Beauveria bassiana for the control of Sunn Pest (Eurygaster integriceps) (Hemiptera: Scutelleridae) and aspects of the insect's daily activity relevant to a mycoinsecticide

A series of investigations was carried out at ICARDA during April-June 2004 and May-June 2005 to investigate the use of the entomopathogenic fungus Beauveria bassiana to control Eurygaster integriceps and to determine aspects of the insect's biology that could be relevant to control with the fungus. Application in 2004 of an oil-based formulation of B. bassiana showed distribution of the spray decreasing from the top of the wheat to the bottom, although the proportion of tillers with coverage at the base was still 80%. Seventy percent of Sunn Pest had received direct hits, with the highest amount on the dorsal surface, followed by the ventral surface, then the legs and antennae. Mortality of field-treated, then laboratory-maintained insects, ranged from 47.6% for insects exposed only to the B. bassiana spray, 74.2% for unsprayed insects exposed to treated wheat and 86.4% for insects receiving direct spray and being exposed to treated wheat. Some field results demonstrated mortality. Mortality assessments following the 2005 treatments were conducted solely in the field, there was no apparent effect upon Sunn Pest numbers. The majority of studies on Sunn Pest biology were carried out on untreated insects outside the trial plots. Different assessment methods resulted in varying estimates of adults and juveniles in the field. The numbers of Sunn Pest found and the proportion of these at the top of the wheat crop, declined after early morning and increased again in the early evening, suggesting sheltering deeper in the crop or in the soil during the middle part of the day. Mobility and location of marked insects was followed in micro-plots and by mark and recapture studies, demonstrating a range of activities, from movement around the crop for a number of metres, including contact with numerous tillers to almost total immobility; Sunn Pest generally avoided movement on the soil. The potential for Sunn Pest control with a mycoinsecticide and how Sunn Pest behaviour is relevant to a mycoinsecticide application are discussed.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Effect of the entomopathogenic fungus,Beauveria bassiana,and its secondary metabolite on detoxifying enzyme activities and acetylcholinesterase (AChE) of the Sunn pest,Eurygaster integriceps (Heteroptera: Scutellaridae)

The effects of Beauveria bassiana spores and its secondary metabolite on insect resistance to an organophosphorus insecticide, fenitrothione and secondary metabolite effects on acetylcholine esterase inhibition were investigated. Findings showed that fungal spores and its secondary metabolite increase total esterase and glutathione S-transferase activities in the hemolymph of infected and treated adults of Eurygaster integriceps. But the fungal secondary metabolite had an adverse effect on AChE activity of adults that decreased its activity level and isoforms of this enzyme in polyacrylamide gel electrophoresis. Fungal infection decreased the susceptibility of E. integriceps adults to fenitrothione, in comparison with uninfected individuals. Possible involvement of detoxifying enzymes in the development of insect resistance to fenitrothione should be considered in combined usage of chemicals and microbial agents for integrated pest management programs.     

Temperature-dependent germination,growth and co-infection of Beauveria spp. isolates from different climatic regions

Biological control agents based on entomopathogenic fungi traditionally contain a single strain that is efficient under certain biotic and abiotic conditions. Since particularly abiotic conditions vary, biological control efficiency may become more resilient at extreme temperatures if two or more fungal strains are combined based on their adaptations to their original environment. Here we evaluated the in vitro temperature-dependent germination and growth rate for six Beauveria spp. isolates originating from either arctic or tropical regions. Isolates of arctic origin showed higher germination and growth rate at 8°C and 12°C than isolates from the tropics while the latter group showed highest germination and in vitro growth at 32°C. Three of the isolates belonging to Beauveria bassiana were further tested in vivo for temperature-dependent infection in the mealworm beetle Tenebrio molitor both individually and combined. The same amounts of conidia were used in all bioassays. Virulence was isolate dependent at all temperatures with no additional effect at the low (12°C) and high (32°C) temperatures of combinations of arctic and tropical isolates. The results therefore indicate that adaptations to abiotic conditions in the natural environment do not directly reflect the effect of biotic environment (such as host infection) under similar conditions. Selection of isolates for biocontrol agents should not be based solely on in vitro experiments, while isolate selection based on virulence should also include considerations of the abiotic conditions the isolates are expected to function.     

Temperature and Ca2+ ion as modulators in cellular immunity of the Sunn pest Eurygaster integriceps Puton (Heteroptera: Scutelleridae)

Environmental conditions in addition to divalent cations may affect the interactions between pathogens and insects. Elucidation of factors which modulate insect immune responses could be a significant part of investigations in this area. In this study, adults of Eurygaster integriceps, as the destructive pest of wheat, were kept at different temperatures in addition to injection with different concentrations of Ca²⁺ to find the effect on cellular immune reactions against Beauveria bassiana. Results showed that total and differentiate hemocyte numbers, nodule formation and phenoloxidase activity increased with elevation of temperature so that the higher values were obtained at 30 and 40°C at various intervals. Higher concentrations of Ca²⁺ ion (5 mM) caused an increase in plasmatocyte length and width especially after 60 min. Similar results were observed for nodule formation and phenoloxidase activity of E. integriceps adults after injection by B. bassiana spores and phenoloxidase activity. It is clear from the current study that thermoregulation and Ca²⁺ ion can positively affect the hemocyte numbers especially plasmatocytes and granulocytes, nodule formation and phenoloxidase activity in E. integriceps. The understanding of modulators of the insect immune response may directly influence novel approaches to obtain safe and effective biological control agents.     

Isolation and characterization of microsatellite loci from the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales)

Beauveria bassiana, an entomogenous fungus used for the biological control of pest insects, comprises a globally‐distributed species complex of regionally endemic lineages. In order to study the population genetics of B. bassiana, detail species boundaries, conduct ecological studies of natural populations and track fates of experimentally‐released strains, sensitive genetic markers are required. We describe the isolation and characterization of eight microsatellite loci that amplify successfully from strains representative of the phylogenetic diversity in the B. bassiana complex.     

Susceptibility of apple clearwing moth larvae,Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC₅₀'s of 2.9×10⁵ and 3.4×10⁵ spores/mL, respectively. Larval mortalities caused by the two isolates at 1×10⁶ spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT₅₀'s of 5.5 and 5.1 days, respectively.     

Efficacy of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against Thaumetopoea pityocampa Shiff. (Lepidoptera: Thaumatopoeidae)

Abstract

In this study, Metarhizium anisopliae TR 106 and Beauveria bassiana TR 217 was tested against fourth instar larvae of Thaumetopoea pityocampa. The LT₅₀ and LT₉₀ of 1?×?10⁶ concentration of M. anisopliae against T. pityocampa were 3.60 and 4.11 for direct application, while these were 2.87 and 3.60?days, respectively in leaves application. The LT₅₀ and LT₉₀ of the 1?×?10⁸ concentration of the same isolate were 2.50 and 2.95?days for direct application, and 2.98 and 3.74?days for leaves application. The LT₅₀ of insect and leaves application for 1?×?10⁶ of B. bassiana were 3.75 and 3.49?days, respectively. The LT₉₀ of same concentration for insect application was 4.48?days, while LT₉₀ for leaves application was 4.63?days. Similarly, LT₅₀ of insect and leaves application for 1?×?10⁸ of B. bassiana were 3.03 and 3.31?days, while LT₉₀ were 3.68 and 4.29?days, respectively. Approximate 100% mycosis was observed in all treatments.     

Persistence of Beauveria bassiana sensu lato and Metarhizium anisopliae sensu lato in Vermont (USA) forest soil

Persistence of granular formulations of Beauveria bassiana sensu lato, strains GHA726 and ERL1170, and Metarhizium anisopliae sensu lato, strains ERL824 and ERL701, applied to forest soil was documented. One formulation was prepared by producing fungi on millet and at application adding cooked, dried millet to provide additional nutrients for fungal growth. The other was made by producing fungi on corn starch ‘packing peanuts’, a biodegradable packaging material, adding additional packing peanuts as a nutrient base. The trial was conducted in three sites in a forest predominating in sugar maple (Acer saccharum) in Underhill, VT, USA. In each site, 33 dominant sugar maples were randomly selected. A 1-m² plot located 1 m from the bole of the tree was established. Soil samples were collected before application, and 1 h, 2, 4, 8, 12 and 16 wk post-application. Fungal levels were estimated based on number of colony-forming units (CFUs). A significant increase in CFUs was observed 2 wk post-application in plots treated with isolates GHA726, ERL1170 and ERL824 as a result of fungal reproduction on nutrients in the formulations. Evidence of greater persistence for the B. bassiana than the M. anisopliae isolates was observed, based on CFU levels over time. Isolates formulated with millet proliferated more than those on packing peanuts. Use of entomopathogenic fungi in forest soil has potential for managing soil-dwelling insect pests such as pear thrips, Taeniothrips inconsequens, and the black vine weevil, Otiorhynchus sulcatus, though the type of formulation and the specific isolate used must be selected carefully.     

First record of Beauveria bassiana (Ascomycota: Clavicipitaceae) infecting Cerambyx welensii (Coleoptera: Cerambycidae) and pathogenicity tests using a new bioassay method

The pathogenicity of Beauveria bassiana on Cerambyx welensii is first reported. Three native fungal isolates from Badajoz (Spain) were bioassayed on neonates using cork oak disks and LC₅₀s were 1.16–1.76 × 10⁵ and 0.30–0.61 × 10⁵ spores/ml on the 7th and 14th day post-inoculation. The fungus is potentially a biocontrol agent of C. welensii.     

Survival and fecundity of Dichroplus maculipennis and Ronderosia bergi (Orthoptera: Acrididae: Melanoplinae) following infection by Beauveria bassiana (Ascomycota: Hypocreales) under laboratory conditions

This study examined the effects of strain Beauveria bassiana (LPSC 1067) on nymphal development time, fecundity and adult survival in Dichroplus maculipennis and Ronderosia bergi under laboratory conditions. It was observed that infection with 1×10³ conidia/ml altered nymphal development time, fecundity and adult survival in both species. Mortality of D. maculipennis during third through the last instar (sixth) was significantly higher among treated nymphs (66±3.8%) than in controls (15±1.7%). Similarly, mortality in R. bergi during third through the last instar (fifth) was higher in treated nymphs (71±2.8%) than in controls (19±1.5%). Nymphal development times of both infected D. maculipennis and R. bergi were longer than controls. On the other hand, among survivors of both the species, control adults lived longer than infected adults. Finally, control grasshoppers of both species were much more successful reproductively than infected grasshoppers.     

Effect of combination treatment with entomopathogenic fungi Beauveria bassiana and Nomuraea rileyi (Hypocreales) on Spodoptera litura (Lepidoptera: Noctuidaeae)

In biocontrol of insect pests, efficacy of treatment with multiple pathogens has not been frequently investigated but may have potential for effective management. The possible advantage of a combination treatment with two entomopathogenic fungi - Beauveria bassiana and Nomuraea rileyi - was assessed in laboratory bioassays on second instar Spodoptera litura. From among the fungal isolates of an epizootic population, two isolates of each fungus differing in virulence to S. litura were chosen, one highly virulent and the other with low virulence. The bioassays were carried out at either a continuous temperature of 25±1°C or at a temperature cycle of 32±2°C 8 h/21±2°C 16 h to mimic the field temperatures during the epizootic. Treatments with the two fungi were done both simultaneously and sequentially. In combination treatments at 25±1°C, in all isolate combinations, a majority of the larvae showed N. rileyi induced mycosis; the percentage mortality and speed of kill of insects in these treatments was similar to the N. rileyi isolate used in the combination treatments. At the temperature cycle of 32±2°C 8 h/21±2°C 16 h, in all combination treatments, all the dead insects exhibited B. bassiana mycosis; the mortality pattern was similar to the B. bassiana isolate used in the combination treatments. The adverse effect of high temperature on virulence of N. rileyi was however, not evident in in vitro growth assays. Combination treatment with both fungi did not have a synergistic effect on insect mortality.     

Trials of Beauveria bassiana, Paecilomyces fumosoroseus and imidacloprid for management of Trialeurodes vaporariorum (Homoptera: Aleyrodidae) on greenhouse grown lettuce

Mineral oil-based emulsifiable preparations of Beauveria bassiana (Bb) and Paecilomyces fumosoroseus (Pfr) conidia were separately applied alone or together with low rates of imidacloprid 10% WP at 4.7% (Im 1), 14.0% (Im 2), and 23.3% (Im 3) of its recommended application rate, respectively, against the greenhouse whitefly, Trialeurodes vaporariorum, on lettuce grown in the greenhouse. Besides eight fungal treatments, the three low application rates of imidacloprid in the oil-based carrier and a blank control (CK) were also included as treatments of the trials conducted in 2002 and 2003. For the 12 treatments of each trial with three replicates, 1,000-fold aqueous dilutions were sprayed twice on transplanted lettuce at a 15-day interval at a rate of ∼1.43×10¹³ conidia ha^-1 for each fungal treatment or at one of the low rates of imidacloprid using a backpack hand-operated hydraulic sprayer. Based on whitefly densities, mortalities, relative efficacies and percent density declines estimated from whitefly counts made at 5-day intervals, all B. bassiana and P. fumosoroseus sprays were highly effective against T. vaporariorum compared to CK and Im 1-3 in both trials. In trial 1, the estimates of whitefly density decline and relative efficacy ranged from 44 and 72% (Bb) to 79 and 90% (Pfr+Im 2-3) on day 10 after the first spray and exceeded 94% for all fungal treatments 10 days after the second spray. Similar trends in whitefly control were also achieved in the corresponding treatments of trial 2. A more desirable and faster control resulted from fungal sprays containing more imidacloprid, but none of the three low imidacoprid rates alone suppressed the whitefly population more effectively than any fungal treatment despite a varying degree of efficacy. Moreover, P. fumosoroseus tended to be more effective against T. vaporariorum than B. bassiana when applied alone or together with the same low rate of imidacloprid but the difference in whitefly control eventually achieved was not significant between the two fungal agents. Thus, the emulsifiable preparations of both fungal agents can be considered as alternatives for whitefly management and for a slightly faster control can be applied together with imidacloprid at around 15% of its common application rate. An economic balance between the efficacy of whitefly control and the ease and cost of conidial production is needed when both fungi are incorporated into whitefly management.     

Molecular identification and characterization of wine yeasts isolated from Tenerife (Canary Island, Spain)

AIMS: The present study was aimed at the identification, differentiation and characterization of indigenous yeasts isolated from Tenerife vineyards (viticulture region that has never been characterized before). Microbiota were studied from 14 samples taken during fermentations carried out in the 2002 vintage, from 11 wineries belonging to five wine regions on Tenerife Island. METHODS AND RESULTS: Yeasts' strains were identified and characterized through restriction analysis of the 5.8S-internal transcribed spacer region and the mitochondrial DNA. At the beginning of alcoholic fermentation, 26 yeast species were found, where 14 species were present in significant frequencies in only one sample. Likewise, the Saccharomyces cerevisiae strains isolated are very specific, as they were only present in one wine region. CONCLUSIONS: There were isolated specific yeasts from each region on Tenerife Island. The founded yeasts may be responsible for distinctive and interesting properties of the studied wines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms part of an extensive taxonomic survey within the ecological framework of vineyards in Tenerife. This investigation is an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the grape growing regions of this island. The results obtained demonstrate the value of using molecular genetic methods in taxonomic and ecological surveys. The results also shed some light on the ecology and oenological potential of S. cerevisiae strains isolated from this unique environment.     

Laboratory evaluation of the virulence of Beauveria bassiana isolates to the sorghum shoot borer Chilo partellus Swinhoe (Lepidoptera: Pyralidae) and their characterization by RAPD-PCR

Beauveria bassiana has long been used as a mycopesticide. It has a wide host range; isolates have been reported to differ in host range and virulence to a given insect species. Identification of a molecular marker linked to a virulent phenotype to a target pest would be useful in screening for isolates effective against it. Twenty B. bassiana isolates were tested for their virulence to the second instar larvae of Chilo partellus Swinhoe in laboratory bioassays and their DNA fingerprints were generated by RAPD-PCR. Three arbitrary categories of aggressiveness were chosen; isolates that caused >70%, between 70 and 40% and <40% larval mortality were grouped as highly, medium and less aggressive types, respectively. In the random amplified polymorphic DNA (RAPD) analysis a 30% variability was observed among the isolates; which clustered into three major groups. The groups based on virulence rating did not match with the RAPD clusters. One of the highly aggressive isolates clustered with less aggressive isolates in one cluster and the other grouped along with the medium aggressive isolates in a different cluster. The B. bassiana isolates were classified phenotypically based on the taxonomic order of the original insect host and the climatic zone (tropical/temperate) from which they were isolated. No correlation between the aggressiveness of the isolate and the relatedness of the original insect host to the tested insect was observed; both the highly aggressive isolates were from coleopteran insects. A correlation was found between the RAPD grouping and the phenotypic classification of the isolates. All the lepidopteran isolates grouped into one major cluster, most sub clusters were constituted by isolates from the same climatic zone.     

Molecular phylogeny of Pseudokeronopsis (Protozoa, Ciliophora, Urostylida), with reconsideration of three closely related species at inter- and intra-specific levels inferred from the small subunit ribosomal RNA gene and the ITS1-5.8S-ITS2 region sequences

In order to address the confused taxonomy of morphotypes within the genus Pseudokeronopsis , the small subunit ribosomal RNA (SS rRNA) gene and the internal transcribed spacer (ITS1)-5.8S-ITS2 region for 13 populations/clones of morphologically similar Pseudokeronopsis species, Pseudokeronopsis flava , Pseudokeronopsis rubra and Pseudokeronopsis carnea , were sequenced and compared at inter- and intra-specific levels. Different geographical/time interval isolates belonging to the same species were slightly divergent, while clones from the same strain had identical SS rRNA gene sequences. Compared with the SS rRNA gene sequence, the sequence of the ITS1-5.8S-ITS2 region of Pseudokeronopsis revealed higher diversity, with nucleotide dissimilarities of 3.9–4.9% at the intra-specific level and 8.0–10.5% at the inter-specific level. There were several minor differences among ITS2 secondary structures of three Pseudokeronopsis species, while three Pseudok. carnea populations were identical. Phylogenetic analyses using multiple algorithms and different species selections confirm that Pseudok. flava , Pseudok. rubra and Pseudok. carnea form a monophyletic group within the urostylids. The result also supports the closest relationship of Pseudokeronopsis and Pseudourostyla that forms the so-called Acaudalia Berger (2006) .     

Virulence,proteolytic and lipolytic activities of Brazilian Beauveria bassiana s.l. isolates (Hypocreales: Clavicipitaceae) to Rhipicephalus microplus ticks (Acari: Ixodidae)

Beauveria bassiana s.l. is a cosmopolitan fungus used in the control of different species of arthropods. The current study explored the virulence for ticks, proteolytic and lipolytic activities of 10 Brazilian B. bassiana s.l. isolates. For this purpose, Rhipicephalus microplus biological parameters was evaluated after immersion of the engorged females in fungal suspension (10⁸ conidia mL^?1) and the enzymatic activities were performed posteriorly the fungal growth in minimal medium. After the biological assays, five isolates changed all parameters analysed with highest efficacy of approximately 61% (CG 206) and 66% (CG 481). However, we observed that the most virulent isolates did not show the highest enzymatic activities. Interestingly, CG 500, considered an isolate of intermediate efficacy, demonstrated higher enzymatic activities than the other isolates in four of five analyses (total protease, Pr1, Pr2 and lipase; p?相似文献   

Coincident isolation of a novel homoisoflavonoid from Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae)

We report on the first phytochemical investigation of a member of the African genus Resnova (Hyacinthoideae: Hyacinthaceae). From the dichloromethane extract of the bulbs of both Resnova humifusa and Eucomis montana (Hyacinthoideae: Hyacinthaceae) a novel 3-benzyl-4-chromanone homoisoflavonoid, 5,6-dimethoxy-7-hydroxy-3-(4′-hydroxybenzyl)-4-chromanone, was isolated. A further 11 known homoisoflavonoids were also identified, the 12 in total presenting a clear biosynthetic sequence. Eight of the 12 compounds found were common to both species.     

Complete mitochondrial genomes from four subspecies of common chaffinch (Fringilla coelebs): New inferences about mitochondrial rate heterogeneity,neutral theory,and phylogenetic relationships within the order Passeriformes

We describe whole mitochondrial genome sequences from four subspecies of the common chaffinch (Fringilla coelebs), and compare them to 31 publicly available mitochondrial genome sequences from other Passeriformes. Rates and patterns of mitochondrial gene evolution are analyzed at different taxonomic levels within this avian order, and evidence is adduced for and against the nearly neutral theory of molecular evolution and the role of positive selection in shaping genetic variation of this small but critical genome. We find evidence of mitochondrial rate heterogeneity in birds as in other vertebrates, likely due to differences in mutational pressure across the genome. Unlike in gadine fish and some of the human mitochondrial work we do not observe strong support for the nearly neutral theory of molecular evolution; instead evidence from molecular clocks, distribution of dN/dS ratios at different levels of the taxonomic hierarchy and in different lineages, McDonald–Kreitman tests within Fringillidae, and site-specific tests of selection within Passeriformes, all point to a role for positive selection, especially for the complex I NADH dehydrogenase genes. The protein-coding mitogenome phylogeny of the order Passeriformes is broadly consistent with previously-reported molecular findings, but provides support for a sister relationship between the superfamilies Muscicapoidea and Passeroidea on a short basal internode of the Passerida where relationships have been difficult to resolve. An unexpected placement of the Paridae (represented by Hume's groundpecker) within the Muscicapoidea was observed. Consistent with other molecular studies the mtDNA phylogeny reveals paraphyly within the Muscicapoidea and a sister relationship of Fringilla with Carduelis rather than Emberiza.