Effects of combining <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Nosema pyrausta</Emphasis> on the mortality of <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>

We tested the combined effect of the fungus Beauveria bassiana and the microsporidium Nosema pyrausta on the European corn borer larvae, Ostrinia nubilalis, in the laboratory. The first instar of O. nubilalis larvae was the most sensitive to the B. bassiana infection followed by the fifth, second, third, and fourth instar (LC₅₀s were 4.91, 6.67, 7.13, 9.15, and 6.51 × 10⁵ conidia/ml for the first to fifth instars, respectively). Mortality of each instar increases positively with concentration of conidia. When B. bassiana and N. pyrausta were used in combination, mortality increased significantly in all instars. Relative to the B. bassiana treatment alone, the B. bassiana + N. pyrausta treatment decreased the LC₅₀s by 42.16%, 37.63%, 21.60%, 27.11%, and 33.95% for the first to fifth instars, respectively. The combined effects of the two pathogens were mostly additive. However, at the two highest concentrations the pathogens interacted synergistically in the first and second instar. Individuals that survived the B. bassiana and B. bassiana + N. pyrausta treatments and developed into adults had significantly shorter lifespans and females oviposited fewer eggs than non-exposed insects. The effects on the longevity and the egg production were most pronounced at high concentration of B. bassiana conidia.     

Physiological effects upon <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae) infected with <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Ascomycota: Hypocreales)

Unfed adult Amblyomma americanum were exposed to the entomopathogenic fungus Beauveria bassiana. Ticks exposed to the fungus exhibited reduced survival and increased water loss as indicated by change in weight. Treated ticks survived 7.2 ± 0.22 days (mean ± SE) and controls survived 17.9 ± 0.73 days (P = 0.01; df = 57). At death, ticks exposed to the fungus had lost 25.2 ± 0.84% of their starting weight; control ticks had lost 14.1 ± 0.85% of their starting weight (P = 0.01; df = 96). Water loss was highest immediately following inoculation, although losses continued to be higher than in uninoculated ticks. This suggests that fungal penetration causes sufficient cuticle damage to cause desiccation, although other water-loss avenues exist, including increased time of spiracular opening. Additionally this study did not eliminate the possibility of a negative impact on water vapor uptake. This is the first study to investigate the effect of an entomopathogenic fungus on the water balance of a tick.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Susceptibility of <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> ticks to acaricides in Ghana

The susceptibility of unfed and fed stages of larvae, nymphs and adult females of Amblyomma variegatum ticks were tested using Shaws filter paper dip method against four acaricides; chlorfenvinphos and dioxathion, chlorfenvinphos, gamma benzene hexachloride and amitraz at four different concentrations including the recommended dose rates. Based on their lethal concentrations (LC₅₀ & LC₉₀) chlorfenvinphos and dioxathion combined and chlorfenvinphos alone placed first and second, respectively, in all stages except at the unfed nymphal stage where gamma benzene hexachloride topped with a LC₅₀ of 0.001629, while chlorfenvinphos and dioxathion combined and chlorfenvinphos alone had LC₅₀ of 0.001794 and 0.002258, respectively. Amitraz appeared to have a quick knock-down effect on larvae and nymphs but at the recommended dose rate, showed no mortality of the ticks at that stage. However, at a concentration of 0.040%, amitraz showed a 100% inhibition of oviposition and hatching of laid eggs. Gamma benzene hexachloride produced only 66% inhibition of oviposition while chlorfenvinphos and dioxathion combined and chlorfenvinphos alone produced 100% inhibition of oviposition at their recommended dose rates. Fed nymphs were more susceptible than the unfed nymphs. Eggs laid by engorged female ovipositing ticks, applied with gamma benzene hexachloride, hatched.This revised version was published online in May 2005 with a corrected cover date.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Molecular detection of <Emphasis Type="Italic">Ehrlichia ruminantium</Emphasis> infection in <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3–15.1%) than in the easterly divisions (Central River and Upper River; range 1.6–7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> (Ascomycota: Hypocreales) as a management agent for free-living <Emphasis Type="Italic">Amblyomma americanum</Emphasis> (Acari: Ixodidae) in Ohio

Unfed adult Amblyomma americanum were exposed to the entomopathogenic fungus Beauveria bassiana and placed in the field. Ticks exposed to the fungus experienced higher mortality than those in the control group (P = 0.001). It is recommended that for inclusion in a tick management program, the fungus be applied in the late evening during the months March through June as this is when ticks are most active and conditions suitable for fungal activity occur.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

<Emphasis Type="Italic">Coxiella</Emphasis> Symbionts in the Cayenne Tick <Emphasis Type="Italic">Amblyomma cajennense</Emphasis>

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.     

Biochemical and molecular characterization of wild-type and fused protoplasts of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Protoplasts were isolated from two isolates each of Beauveria bassiana and Metarhizium anisopliae using lysing enzymes. Intra- and intergeneric protoplast fusion has been carried out using 40% polyethylene glycol. The fused protoplasts of B. bassiana and M. anisopliae have been regenerated on Czapek–Dox agar media, and a total of four fusants were selected for further studies. An increase in proteinase and chitinase enzyme activity was recorded with all fusants as compared to the wild-type isolates. To understand the nature of recombination process, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were carried out on genomic DNA of fused and wild-type isolates. The present study demonstrates the scope and significance of the protoplast fusion technique as a rapid consistent method for identification of B. bassiana and M. anisopliae fused and wild-type isolates based on the banding pattern of RAPD and RFLP that can be reliably used ahead for further applications on these species.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

Natural Infections Caused by the Fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> as a Pathogen of <Emphasis Type="Italic">Musca domestica</Emphasis>in the Neotropic

A survey for entomopathogenic fungi of Musca domestica adults was conducted in poultry houses in La Plata, Buenos Aires province, Argentina, during the years 2002 and 2003. Adult house flies were found infected with the fungus Beauveria bassiana (Bals.) Vuill. (Deuteromycotina: Hyphomycetes) from field collections, with a natural infected prevalence between 0.4–1.45%. This is the first record of natural infections of house flies caused by B. bassiana for the neotropics. Pathogenicity assays under laboratory conditions showed 94% adult mortality at 14 days post challenge. CIC fellow     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Phenotypic variability of environmental isolates of the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

A total of 35 isolates of Beauveria bassiana (Bals.) Vuill. isolates obtained from various insects of Novosibirsk oblast were investigated. The fungal morphotypes were found to include cultures of high, medium, and low virulence. Low correlation (r < 0.48) was observed between virulence and the morphophysiological characteristics of the isolates (lipase and protease activity, biomass, radial growth rate, conidia productivity, and relief). Isolates exhibiting high virulence to insects of a certain order proved to be virulent to the insects of other orders. A high correlation (r > 0. 74) was revealed between the virulence of the isolates to the potato beetle Leptinotarsa decemlineata Say and the locusts Calliptamus barbarus Costa and Locusta migratoria L. Isolates obtained from insects of the same species in the same site may differ significantly in virulence.     

Substrate influence on physiology and virulence of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> acting on larvae and adults of <Emphasis Type="Italic">Tenebrio molitor</Emphasis>

Two isolates of Beauveria bassiana, wild type (wt) and its mutant type (mt) were compared in terms of growth patterns on culture plates containing media based on wheat bran, grasshopper exoskeletons, colloidal chitin or Sabouraud-dextrose agar (SDA). Germination for the mt isolate was up to 33% faster in all media. Influence of media on virulence was determined against larvae and adults of Tenebrio molitor. Mortality higher than 90% was reached for adults after 6 days using conidia from all media. For larvae, a mortality of 80% was reached after 11 days with conidia collected from SDA medium and between 15 and 35% with conidia from other media. In SDA medium, conidial yield was almost ten times higher for the mt isolate compared to the wt isolate; however, virulence traits were similar against either larvae or adults. These results may influence commercial preparations of entomopathogenic fungi based on conidia.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

Impact of sequential exposure of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and imidacloprid against susceptible and resistant strains of <Emphasis Type="Italic">Musca domestica</Emphasis>

Insecticide resistance in the housefly Musca domestica is hampering pest management. However, entomopathogens, possibly in combination with insecticides, may have control potential against resistant houseflies. This study investigates the combination of the entomopathogenic fungus Beauveria bassiana and the neonicotinoid insecticide, imidacloprid against a susceptible and a resistant housefly strain, respectively under laboratory conditions. The fungus and insecticide were tested alone and in combinations at LC₃₀. Significant and synergistic interactions between B. bassiana and imidacloprid were observed with increased mortality rates of the combined treatment as compared to individual treatment in housefly strains 772a (susceptible) and 766b (resistant). Significant differences in the GST and P450 activities for both strains were found. Female 766b flies caused 15- to 237-fold increases in gene expression of xenobiotic response genes for B. bassiana and 23- to 120-fold changes for imidacloprid. The combination of B. bassiana and imidacloprid caused significant synergistic interaction when applied against two housefly strains irrespective of order of application. The effect was highest when the insecticide was applied first. The resistant housefly strain had elevated detoxification enzymes and higher expression of detoxification genes, but showed the same level of susceptibility to the combined fungus/insecticide treatment as the susceptible strain.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.