RESUMEN
Beauveria bassiana (Bb) is a widespread entomopathogenic fungus widely used in agriculture for crop protection. Other than pest control, fungi belonging to the B. bassiana complex represent an important microbial resource in agroecosystems, considering their multiple interactions with other microorganisms as antagonists of phytopathogens, or with plants as endophytic colonizers and growth promoters. Here, we characterised field collected or commercial isolates of B. bassiana relative to the environmental factors that affect their growth. We further compared the metabolome, the entomopathogenic potential and biocontrol activity of the tested isolates respectively on the insect pest Spodoptera littoralis or against the fungal plant pathogen Fusarium oxysporum. Our analysis revealed that the B. bassiana complex is characterised by a high level of inter-isolate heterogeneity in terms of nutritional requirements, establishment of intra- or inter-kingdom interactions, and the nature of metabolites produced. Interestingly, certain B. bassiana isolates demonstrated a preference for low nutrient plant-derived media, which hints at their adaptation towards an endophytic lifestyle over a saprophytic one. In addition, there was a noticeable variation among different B. bassiana isolates in their capacity to kill S. littoralis larvae in a contact infection test, but not in an intrahaemocoelic injection experiment, suggesting a unique level of adaptability specific to the host. On the other hand, most B. bassiana isolates exhibited similar biocontrol efficacy against the soil-dwelling ascomycete F. oxysporum f. sp. lycopersici, a pathogen responsible for vascular wilt disease in tomato plants, effectively averting wilting. Overall, we show that the effectiveness of B. bassiana isolates can greatly vary, emphasising the importance of isolate selection and nutritional adaptability consideration for their use in sustainable agriculture.
Asunto(s)
Beauveria , Fusarium , Larva , Control Biológico de Vectores , Spodoptera , Beauveria/fisiología , Beauveria/aislamiento & purificación , Beauveria/metabolismo , Animales , Spodoptera/microbiología , Larva/microbiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Agricultura , Metaboloma , Endófitos/aislamiento & purificación , Endófitos/metabolismo , Endófitos/fisiología , Endófitos/clasificaciónRESUMEN
Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: ⢠102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. ⢠Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. ⢠MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.
Asunto(s)
Beauveria , Gossypium , Hemípteros , Control Biológico de Vectores , Filogenia , Beauveria/genética , Beauveria/patogenicidad , Beauveria/clasificación , Beauveria/aislamiento & purificación , Animales , Hemípteros/microbiología , Virulencia , Gossypium/microbiología , Ninfa/microbiología , Esporas Fúngicas/crecimiento & desarrollo , Variación GenéticaRESUMEN
It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.
Asunto(s)
Beauveria , Variación Genética , Filogenia , ARN Ribosómico 28S , Beauveria/genética , Beauveria/clasificación , Beauveria/aislamiento & purificación , Argentina , ARN Ribosómico 28S/genética , Animales , ADN de Hongos/genética , Insectos/microbiología , Análisis de Secuencia de ADN , Datos de Secuencia Molecular , Intrones , ADN Ribosómico/genética , Análisis por ConglomeradosRESUMEN
The genus Beauveria comprises economically important entomopathogenic fungi, widely used for biological control in agriculture. Interest in these organisms in Costa Rica prompted surveys and establishment of collections in the past two decades. However, there was neither a formal identification nor a characterization of the isolates. With that purpose, the morphology and genetic variation by microsatellites and partial sequencing of Bloc, TEF-1α and RPB2 regions were studied for 32 isolates of Beauveria, which included 26 from Costa Rica, five from Puerto Rico and one from Honduras. The isolates were identified as B. bassiana (29) and B. caledonica (3). Ninety-three percent of B. bassiana isolates belonged to a monophyletic group of African and Neotropical isolates. A total of 105 alleles were recorded with 11 SSR markers, and the results suggested high diversity within the collection. Mantel tests showed low association between geographic origin and the variation among isolates.
Asunto(s)
Beauveria/clasificación , Genes Fúngicos , Variación Genética , Beauveria/citología , Beauveria/genética , Beauveria/aislamiento & purificación , Costa Rica , Honduras , Filogenia , Puerto RicoRESUMEN
A simple method for fungal epiphyte microscopic observations and preservation is described. A two-part clear casting resin, cotton leaves and two species of fungi were used to validate this protocol. We obtained very detailed images of fungal structures using this approach in addition to retaining the impressions for future reference.
Asunto(s)
Beauveria/aislamiento & purificación , Endófitos/aislamiento & purificación , Microscopía/métodos , Hojas de la Planta/microbiología , Sordariales/aislamiento & purificación , Beauveria/citología , Endófitos/clasificación , Endófitos/genética , Gossypium/microbiología , Microscopía/instrumentación , Hojas de la Planta/química , Resinas Sintéticas/química , Sordariales/citologíaRESUMEN
Entomopathogenic fungi are important agents for mosquito vector control. We report on the utility of a simple method to detect fungi on living larvae of Aedes aegypti that had been exposed to a fungal entomopathogen. Four species of the hypocrealean genera Metarhizium, Beauveria, Tolypocladium and Culicinomyces, known for their larvicidal activity against mosquito species, were tested. Living larvae previously exposed to a suspension of different conidial concentrations were set directly into the surface water film on non-nutritive agar supplemented with chloramphenicol, thiabendazole and crystal violet and then incubated. Except for C. clavisporus ARSEF 964 (which developed and produced conidia mostly inside the cadaver rather than on its surface in the present study), this method favored external fungal development and conidiogenesis on larvae of different instars after death. The dead larva on the water agar represents the unique and specific source of nutrition for the fungus that killed it. The technique facilitates the detection and posterior isolation of entomopathogenic fungi, and offers a compact, convenient, and rapid means to survey larval mosquito populations for fungal pathogens at the field.
Asunto(s)
Aedes/microbiología , Entomología/métodos , Hypocreales/aislamiento & purificación , Control de Mosquitos/métodos , Parasitología/métodos , Aedes/crecimiento & desarrollo , Animales , Beauveria/aislamiento & purificación , Larva/crecimiento & desarrollo , Larva/microbiología , Metarhizium/aislamiento & purificaciónRESUMEN
Entomopathogenic fungi are an important factor in the natural regulation of arthropod populations. Moreover, some can exist as an endophyte in many plant species and establish a mutualistic relationship. In this study, we have investigated the endophytic growth of Beauveria bassiana within different tissues of Phaseolus vulgaris in the presence and absence of Tetranuychus urticae. After the colonization of the B. bassiana within the internal tissues of P. vulgaris. The susceptibility of T. urticae appeared to depend on the life stage where high, moderate, and low mortalities were recorded among adults, nymphs, and eggs, respectively. In addition, this study provided, for the first time, molecular insight into the endophytic growth of B. bassiana by analyzing the expression of several genes involved in the development of the entomopathogenic fungi at 0-, 2-, and 7- days post-inoculation. B. bassiana displayed preferential tissue colonization within P. vulgaris that can be put into the following order based on the detection rate: leaf > stem > root. After analyzing the development-implicated genes (degrading enzymes, sugar transporter, hydrophobins, cell wall synthesis, secondary metabolites, stress management), the most remarkable finding is the detection of behavioral change between parasitic and endophytic Beauveria during post-penetration events. This study elucidates the tri-trophic interaction between fungus-plant-herbivore.
Asunto(s)
Beauveria/crecimiento & desarrollo , Beauveria/genética , Endófitos/crecimiento & desarrollo , Endófitos/genética , Phaseolus/microbiología , Phaseolus/parasitología , Tetranychidae/fisiología , Animales , Beauveria/aislamiento & purificación , ADN de Hongos/análisis , Endófitos/aislamiento & purificación , Regulación Fúngica de la Expresión Génica , Estadios del Ciclo de Vida , Control Biológico de Vectores , Tetranychidae/crecimiento & desarrollo , Tetranychidae/patogenicidadRESUMEN
The p53-like transcription factor (TF) NDT80 plays a vital role in the regulation of pathogenic mechanisms and meiosis in certain fungi. However, the effects of NDT80 on entomopathogenic fungi are still unknown. In this paper, the NDT80 orthologue BbTFO1 was examined in Beauveria bassiana, a filamentous entomopathogenic fungus, to explore the role of an NDT80-like protein for fungal pest control potential. Disruption of BbTFO1 resulted in impaired resistance to oxidative stress (OS) in a growth assay under OS and a 50% minimum inhibitory concentration experiment. Intriguingly, the oxidation resistance changes were accompanied by transcriptional repression of the two key antioxidant enzyme genes cat2 and cat5. ΔBbTFO1 also displayed defective conidial germination, virulence and heat resistance. The specific supplementation of BbTFO1 reversed these phenotypic changes. As revealed by this work, BbTFO1 can affect the transcription of catalase genes and play vital roles in the maintenance of phenotypes associated with the biological control ability of B. bassiana.
Asunto(s)
Beauveria/metabolismo , Proteínas Fúngicas/metabolismo , Insectos/microbiología , Factores de Transcripción/metabolismo , Virulencia/genética , Animales , Beauveria/aislamiento & purificación , Beauveria/patogenicidad , Catalasa/genética , Catalasa/metabolismo , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Mutación , Estrés Oxidativo/genética , Fenotipo , Filogenia , Estrés Fisiológico , Temperatura , Factores de Transcripción/clasificación , Factores de Transcripción/genéticaRESUMEN
Entomopathogenic fungi (EPF) are important soil-dwelling entomopathogens, which can be used as biological control agents against pest insects. EPF are capable of causing lethal epizootics in pest insect populations in agroecosystems. During a survey of the orchard soil at an organic farm, different EPF species were collected and identified to species level, using both morphological and molecular techniques. The EPF were trapped from soil samples taken from an apricot orchard. The traps, which were baited in the laboratory, used susceptible host insects, including the last-instar larvae of Galleria mellonella (wax moth larvae) and Tenebrio molitor (mealworm larvae). The potential pathogenicity of the local Metarhizium majus isolate was tested and verified using susceptible laboratory-reared last-instar T. molitor larvae. The identification of the M. majus isolated from South African soil was verified using both morphological and molecular techniques. The occurrence of M. majus in the South African soil environment had not previously been reported.
Asunto(s)
Metarhizium/genética , Metarhizium/aislamiento & purificación , Metarhizium/metabolismo , Animales , Beauveria/aislamiento & purificación , Beauveria/patogenicidad , Agentes de Control Biológico/farmacología , Insectos/microbiología , Larva/microbiología , Control Biológico de Vectores/métodos , Suelo , Microbiología del Suelo , SudáfricaRESUMEN
Soil-borne pathogens and weeds could synergistically affect vegetable growth and result in serious losses. The investigation of antagonistic metabolites from a marine-derived entomopathogenic fungus, Beauveria felina, obtained polyhydroxy steroid (1), tricyclic diterpenoid (2), isaridin (3), and destruxin cyclodepsipeptides (4-6). The structures and absolute configurations of new 1-3 were elucidated by extensive spectroscopic and X-ray crystallographic analyses, as well as electronic circular dichroism (ECD) calculations. Compounds 1 and 2 showed antifungal activities against carbendazim-resistant strains of Botrytis cinerea, with the minimum inhibitory concentration (MIC) values ranging from 16 to 32 µg/mL, which were significantly better than those of carbendazim (MIC = 256 µg/mL). Compound 5 exhibited significant antagonistic activity against the radicle growth of Amaranthus retroflexus seedlings, which was almost identical to that of the positive control (2,4-dichlorophenoxyacetic acid). The structure-activity differences of 4-6 suggested that the Cl atom in HMPA1 and ß-Me in Pro2 should be the key factors to their herbicidal activities. Besides, compounds 3-6 showed moderate nematicidal activities against Meloidogyne incognita. These antagonistic effects of 1-6 were first reported and further revealed the synergistically antagonistic potential of B. felina to be developed into the biopesticide.
Asunto(s)
Antifúngicos/farmacología , Antinematodos/farmacología , Beauveria/química , Beauveria/metabolismo , Animales , Antifúngicos/química , Antifúngicos/metabolismo , Antinematodos/química , Antinematodos/metabolismo , Beauveria/aislamiento & purificación , Botrytis/efectos de los fármacos , Botrytis/crecimiento & desarrollo , Cristalografía por Rayos X , Depsipéptidos/química , Depsipéptidos/metabolismo , Depsipéptidos/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Agua de Mar/microbiología , Metabolismo Secundario , Tylenchoidea/efectos de los fármacos , Tylenchoidea/crecimiento & desarrolloRESUMEN
BACKGROUND: Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome. RESULTS: There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in. CONCLUSIONS: Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.
Asunto(s)
Beauveria/genética , Beauveria/patogenicidad , Variación Genética , Beauveria/aislamiento & purificación , Análisis por Conglomerados , Genómica , Mutación INDEL , Polimorfismo de Nucleótido Simple , Dominios Proteicos , Virulencia/genética , Secuenciación Completa del GenomaRESUMEN
Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A bar-specific short guide RNA was used to target and cause a double-strand break in bar, and a donor DNA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.
Asunto(s)
Beauveria/citología , Beauveria/enzimología , Pared Celular/enzimología , Proteínas Fúngicas/metabolismo , Sintasas Poliquetidas/metabolismo , Animales , Secuencia de Bases , Beauveria/aislamiento & purificación , Beauveria/patogenicidad , Sistemas CRISPR-Cas/genética , Pared Celular/ultraestructura , Reparación del ADN por Unión de Extremidades/genética , Fluorescencia , Edición Génica , Prueba de Complementación Genética , Sitios Genéticos , Insectos/microbiología , Viabilidad Microbiana , Mutagénesis/genética , Mutación/genética , Fagocitosis , Esporas Fúngicas/fisiología , Esporas Fúngicas/ultraestructuraRESUMEN
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 1000â¯m to 2200â¯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.
Asunto(s)
Beauveria , Animales , Beauveria/genética , Beauveria/aislamiento & purificación , Beauveria/metabolismo , Beauveria/patogenicidad , Bioensayo , Escarabajos/microbiología , Hemípteros/microbiología , Kirguistán , Control Biológico de Vectores , Filogenia , ARN Ribosómico 18S/genéticaRESUMEN
OBJECTIVE: Lobesia botrana, the European grapevine moth, affects Vitis vinifera L. and other species of economic importance in a number of countries through damage caused by its larvae in berries and associated secondary diseases such as Botrytis cinerea. Control of the moth in urban areas is difficult due to poor chemical management of infested plants in houses. Additionally, in winter, L. botrana is in its pupal stage covered with a cocoon that prevents the penetration of chemical pesticides. For this reason, the objective of this work was to control the pupal stage with a formulation based on the entomopathogenic fungus Beauveria pseudobassiana in urban areas. RESULTS: The strain RGM 1747 was identified as B. pseudobassiana by multilocus sequence analysis. The biocontrol activity of this formulated fungus against the infestation of vines with breeding pupae without cocoons showed 100% infection 21 days after inoculation under winter conditions. Finally, the biocontrol activity of the formulated fungus against natural infestations of L. botrana in winter in urban areas reached an efficacy of 51%. This result suggests that the B. pseudobassiana formulation is able to penetrate the cocoon and contributes to the integrated pest management of L. botrana.
Asunto(s)
Beauveria/fisiología , Mariposas Nocturnas/microbiología , Control Biológico de Vectores , Pupa/microbiología , Estaciones del Año , Vitis/parasitología , Animales , Beauveria/aislamiento & purificación , Chile , FilogeniaRESUMEN
Information on the biology and ecology of Beauveria bassiana in different habitats could provide essential knowledge in their development as biocontrol agents of insect pests. In this study, phylogenetic and genotypic information was used to evaluate the genetic diversity of B. bassiana within semi natural and agricultural habitats in Karnataka State of South India and assessed their extracellular chitinase activity and pathogenicity against cowpea aphid, Aphis craccivora. Multilocus phylogeny and microsatellite genotyping of B. bassiana conjointly resolved three phylogenetic species, Bb_1, Bb_2, and Bb_3, in semi natural and agricultural habitats. None of the three phylogenetic species of B. bassiana were associated with crop plants in agroecosystem or insect hosts in semi natural habitat. All the three phylogenetic species were detected with four genotypes each. All isolates of B. bassiana were pathogenic to A. craccivora in greenhouse bioassays. Isolate GKVK 01_13 caused a significantly high mortality of aphids and detected with an increased level of chitinase activity. The study results suggest that application of indigenous virulent strain of B. bassiana could provide effective control of native insect pest A. craccivora.
Asunto(s)
Áfidos/microbiología , Beauveria/genética , Beauveria/patogenicidad , Microbiología del Suelo , Animales , Áfidos/fisiología , Beauveria/clasificación , Beauveria/aislamiento & purificación , Ecosistema , Variación Genética , India , Control Biológico de Vectores , Filogenia , VirulenciaRESUMEN
Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.
Asunto(s)
Beauveria/aislamiento & purificación , Granjas , Metarhizium/aislamiento & purificación , Microbiología del Suelo , Animales , Australia , Beauveria/clasificación , Beauveria/genética , Biodiversidad , ADN Intergénico/genética , Genes Fúngicos , Hypocreales/clasificación , Hypocreales/aislamiento & purificación , Insectos/microbiología , Larva/microbiología , Metarhizium/clasificación , Metarhizium/genética , Factor 1 de Elongación Peptídica/genética , FilogeniaRESUMEN
The entomopathogenic fungus Beauveria bassiana is used widely as a biological control agent against a wide range of insect pests globally. In this study, 44 Beauveria isolates from the state of Colima, Mexico harbored in the "Colección de Hongos Entomopatógenos" of the "Centro Nacional de Referencia de Control Biológico" and from different substrates, insect-hosts, and localities were characterized with molecular markers. All isolates were identified using a Bayesian phylogenetic analysis of translation elongation factor 1-α (TEF) and nuclear intergenic Bloc region. Forty-three isolates were identified as B. bassiana and grouped into two sub-clades, i.e., AFNEO_1 (nâ¯=â¯22; previously defined as a clade with African and Neotropical origin) and Bb clade (nâ¯=â¯21; closely associated with ex-type strain ARSEF 1564), and one isolate was identified as B. pseudobassiana. The fixation index (FSTâ¯=â¯0.493) established the genetic differentiation between AFNEO_1 and Bb clades. High genotype richness and genetic diversity of AFNEO_1 and Bb clades were revealed in sequence analysis of Bloc region and SSR genotyping. Moreover, the AFNEO_1 and Bb clades were confirmed as two independent clonally structured assemblages. Finally, the AMOVA detected no significant association between any combination of substrate, insect-host or geographical origin. High genetic variation of B. bassiana in Colima, Mexico could suggest a functional diversity among isolates that may include those effective against a specific insect pest.
Asunto(s)
Beauveria , Variación Genética , Insectos/microbiología , Animales , Beauveria/clasificación , Beauveria/genética , Beauveria/aislamiento & purificación , ADN Intergénico/genética , Ambiente , Marcadores Genéticos , Genotipo , Geografía , Especificidad del Huésped , Proteínas de Insectos/genética , México , Factor 1 de Elongación Peptídica/genética , Control Biológico de Vectores , FilogeniaRESUMEN
Storage proteins in the 30â¯K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30â¯K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30â¯K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311â¯bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4â¯h and the time for death of all infected larvae was postponed by 10â¯h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.
Asunto(s)
Antifúngicos/farmacología , Beauveria/efectos de los fármacos , Bombyx/microbiología , Proteínas de Insectos/metabolismo , Proteínas Recombinantes/farmacología , Animales , Antifúngicos/metabolismo , Beauveria/crecimiento & desarrollo , Beauveria/aislamiento & purificación , Beauveria/metabolismo , Bombyx/inmunología , Clonación Molecular , Resistencia a la Enfermedad/genética , Escherichia coli , Genes de Insecto/inmunología , Genes de Insecto/fisiología , Hemolinfa/metabolismo , Proteínas de Insectos/genética , Larva/metabolismo , Larva/microbiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismoRESUMEN
The use of Beauveria bassiana in biological control of agricultural pests is mainly hampered by environmental factors, such as elevated temperatures and low humidity. These limitations, further amplified in a global warming scenario, could nullify biological control strategies based on this fungus. The identification of thermotolerant B. bassiana isolates represents a possible strategy to overcome this problem. In this study, in order to maximize the probability in the isolation of thermotolerant B. bassiana, soil samples and infected insects were collected in warm areas of Syria. The obtained fungal isolates were tested for different biological parameters (i.e., growth rate, sporulation and spore germination) at growing temperatures ranging from 20°C to 35°C. Among these isolates (eight from insects and 11 from soil samples), the five with the highest growth rate, spore production and germination at 30°C were tested for their entomopathogenicity through in vivo assays on Ephestia kuehniella larvae. Insect mortality induced by the five isolates ranged from 31% to 100%. Two isolates, one from Phyllognathus excavatus and one from soil, caused 50% of the larval mortality in less than four days, reaching values exceeding 92% in ten days. These two isolates were molecularly identified as B. bassiana sensu stricto by using three markers (i.e., ITS, Bloc and EF1-α). Considering these promising results, further studies are ongoing, testing their efficiency in field conditions as control agents for agricultural insect pests in Mediterranean and Subtropical regions.
Asunto(s)
Beauveria/fisiología , Insectos/microbiología , Control Biológico de Vectores/métodos , Animales , Beauveria/clasificación , Beauveria/genética , Beauveria/aislamiento & purificación , Proteínas Fúngicas/genética , Insectos/crecimiento & desarrollo , Larva/microbiología , Factor 1 de Elongación Peptídica/genética , Filogenia , Microbiología del Suelo , Siria , Termotolerancia , Clima TropicalRESUMEN
Biotyper analysis of Nannizziopsis guarroi, a fatal fungal pathogen in lizards, was described recently. Hypocrealean fungal infections in captive reptiles appear with an increasing frequency during the last decade. Therefore, the aim of this study was to proof Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) as diagnostic tool for the identification of reptile pathogenic hypocrealean fungi. Ten fungal isolates obtained from nine reptiles with fungal glossitis, disseminated visceral mycosis, pneumomycosis, and fungal keratitis were analyzed. Phylogeny consisted of fragments of the large subunit of nuclear encoded ribosomal DNA (D1/D2, LSU) and the internal transcribed spacer region 1 of nuclear encoded ribosomal DNA (ITS1) as well as the protein coding gene translation elongation factor 1 alpha (TEF). Results revealed unanimously two Metarhizium granulomatis genotypes in a total of three isolates, various M. viride genotypes (n = 3), two different Purpureocillium lilacinum isolates as well as one isolate of each P. lavendulum and Beauveria bassiana. Purpureocillium lilacinum and B. bassiana are likewise frequently employed as a mycoinsecticide and mycoacaricide in agriculture on a worldwide scale and have occasionally been reported in man, causing fungal keratitis, sclerokeratitis, nosocomial infections in immunosuppressed patients, as well as cavitary pulmonary disease and cutaneous hyalohyphomycosis in immunocompetent patients. According to the results establishment of Biotyper analysis for faster differentiation of reptile-associated fungal pathogens is entirely justified.