxonomically informative morphology. This has impeded assessment of species diversity in this genus and investigation of their natural history. A gene-genealogical approach was used to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species. Analyses were based on nuclear ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-alpha) sequences for 86 exemplar isolates from diverse geographic origins, habitats and insect hosts. Phylogenetic trees were inferred using maximum parsimony and Bayesian likelihood methods. Six well supported clades within Beauveria, provisionally designated A-F, were resolved in the EF1-alpha and combined gene phylogenies. Beauveria bassiana, a ubiquitous species that is characterized morphologically by globose to subglobose conidia, was determined to be non-monophyletic and consists of two unrelated lineages, clades A and C. Clade A is globally distributed and includes the Asian teleomorph Cordyceps staphylinidaecola and its probable synonym C. bassiana. All isolates contained in Clade C are anamorphic and originate from Europe and North America. Clade B includes isolates of B. brongniartii, a Eurasian species complex characterized by ellipsoidal conidia. Clade D includes B. caledonica and B. vermiconia, which produce cylindrical and comma-shaped conidia, respectively. Clade E, from Asia, includes Beauveria anamorphs and a Cordyceps teleomorph that both produce ellipsoidal conidia. Clade F, the basal branch in the Beauveria phylogeny includes the South American species B. amorpha, which produces cylindrical conidia. Lineage diversity detected within clades A, B and C suggests that prevailing morphological species concepts underestimate species diversity within these groups. Continental endemism of lineages in B. bassiana s.l. (clades A and C) indicates that isolation by distance has been an important factor in the evolutionary diversification of these clades. Permutation tests indicate that host association is essentially random in both B. bassiana s.l. clades A and C, supporting past assumptions that this species is not host specific. In contrast, isolates in clades B and D occurred primarily on coleopteran hosts, although sampling in these clades was insufficient to assess host affliation at lower taxonomic ranks. The phylogenetic placement of Cordyceps staphylinidaecola/bassiana, and C. scarabaeicola within Beauveria corroborates prior reports of these anamorph-teleomorph connections. These results establish a phylogenetic framework for further taxonomic, phylogenetic and comparative biological investigations of Beauveria and their corresponding Cordyceps teleomorphs.
Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.
An increasing number of recent studies demonstrate that entomopathogenic fungi, often solely considered as insect pathogens, play additional roles in nature, including endophytism, plant disease antagonism, plant growth promotion, and rhizosphere colonization. These newly emerging, but not yet fully understood, ecological roles hint at the possibility that we have been overlooking important attributes in our quest to develop fungal entomopathogens exclusively as inundative biopesticides against insect and other arthropod pests. Such additional roles recently-discovered to be played by entomopathogenic fungi provide opportunities for the multiple use of these fungi in integrated pest management (IPM) strategies. Of particular interest is the ability displayed by various genera of entomopathogenic fungi to colonize a wide variety of plant species in different families, both naturally and artificially following inoculation, and confer protection against not only insect pests but also plant pathogens. This article reviews the literature currently available on the endophytic colonization of different host plants by fungal entomopathogens, and summarizes the negative effects of such colonization on insect pests and plant pathogens that have been reported to date. It also addresses the possible mechanisms of protection conferred by endophytic fungal entomopathogens and explores the potential use of these fungi as dual microbial control agents against both insect and pathogen pests. Moreover, interactions amongst endophytic fungal entomopathogens and other endophytes are discussed. Finally, current limitations and future research directions for the innovative use of endophytic fungal entomopathogens as dual microbial control agents are summarized.
The entomopathogenic fungi, Beauveria bassiana IRAN 187C and Metarhizium anisopliae IRAN 437C are the two most virulent isolates against the house fly, Musca domestica L. Effects of temperatures (15, 20, 25, 30, 35°C) and relative humidity levels of (45 and 75%) on virulence of the fungi were detected by immersion and inoculated larval bed methods for larvae and baiting method for adults. The results indicated no significant difference in larval mortality and larval cadaver sporulation at temperatures of 25-30°C and 45-75% RH for Ma 437 C. But Bb 187C caused higher mortality and cadaver sporulation at this thermal range and 75% RH. There was no significant difference in adult mortality between 20, 25 and 30°C for Ma 437 C and between 25 and 30°C for Bb 187 C. Mortality and fly cadaver sporulation were higher at the mentioned thermal range at 75% than 45% RH for both isolates. LT50 values decreased with temperature increasing between 15-30°C but increased again at 35°C. M. anisopliae caused more mortality and cadaver sporulation in wider ranges of temperature and humidity than B. bassiana. So, this isolate would be good candidate for microbial control of the house fly.
Experiments on two isolates of Beauveria bassiana (Balsamo) Vuillemin (IMI 389521 and IMI 386243) investigated whether modifying the solid-substrate phase of mass-production, namely the par-boiling, water and washing regimes of the rice substrate, affected conidial yield and germination. Germination was assessed on progressively drier media (0.987, 0.978 and 0.968 aw); the effects were also studied before and after a period of short-term cool storage. There was little difference in conidial yield between treatments and/or isolates. The only significant difference was IMI 389521 on unwashed then par-boiled rice with 600 ml water/kg rice, which yielded more conidia than IMI 386243 on unwashed and not par-boiled rice. There were significant treatment effects on the germination of both isolates. In particular, the germination of conidia produced on rice that had been washed under running tap-water for 5 min and not par-boiled, was lower than all other treatments, for both isolates. Highest conidial germination for IMI 389521 was from the unwashed then par-boiled rice with 600 ml water/kg, at 96%; highest germination for IMI 386243 was from the unwashed and not par-boiled rice, at 72%. As the growth media was made progressively drier there was no change in the germination levels of IMI 389521, for any of the rice treatments. In contrast, germination of two treatments of IMI 386243, namely washed under running tap-water for 5 min and not par-boiled, and unwashed then par-boiled with 300 ml water/kg, fell significantly (by 29 and 35%, respectively). After 8-weeks storage at 5 °C, germination had fallen significantly for three of the four treatments of IMI 386243 and for one treatment of IMI 389521; the effect was greatest for IMI 386243 produced on unwashed and not par-boiled rice, with a 62% loss in germination. Inherent differences between isolates and limitations of the methodology are discussed.
Pathogenic fungi are important natural regulators of insect populations. However, many attempts to harness their potential for pest control have met with comparatively minor commercial success. Studies on mechanisms of pathogenesis have yet to contribute to the development of more efficient mycoinsecticides, but new insights into the pathogenic process are laying the groundwork. Significant progress has been made in particular in understanding enzymes involved with the penetration of host cuticle and the role of insecticidal toxins. Insect cuticle comprises up to 70% protein and it is not surprising that extracellular fungal proteases appear to be particularly important in the penetration process. Subtilisins, chymotrypsins, trypsins and metalloproteases, usually with multiple isoforms of each, provide an impressive backed-up arsenal. Pathogenic fungi produce a wide variety of toxic metabolites, which vary from low molecular weight products of secondary metabolism to complex cyclic peptides and proteolytic enzymes. Comparatively few compounds have been found in diseased insects, in quantities sufficient to account for symptoms of mycosis. An exception, a family of cyclic peptides called the destruxins, are dealt with in detail. The potential for synergy between toxins is explored also.
Premise of the study:
The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. Metarhizium robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M. robertsii were examined after inoculation with fungal conidia.
Methods:
We used light and confocal microscopy to ascertain the plant endophytic association with GFP-expressing M. robertsii. Root lengths, root hair density, and lateral roots emerged were also observed.
Key results:
Initially, M. robertsii conidia adhered to, germinated on, and colonized roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M. robertsii on the root (within 1-2 d). Plants inoculated with M. robertsii ΔMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M. robertsii did not stimulate root hair proliferation. Longer-term (60 d) associations showed that M. robertsii endophytically colonized cortical cells within bean roots. Metarhizium appeared as a mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant.
Conclusions:
These results suggest that M. robertsii is not only rhizosphere competent but also displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs.
The germination of Penicillium expansum and Aspergillus niger spores was monitored microscopically on malt extract agar at isothermal conditions ranging from 0 to 33°C and 5 to 41.5°C, respectively. The obtained germination data, expressed as percentage of germination (% P) versus time, were fitted to the modified Gompertz equation for the estimation of the germination kinetic parameters (lag time, λ(g), and germination rate, μ(g)), which were further modeled as a function of temperature via the use of Cardinal Models with Inflection (CMI). The effect of temperature on these parameters was similar with that previously reported for mycelium growth kinetics of the tested isolates. The germination of spores was also studied at various dynamic time-temperature conditions including single or sequential temperature shifts. The germination of spores at fluctuating temperatures was predicted using the modified Gompertz equation in conjunction with the CMI models for λ(g) and μ(g) and based on the assumptions that i) a temperature shift does not result in any additional λ(g) and, thus, the total lag can be calculated by adding relative parts of the lag time, and ii) after a temperature shift the germination rate μ(g) adapts instantaneously to the new temperature. The comparison between predicted and observed data showed that the germination of spores is strongly affected by the extent of the temperature shift, the percentage of germinated spores at the time of the shift and the fungal species. Apart from the scientific interest in understanding the dynamics of spores' germination, the models developed in this study can be used as tools in effective quality management systems for fungi control in foods.
Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.
Laboratory experiments were done to measure the pathogenicity of 10 autochthonous isolates of Beauveria bassiana (Balsamo) Vuill. and of five Metarhizium anisopliae (Metsch.) Sorok. toward puparia and adults of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Although all isolates applied via inoculation of the fungal suspensions on the ventral surface of the abdomen were pathogenic to adults, with mortality rates ranging from 30 to 100% and average survival times (ASTs) from 6.5 to 8.6 d, when C. capitata puparia were immersed in the conidial suspensions, only B. bassiana Bb-1333 and EABb 01/103-Su and M. anisopliae EAMa 01/58-Su isolates caused >50% mortality of puparia. In a second series of bioassays conducted on five selected isolates, adults were sprayed with four 10-fold concentrations ranging from 1.0 x 10(5) to 1.0 x 10(8) colony-forming units (cfu)/ml. The median lethal concentrations (LC50) of the four most virulent isolates ranged from 4.9 x 10(5) to 2.0 x 10(6) cfu/ml with estimated time to kill 50% of the insects ranging from 4.6 to 5.3 d. The effect of a sublethal dose (ca. LD50) of either B. bassiana EABb 01/103-Su or M. anisopliae EAMa 01/58-Su isolate was studied by reciprocal crossing. Treatment with B. bassiana reduced fecundity and fertility at 6, 8, and 10 d after treatment, with fecundity and fertility reductions ranging from 20.0 to 71.2% and from 33.6 to 60.0%, respectively. These reductions occurred in pairing combinations of treated females with either treated or nontreated males. M. anisopliae was more effective in reducing fecundity and fertility at 6 d after treatment, with the reduction varying from 58.4 to 72.1% and from 28.6 to 45.9%, respectively. In addition, the first oviposition was significantly delayed for 1 d in females treated by either fungal species. The above-mentioned five selected isolates were assayed against C. capitata puparia treated as late third instars in sterilized soil at 25'C under three moisture conditions (-0.1, -0.01, and -0.0055 MPa). At -0.01 MPa, all isolates were low pathogenic to C. capitata puparia, whereas significant differences in the puparia mortality occurred between isolates at -0.1 and -0.0055 MPa. The highest pupal mortalities ranged from 52.5 to 70.0%, as a function of soil moisture and were caused by EAMa 01/58-Su and Bb-1333 isolates.
USDA-ARS Plant Protection Research Unit US Plant, Soil & Nutrition Laboratory Tower Road Ithaca
- Richard A Humber
Humber, Richard A., 2005. Entomopathogenic Fungal Identification updated November
2005. USDA-ARS Plant Protection Research Unit US Plant, Soil & Nutrition
Laboratory Tower Road Ithaca, NY 14853-2901.
The insect-pathogenic fungus Metarhizium robertsii
- R K Sasan
- M J Ridochka
Sasan, R.K., Ridochka, M.J., 2012. The insect-pathogenic fungus Metarhizium robertsii.
101-107.
Laboratory evaluation of entomopathogenic fungi Baeuveria bassiana and Metarhizium anisopliae against pupal and adults of Ceratitis capitata (Diptera: Tephritidae)
- Quesada-Moraga