Chemotaxonomic discrimination among the fungal genera Tolypocladium, beauveria and Paecilomyces

Fungi of the genera Beauveria and Paecilomyces were found to produce cyclotetradepsipeptides, beauverolides. Production of beauverolides was not detected at the genus Tolypocladium. Analysis of beauverolides therefore provides a very simple chemotaxonomic test which seems to be suitable for fast discrimination between the genera Beauveria vs Tolypocladium and complementing morphological examination. A GC-MS study of β-hydroxy acid distribution in the beauverolide hydrolyzates revealed that all strains prdouce γ-methyl-β-hydroxy acids only. Their occurrence thus cannot be used as a taxonomic marker of different species within the genera Beauveria and Paecilomyces.     

Mitochondrial evolution in the entomopathogenic fungal genus Beauveria

Species in the fungal genus Beauveria are pathogens of invertebrates and have been commonly used as the active agent in biopesticides. After many decades with few species described, recent molecular approaches to classification have led to over 25 species now delimited. Little attention has been given to the mitochondrial genomes of Beauveria but better understanding may led to insights into the nature of species and evolution in this important genus. In this study, we sequenced the mitochondrial genomes of four new strains belonging to Beauveria bassiana, Beauveria caledonica and Beauveria malawiensis, and compared them to existing mitochondrial sequences of related fungi. The mitochondrial genomes of Beauveria ranged widely from 28,806 to 44,135 base pairs, with intron insertions accounting for most size variation and up to 39% (B. malawiensis) of the mitochondrial length due to introns in genes. Gene order of the common mitochondrial genes did not vary among the Beauveria sequences, but variation was observed in the number of transfer ribonucleic acid genes. Although phylogenetic analysis using whole mitochondrial genomes showed, unsurprisingly, that B. bassiana isolates were the most closely related to each other, mitochondrial codon usage suggested that some B. bassiana isolates were more similar to B. malawiensis and B. caledonica than the other B. bassiana isolates analyzed.     

Correlation of fruit tree rhizosphere soils with entomopathogenic fungi

Entomopathogenic fungi are microorganisms that control the density of host insects in nature; they are being studied as environmentally friendly alternatives to chemical insecticides for controlling insect pests. The main habitat of entomopathogenic fungi is soil, and the correlation between the distribution of entomopathogenic fungi and the physicochemical characteristics of soils planted with different trees, including vine (outdoor, greenhouse, and greenhouse shine musket), apple, peach, and pear, were analyzed. The entomopathogenic fungi of the genera Beauveria, Metarhizium, and Purpureocillium investigated in this study were all found in soil samples from vine-greenhouse, apple, and peach trees. Purpureocillium and Beauveria abundances were positively correlated with soil properties; however, Metarhizium abundances were not correlated with soil properties. The Metarhizium isolates discovered in this study showed pathogenicity to cotton aphids (an agricultural pest) and can be employed as sources for biological studies in the future. This study provides data on the diversity and abundance of entomopathogenic fungi related with soil properties, as well as the molecular, biological, and insecticidal characteristics of Metarhizium isolates.     

Effect of talc-formulated entomopathogenic fungus Beauveria against leaffolder (Cnaphalocrosis medinalis) in rice

Thirteen Beauveria strains were isolated from the soil and infected insects. Among the various isolates, B₂ isolate (Arachalore) showed a higher percentage of mortality against C. medinalis (73.3%) under in vitro conditions. Conidial concentration of 1 × 10⁸ of the B2 strain registered maximum mortality of 76.7%. The least LT₅₀ value of 4.4 days was registered in B2 isolate with the spore concentration of 1 × 10⁸ and the LC₅₀ value was 3.4 × 10⁴. Beauveria strains altered the feeding behavior of C. medinalis, reduced the pupal weight, prolonged the pupation period, malformed the pupa and adult under in vitro. The efficacy of the talc-based bioformulation of Beauveria (B2) strain was tested as seed treatment + seedling dip + soil application + foliar spray against rice leaffolder under in vitro and greenhouse conditions. The percentage damage was significantly less (5.5) in B2 as compared to untreated healthy control (25.8). In addition, the same treatment increased the activities of defense-related enzymes, namely peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, chitinase, and phenolics in rice.     

Base composition of DNA from symbiotic dinoflagellates: a tool for phylogenetic classification

DNA of eight endosymbiotic dinoflagellates (zooxanthellae) from seven different host species has been analyzed as to its thermal characteristics and base composition by means of spectrophotometry and high performance liquid chromatography. All algae under investigation contain both methylcytosine and hydroxymethyluracil in addition to the bases typical of nuclear DNA. As a result, melting temperatures are decreased, suggesting lower contents of guanine plus cytosine than actually present. True percentages of guanine plus cytosine plus methylcytosine range from about 43 to 54 mol%. They are unique for the symbionts from different hosts, indicating phylogenetic separation of the taxa comparised within the genus Symbiodinium.Abbreviations dA deoxyadenosine - dC deoxycytidine - dG deoxyguanosine - dT deoxythymidine - m5dC 5-methyldeoxycytidine - hmdU 5-hydroxymethyldeoxyuridine - rC ribocytidine - Br8G bromine-80guanosine - A adenine - C cytosine - G guanine - T thymine - m5C 5-methylcytosine - hmU 5-hydroxymethyluracil - G+C guanine plus cytosine plus 5-methylcytosine - HPLC high performance liquid chromatography - T _m temperature at the midpoint of hyperchromic shift - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - EDTA ethylenediamine-tetraacetic acid, disodium salt - TRIS tris-(hydroxymethyl)-aminomethane - 1×SSC standard saline citrate (0.15 M NaCl+0.015 M trisodium citrate, pH 7.0)     

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle,Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae)

Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×10⁶ conidia mL^–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.     

CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi,especially Metarhizium acridum,from soil

The selective media most commonly used for isolating hyphomycetous species of entomopathogenic fungi from non-sterile substrates rely on N-dodecylguanidine monoacetate (dodine) as the selective fungicide. Although these media are effective for isolating many species of Metarhizium and Beauveria from soil, they are inefficient media for isolation of an important Metarhizium species, Metarhizium acridum, from non-sterile soil. Our current study was directed to formulating a dodine-free selective medium that is efficient for isolating naturally occurring Beauveria spp. and Metarhizium spp., especially M. acridum, from soil. The selective medium (designated CTC medium) consists of potato dextrose agar plus yeast extract (PDAY) supplemented with chloramphenicol, thiabendazole and cycloheximide. In comparisons with selective media previously reported in the literature, the CTC medium afforded colonies that were larger and had both earlier and more abundant conidiation of entomopathogenic fungi, features which greatly facilitated identification of the emerging entomopathogenic fungi. In addition to efficient re-isolation of M. acridum, this medium also is an effective tool for selective isolation of Metarhizium brunneum, Metarhizium robertsii, Beauveria bassiana and Beauveria brongniartii from non-sterile field-collected soil samples inoculated (spiked) with fresh conidia in the laboratory.     

Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution

Dual biological control, of both insect pests and plant pathogens, has been reported for the fungal entomopathogens, Beauveria bassiana (Bals.-Criv.) Vuill. (Ascomycota: Hypocreales) and Lecanicillium spp. (Ascomycota: Hypocreales). However, the primary mechanisms of plant disease suppression are different for these fungi. Beauveria spp. produce an array of bioactive metabolites, and have been reported to limit growth of fungal plant pathogens in vitro. In plant assays, B. bassiana has been reported to reduce diseases caused by soilborne plant pathogens, such as Pythium, Rhizoctonia, and Fusarium. Evidence has accumulated that B. bassiana can endophytically colonize a wide array of plant species, both monocots and dicots. B. bassiana also induced systemic resistance when endophytically colonized cotton seedlings were challenged with a bacterial plant pathogen on foliage. Species of Lecanicillium are known to reduce disease caused by powdery mildew as well as various rust fungi. Endophytic colonization has been reported for Lecanicillium spp., and it has been suggested that induced systemic resistance may be active against powdery mildew. However, mycoparasitism is the primary mechanism employed by Lecanicillium spp. against plant pathogens. Comparisons of Beauveria and Lecanicillium are made with Trichoderma, a fungus used for biological control of plant pathogens and insects. For T. harzianum Rifai (Ascomycota: Hypocreales), it has been shown that some fungal traits that are important for insect pathogenicity are also involved in biocontrol of phytopathogens.     

Efficacy and molecular studies of a Lebanese isolate of Beauveria for control of Thaumetopoea wilkinsoni (Lepidoptera: Thaumetopoeidae)

A Lebanese isolate of a Beauveria species originally isolated from a hymenopterous insect pest was found to be very effective against the pine processionary moth Thaumetopoea wilkinsoni. In laboratory bioassays, high mortality rates of the five larval stages were attained with spore concentrations of about 5000–50,000 spores/larva. The efficacy of kill on the first three larval stages was similar to the growth regulator diflubenzuron, but it was significantly higher on the fourth and fifth larval stages. Sequences of the DNA lyase gene and the EF-1α gene were used for molecular characterisation of this Beauveria isolate. The DNA lyase gene showed more polymorphism than the previously reported ITS region and EF-1α gene. This constitutes the first report on the possibility of using the DNA lyase gene as a molecular tool in fungal taxonomy.     

Discrimination of Chinese Beauveria strains by DGGE genotyping and taxonomic identification by sequence analysis of the Bloc nuclear intergenic region

Anamorphic Beauveria are cosmopolitan entomopathogenic fungi that parasitize a broad range of insect species in virtually all terrestrial habitats. A diversity survey of 189 exemplar strains of Beauveria from the RCEF culture collection, representative of its taxonomic diversity, geographic distribution and insect host range in China, was conducted based on a combination of DGGE genotyping and nucleotide sequence analysis of the Bloc nuclear intergenic region. The DGGE assays detected 42 electrophoretically distinct haplotypes, with each haplogroup including 1–13 individuals. Nucleotide sequence analysis established that all haplogroups were uniquely distinguished by one or more nucleotide differences and that isolates from the same DGGE haplogroup share sequence identity. A phylogenetic analysis inclusive of this Bloc haplotype diversity assigned the Chinese Beauveria strains to six species lineages corresponding to B. bassiana sensu lato. (Bals.) Vuill, B. brongniartii (Sacc.) Petch, B. australis S.A. Rehner & Humber, B. asiatica S.A. Rehner & Humber, B. pseudobassiana S.A. Rehner & Humber and B. caledonica Bissett & Widden. B. australis is reported for the first time in China. This study represents the first phylogenetic survey of Beauveria species diversity in China, and demonstrates a simple and effective screening strategy to facilitate the identification of Beauveria genotypes.     

Influence of sodium chloride on inhibition of Desulfovibrio by a surfactant

Summary The abnormally high resistance of certain marine strains of the sulphate-reducing bacteria to the bacteriostatic effects of cetyltrimethylammonium bromide (CTAB) has been studied. A strain of the NaCl-requiring species Desulfovibrio salexigens and a halotolerant strain of D. desulfuricans both showed a marked increase in resistance to CTAB (about one-hundredfold for the former organism) with increasing salinity of the growth medium: this was attributed to enhanced micelle formation by CTAB in presence of NaCl.     

Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">B. brongniartii</Emphasis>

Background  

The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus.     

Comparative Analysis of the Biological Activity and Chromatographic Profiles of the Extracts of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">B. pseudobassiana</Emphasis> Cultures Grown on Different Nutrient Substrates

Soil, enodphytic, and insect-pathogenic micromycetes of the genus Beauveria are widespread in nature and are important producers on mycoinsecticides, enzymes, and pharmacologically usable and toxic compounds. The goal of the work was to determine chemodiagnostic approaches to differentiation of Beauveria cryptic species using the strains B. bassiana BBL and B. pseudobassiana BCu22 by comparing their toxicological properties (insecticidal, antimicrobial, phytotoxic, cytotoxic, and esterase-inhibition activity) and metabolite profiles (TLC and HPLC/DAD patterns) of the extracts from the cultures of these fungi growing on different loose substrates, on solid and liquid media. It was shown that when the strains were cultured in liquid media (SDAY and Adámek medium) and on solid substrates (millet and Czapek agar medium), they could be differentiated by the extract yield and chromatographic profiles, as well as by their insecticidal, antifungal, and cytotoxic activity. Thus, antifungal properties were more pronounced in B. pseudobassiana BCu22 grown in liquid Adámek and SDAY media, while cytotoxic properties were more notable in B. bassiana BBL grown in Adámek medium and on millet. Insecticidal properties of the extracts from these cultures varied depending on the substrate composition. Since the extracts of the studied fungi exhibited a broad spectrum of biological activity, the toxic properties of Beauveria spp. should be considered in the course of assessment of safety of these fungi as bioinsecticides.     

Diversity and pharmaceutical screening of fungi from benthic mats of Antarctic lakes

During the MICROMAT project, the fungal diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics and anticancers. In all, 160 filamentous fungi belonging to fifteen different genera and 171 yeasts were isolated from 11 lakes, classified and cultivated in different media and at different temperatures. Filamentous fungi were then screened to discover novel antimicrobial and cytotoxic compounds. A total of 1422 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 47 (29%) filamentous fungi showed antimicrobial activity; most of them inhibited the growth of gram-positive Staphyloccus aureus (14%), gram-negative E. coli (10%), and of yeasts Candida albicans (11%) and Cryptococcus neoformans (8%). Less activity was detected against representatives of enterobacteria and filamentous fungi. The most productive in terms of bioactivities were cold-tolerant cosmopolitan hyphomycetes such as Penicillium, Aspergillus, Beauveria and Cladosporium. Two bioactive bis-anthraquinones (rugulosin and skyrin) were identified by LC–MS as the main products in a strain of Penicillium chrysogenum isolated from a saline lake in the Vestfold Hills. LC–MS fractionation of extracts from two diverse species of Aspergillus, that exhibited relatively potent antimicrobial activities, evidenced a chemical novelty that was further investigated. To our knowledge, this is the first report of new antibiotics produced by fungi from benthic microbial mats from Antarctic lakes. It can be concluded that these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as drugs.     

Immunological characterization of the entomopathogenic hyphomycetes Beauveria and Metarhizium

Immunological comparison among Beauveria species and Metarhizium strains isolated from insect larvae were made using immunoelectrophoresis. Antigens consisted of somatic or metabolic buffer-soluble proteins. Antisera were produced in rabbits against each of the 6 fungal strains. Each antigen was tested against the homologous antiserum non-adsorbed or cross-adsorbed with one other antigen and against the heterologous antisera. The immunoelectrophoresis slides were analyzed in terms of the number of precipitin arcs obtained with cross-reacting and reference antigens, with any one antiserum. Immunoelectrophoregrams were also considered.These tests revealed numerous antigenic similarities between different isolates. Moreover, results indicate that distinct and reproducible differences exist, both between two species of Beauveria and between four strains of M. anisopliae. The groupings of strains obtained from immunoelectrophoretic data correspond well with those based on spore morphology. Some methodological aspects are discussed. The present results are in agreement with our preliminary studies. They confirm that immunoelectrophoresis proves to be a very useful aid for the characterization of the Hyphomycetes candidates for biological control of insect pests.

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Résumé Beauveria bassiana et B. brongnianii d'une part et 4 souches de Metarhizium anisopliae d'autre part, ont été analysés par immunoélectrophorèse de leurs protéines cellulaires ou métaboliques. Chaque antigène a été éprouvé contre le sérum homologue non adsorbé ou adsorbé avec un antigène hétérologue et contre les sérums hétérologues. Les parentés et les différences entre les 2 Beauveria et entre les 4 Metarhizium ont été définies quantitativement par le nombre d'arcs de précipitation communs ou specifiques. Nous avons aussi considéré les immunoelectrophorégrammes des divers isolats.Les résultats des réactions croisées montrent d'importantes communautés antigéniques entre les pathotypes étudiés, toutefois l'utilisation de l'immunoadsorption a permis de mettre en évidence les fractions spécifiques aussi bien entre les 2 Beauveria qu'entre les 4 souches de l'espèce M. anisopliae. Les distinctions établies sur les données sérologiques correspondent aux différences morphologiques sporales entre isolats. Ces résultats précisent et confirment nos études préliminaires en démontrant l'intérêt de la caractérisation immunoélectrophorétique des Hyphomycètes candidats à la lutte biologique contre les insectes nuisibles.

     

Efficacy of Beauveria bassiana applications on coffee berry borer across an elevation gradient in Hawaii

ABSTRACT

The effect of three rates of a commercial formulation of Beauveria bassiana Strain GHA was evaluated against the coffee berry borer (CBB) Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), at three commercial coffee farms located at different altitudes on the island of Hawaii. H. hampei infestation and natural prevalence of B. bassiana increased with the elevation. At 145 metres above sea level (Farm 1), beetle infestation was 3.9%; at 538?m (Farm 2), beetle infestation was 12.2%; and at 768?m (Farm 3) infestation was 22.3%. The prevalence of natural B. bassiana killing CBB was 5.5% on Farm 1, 3.3% on Farm 2 and 23.1% on Farm 3. Monthly applications of B. bassiana resulted in no significant differences in levels of CBB infestation among treatments. Similarly, rates of infested berries with visually detectable signs of B. bassiana were similar among the B. bassiana treatments, ranging from 0.44% to 4.24%, and those percentages were larger than the treatments without B. bassiana. The percentage of females killed by Beauveria ranged from 69% to 95%. Effect of dose of BotaniGard^® ES was reduced when beetles were in C position compared to A and B positions. B. bassiana can be an important component of an integrated pest management program for CBB.     

A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae)

Introduction – It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. Objective – To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). Methodology – Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. Results – The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT‐PCR analysis. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from various recalcitrant Euphorbiaceae members. Copyright © 2010 John Wiley & Sons, Ltd.     

添加CTAB促进吸水链霉菌产谷氨酰胺转胺酶

研究了添加十六烷基三甲基溴化铵（CTAB）对吸水链霉菌（Streptomyces hygroscopicus）合成谷氨酰胺转胺酶的影响。结果表明,添加CTAB可以提高发酵过程中谷氨酰胺转胺酶的酶活,摇瓶培养中,CTAB的最佳添加时间和添加量分别为32h和1%,发酵终了时,谷氨酰胺转胺酶酶活最高达5.04u/mL,比对照提高了21.8%。初步研究表明,CTAB的主要作用是促使谷氨酰胺转胺酶的酶原转化为成熟酶,因此,在发酵过程中添加适当浓度的CTAB,可使酶原快速、完全地转化为成熟的MTG,解除酶原的产物抑制作用,促进了细胞产酶。