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.     

Beauveria caledonica is a naturally occurring pathogen of forest beetles

In New Zealand, two introduced scolytid beetles, Hylastes ater and Hylurgus ligniperda (Curculionidae: Scolytinae) are pests in pine plantations. Investigation of the naturally occurring pathogens of these exotic pests revealed that both are attacked by Beauveria caledonica, a species originally isolated and described from soil in Scotland. The isolates in New Zealand were identical in morphology and conserved DNA region (rDNA, elongation factor α) sequence to isolates held in the USDA-ARS insect pathogens culture collection. In bioassay, the B. caledonica isolates were highly pathogenic to adults of H. ligniperda and larvae of Tenebrio molitor. Sporulation was observed on cadavers, confirming the species can utilise the cadavers. As both species were likely to have been introduced to New Zealand from Europe, a search was made for B. caledonica in the northern UK and Ireland. The fungus was found as a naturally-occurring pathogen of the weevil pest, Hylobius abietis (Curculionidae: Scolytinae), developing in spruce and other beetles in forests in both regions.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Association of Entomopathogenic Fungi with Exotic Bark Beetles in New Zealand Pine Plantations

Hylastes ater and Hylurgus ligniperda are introduced pests of re-established Pinus radiata in New Zealand. Both species breed under the bark of stumps in recently harvested areas. Adult maturation feeding on pine seedlings planted in adjacent areas can significantly impact seedling growth, and in severe cases seedlings will die. Entomopathogenic fungi are important natural mortality factors in bark beetle populations, and Beauveria spp. are predominant. Here, we report on the isolation of other fungal species from H. ater in New Zealand. Based on morphological characteristics and sequencing data, two species, Metarhizium flavoviride var. pemphigi and Hirsutella guignardii, were recovered from H. ater. Both are new records for New Zealand and appear to be the first records of these species from bark beetles worldwide.     

Hypocrealean fungi associated with populations of <Emphasis Type="Italic">Ips typographus</Emphasis> in West Carpathians and selection of local <Emphasis Type="Italic">Beauveria</Emphasis> strains for effective bark beetle control

In Slovakia, a diversity of entomopathogenic fungi (Ascomycota, Hypocreales) associated with outbreaks of Ips typographus was studied in 81 localities and as many as 113 in vitro cultures of five entomopathogenic species were isolated from infected individuals: Beauveria bassiana (87 isolates), B. pseudobassiana (14 isolates), B. caledonica (6 isolates), Lecanicillium lecanii (4 isolates) and Isaria farinosa (2 isolates). B. pseudobassiana is recorded in natural populations of I. typographus for the first time. Biological properties of selected Beauveria isolates, including colony growth, biomass production, conidia yield and pathogenicity to I. typographus adults, were studied in a series of laboratory bioassays and much intra- and interspecific variability was detected. B. bassiana isolates produced biomass or conidia at significantly higher rate than B. pseudobassiana and B. caledonica isolates. Two B. bassiana isolates were selected as the most virulent to bark beetle adults, demonstrating a mean LC₅₀ ranging from 0.72 to 2.05?×?10⁶ conidia ml^?1, and were qualified as promising candidates for biocontrol of I. typographus. Their virulence was significantly higher than that of the mycoinsecticides Boverol®, which was used as a reference strain in the virulence bioassays.     

Entomopathogenic fungi as biological control agents for the vector of the laurel wilt disease,the redbay ambrosia beetle,Xyleborus glabratus (Coleoptera: Curculionidae)

The redbay ambrosia beetle (RAB), Xyleborus glabratus, is a wood-boring insect that vectors the fungal pathogen, Raffaelea lauricola, which causes laurel wilt, a lethal disease of avocado. The objective of this study was to determine the susceptibility of RAB to infection and subsequent death by exposure to three commercial strains of entomopathogenic fungi [two strains of Isaria fumosorosea (Ifr 3581 and PFR), and strain GHA of Beauveria bassiana]. RAB females were dipped in fungal spore solutions and their median survivorship times (MST) determined. Contact with any of the biopesticides resulted in death of all RAB females. MSTs of RAB females ranged from 3 days (B. bassiana) to 5 days (I. fumosorosea PFR). B. bassiana killed RAB females faster, followed by Ifr 3581 and PFR. RAB females dipped in B. bassiana suspensions had the highest number of viable spores attached to their bodies, followed by Ifr 3581. Beetles dipped in PFR suspension had significantly less viable spores attached to their bodies. No significant differences were observed in the mortality of beetles exposed to entomopathogenic fungi by dipping in a fungal suspension or walking on treated avocado bolts. Beetles bored into the logs and constructed galleries, but they were found dead inside the galleries a few days after exposure to the entomopathogens. Entomopathogenic fungal infection in dead beetles was confirmed through molecular techniques. This is the first study to demonstrate that entomopathogenic fungi are potential biological control agents against RAB.     

Effect of Norwegian entomopathogenic fungal isolates against Otiorhynchus sulcatus larvae at low temperatures and persistence in strawberry rhizospheres

Biological control of belowground stages of the black vine weevil Otiorhynchus sulcatus F. (Coleoptera: Curculionidae) in strawberries in cool temperate regions using entomopathogens is challenged by low temperatures during the periods when larvae are vulnerable to infections. In a laboratory study we tested six indigenous Norwegian isolates of entomopathogenic fungi (one Beauveria bassiana, three Beauveria pseudobassiana, and two Metarhizium brunneum; Ascomycota: Hypocreales) for their efficacy against O. sulcatus larvae at 6, 12, and 18 °C. At the lowest temperature only Beauveria spp. affected survival of O. sulcatus while all three fungal species reduced larval survival compared to the control treatment at 12 and 18 °C. Two of the Norwegian isolates, one B. pseudobassiana and one M. brunneum, were then evaluated for long-term persistence (>1 year) in the bulk soil and the rhizosphere soil of strawberries in a semi-field experiment. An exotic isolate of M. brunneum sharing origin with a widespread commercial biocontrol agent (F52/Met52 (Novozymes)) was included for comparison. All three isolates showed significantly higher abundances in the rhizosphere soil compared to bulk soil at 153, 366, and 471 days after inoculation, thus indicating rhizosphere competence for B. pseudobassiana. Notably, CFU levels for both Norwegian isolates were much higher than for the exotic M. brunneum isolate. Selection of locally adapted isolates may therefore be of importance when considering biocontrol strategies of belowground pests in strawberry production.     

Comparative laboratory studies on three fungal pathogens of the elm bark beetle Scolytus scolytus: Pathogenicity of Beauveria bassiana,metarhizium anisopliae, and Paecilomyces farinosus to larvae and adults of S. scolytus

The entomogenous fungi Beauveria bassiana (nine isolates), Metarhizium anisopliae (seven isolates), and Paecilomyces farinosus (four isolates) were tested as pathogens of larvae of the elm bark beetle, Scolytus scolytus. Single isolates of B. bassiana and M. anisopliae were also tested against adult beetles. Of the 21 isolates tested as conidial suspensions against larvae, all proved pathogenic. The three most and least virulent isolates were, respectively, isolates of B. bassiana and M. anisopliae. The other isolates fell between these two extremes, with the four P. farinosus isolates all moderately virulent. Spore retention on larvae following inoculation was estimated by washing conidia off the larvae. From the results it was possible to relate larval mortality to the approximate spore dose causing infection at different spore concentrations. Thus, application of spores of the three pathogens at a concentration of 10³ spores/ml resulted in limited mortality. At this concentration, an average of only a single spore was recovered from the inoculated larva. Adult bark beetles also proved susceptible to infection by isolates of B. bassiana and M. anisopliae. They were exposed to discs of elm bark dipped in a conidial suspension. It was estimated that a dose of less than 100 spores could cause infection of beetles following feeding on the elm bark discs.     

Microbial biological control potential of three strains of Beauveria bassiana s. l. against greenhouse shore fly Scatella tenuicosta: Assessment of virulence,mass production capacity,and effects on shore fly reproduction

The microbial biological control potential of three strains of Beauveria bassiana sensu lato originally isolated from the shore fly Scatella tenuicosta (Diptera: Ephydridae) was assessed in a series of laboratory bioassays. Comparisons were made to two commercially-available strains of B. bassiana. Two of the shore fly strains proved 27–67 times more virulent than the commercial strains in terms of LC₅₀ (14–17 vs. 458–942 conidia/mm²) and killed shore flies more rapidly. B. bassiana s. l. strain ST1 exhibited a mass production capacity comparable to the commercial B. bassiana stain GHA, producing 2.8 × 10¹² conidia/kg barley-based solid substrate in ventilated mushroom spawn bags. The shore fly strains of Beauveria sporulated on a higher percentage of killed adult shore flies and produced substantially greater numbers of conidia per cadaver than the commercial strains, indicating that these pathogens are well adapted to this host. Female shore flies treated with strain ST1 survived for only 5 days, with longevity being reduced by 8–10 days compared to control insects. This reduction in survival had a large impact on total lifetime egg production, reducing it by 78–88%, depending on the time of treatment relative to the pre-oviposition period. However, fungal growth within infected female shore flies had no effect on egg production or egg viability until the day before the flies succumbed to mycosis (day 4 post-inoculation). As a consequence, the intrinsic rate of shore fly population increase and population doubling time were little affected by fungal infection (0.4357 vs. 0.4152 and 1.6 vs. 1.7 days for control vs. Beauveria-treated populations, respectively). These findings underscore the challenges involved with use of slow-acting pathogens for control of highly fecund greenhouse pests and the fundamental necessity of integrating these agents into integrated pest management systems.     

Identification of Molecular Variants in Mitochondrial DNAs of Members of the Genera Beauveria, Verticillium, Paecilomyces, Tolypocladium, and Metarhizium

A set of five mitochondrial (mt) probes derived from a strain of Beauveria bassiana was used to evaluate the similarity of mtDNAs from 15 additional isolates of this fungus and five genera of other entomopathogenic fungi. The probes and genes encoded for (shown in parentheses) were pBbmtE2 (NADI, ATP6), pBbmtE3 (ATP6, small rRNA [srRNA]), pBbmtE4 (srRNA, CO3, NAD6), pBbSE1 (NAD6, tRNA^{Val, Ile, Ser, Trp, Pro}, large rRNA [lrRNA]), and pBbXS1 (lrRNA). The probes produced identical hybridization patterns in EcoRI-digested DNA from nearly all isolates of B. bassiana and Beauveria caledonica. Similar patterns were also observed with Beauveria densa. The isolates of B. caledonica and B. densa DNAs could be differentiated from each other and from B. bassiana on the basis of a HindIII digestion and probing with pBbmtE3. Probe pBbmtE2 produced either a 5.0-kb or a 4.1-kb band in all of the B. bassiana isolates. This observation was used to categorize the mtDNA of B. bassiana into two types, designated A and B. Hybridization of the five probes produced distinct banding patterns in Beauveria brongniartii, Tolypocladium cylindrosporum, Tolypocladium nivea, Metarhizium anisopliae, Verticillium lecanii, and Paecilomyces farinosus. Hybridizations carried out with multiple probes simultaneously present produced unique patterns which characterized the B. bassiana group from all other fungi tested. These results are discussed in terms of how mtDNA polymorphisms in B. bassiana may relate to natural population structures, mt transmission in deuteromycetes, and the use of mtDNA polymorphisms in structural analysis of mtDNA.     

Evaluation of new fermentation and formulation strategies for a high endophytic establishment of Beauveria bassiana in oilseed rape plants

Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases.When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1 × 10¹⁰ SCS/g sucrose in shake flasks and 1.8 ± 0.1 × 10¹⁰ SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in.Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1% Triton X-114, 1% molasses, 1% titanium dioxide and 10⁶ spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves.Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.     

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.     

Laboratory and semi-field evaluation of Beauveria bassiana (Ascomycota: Hypocreales) against the lettuce aphid,Nasonovia ribisnigri (Hemiptera: Aphididae)

The lettuce aphid, Nasonovia ribisnigri (Mosley), is an economically important pest of lettuce worldwide. The entomopathogenic fungus Beauveria bassiana strain GHA has recently been reported as a potential biocontrol candidate for use against the lettuce aphid. This study provides information on the mortality inflicted by B. bassiana when applied against different life stages of the lettuce aphid under laboratory conditions and how fungus infection affects the aphid fecundity. In addition, temporal changes in persistence of fungus inoculum applied to foliage of young lettuce plants under semi-field conditions was analysed. Immature life stages were generally the least susceptible to fungal infection and the susceptibility of all stages was dose-dependent, with the highest mortality occurring at the highest dose. B. bassiana significantly affected the rate of nymph production by the lettuce aphid, with the highest effect seen when the alatoid fourth instar of N. ribisnigri was inoculated with B. bassiana. The persistence of B. bassiana conidia on lettuce foliage was not influenced by leaf position. Within 5 days, the cumulative percentage decline in the conidial population was 38% which declined further to 92% and 99% on day 11 and 20 post-spraying, respectively. In accordance, the infectivity to second instar lettuce aphid nymphs of B. bassiana conidia deposited on leaves declined according to an exponential decay model predicting an intercept of 0.59 ± 0.03 (S.E), a reduction in aphid mortality at a rate of 11% with each increasing day after fungal application and a fungus half-life of 6.34 ± 0.69 days.     

Management of Frankliniella occidentalis (Thysanoptera: Thripidae) with granular formulations of entomopathogenic fungi

Western flower thrips (WFT), Franklinella occidentalis, is a major pest of ornamentals. Mycotized millet grains with entomopathogenic fungi applied to soil of potted marigold plants were tested to target the soil-dwelling stages of thrips. Two experimental fungal isolates, (Beauveria bassiana [ARS7060] and Metarhizium anisopliae [ERL1171]), were compared with the registered B. bassiana strain GHA [commercialized as BotaniGard®] and untreated controls in greenhouse caged trials. Mycotized millet grains were mixed into the upper surface of the potting soil in pots of flowering ‘Hero Yellow’ marigolds (4 g/pot). One week after application five mated WFT females were released onto each plant (four plants per cage). At 8 weeks post-infestation, the mean total number of thrips per plant was 81% and 90% less in the ERL1171 and ARS 7060 treatments, respectively, than in the control. The mean numbers of thrips per plant for the control and GHA treatments were not significantly different. Plant damage was 60% less on plants treated with the experimental fungi than the control and GHA treatments. At 10 weeks post-application, 75–90% of WFT collected from the treatments of the experimental fungi were infected with the fungal species applied. These results demonstrate that soil applications of entomopathogenic fungi can reduce WFT populations significantly and prevent major damage.     

Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Oxaloacetate hydrolase gene links the cytoplasmic route of oxalate formation to differentiation and virulence of entomopathogenic fungus Beauveria bassiana

Oxalic acid is used as a functional molecule by most filamentous fungi, and produced via cytoplasmic pathway and mitochondrial pathway. The cytoplasmic pathway of oxalate production from oxaloacetate is a one-step reaction catalyzed by oxaloacetate hydrolase. The entomopathogenic fungus Beauveria bassiana contains a unique oxaloacetate hydrolase gene (BbOAH). The role of cytoplasmic pathway of oxalate production in B. bassiana development and virulence was studied via construction of a targeted gene disruption mutant of BbOAH. Disruption of BbOAH resulted in a slight decrease (~ 30%) in oxalate production, but has no significant influence on fungal growth. The mutant strain displayed a significant delay at early stage of conidial development, and a significant defect in dimorphic transition. Additionally, bioassay using the greater waxmoth as host indicated a slight (~ 20%) decrease in mortality caused by the gene disruption strain. The phenotypic defects of the ΔBbOAH strain could be restored by ectopic integration of BbOAH. Our findings indicate that BbOAH gene connects the cytoplasmic route of oxalate production to fungal development and virulence, although the cytoplasmic route is not indispensable for oxalate production in B. bassiana.     

A population survey of Beauveria bassiana in the microhabitat of the red turpentine beetle,Dendroctonus valens,in Chinese pine forests

Field survey of the entomopathogenic fungus Beauveria bassiana in association with the red turpentine beetle, Dendroctonus valens, was undertaken in three pine plantations in Northern China. In total, 88 strains of B. bassiana sensu lato were isolated from the soil, bark, beetle frass, living adult and cadaver samples and soil was proved to be an important inoculum reservoir for fungal entomopathogens. Of these, 77 isolates were included for genetic diversity analysis by PCR for inter-simple sequence repeats (ISSR). Genetic diversity and population structure analysis of the isolates from three sites and five niches demonstrated high genetic diversity and heterogeneity between and/or within populations. Wright's statistics revealed a high gene flow rate (4.529) among the three populations, especially among the soil-derived isolate subpopulations. Low variation was mainly caused (94.8%) by variation among different substrates, suggesting the importance of microhabitat substrates on genetic diversity of B. bassiana. Phylogenetic variation was not associated with geographic distance.     

The endophytic fungal entomopathogens Beauveria bassiana and Purpureocillium lilacinum enhance the growth of cultivated cotton (Gossypium hirsutum) and negatively affect survival of the cotton bollworm (Helicoverpa zea)

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum, were assessed on the growth of cultivated cotton (Gossypium hirsutum) and development of the cotton bollworm (Helicoverpa zea). In two replicate greenhouse trials, cotton plants were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia and evaluated for effects on both plant dry biomass, number of nodes and number of developing flowers (squares). We similarly treated cotton plants and evaluated H. zea performance using no-choice in planta assays starting at the 2nd larval instar. Treatment with both fungal endophytes resulted in a significant increases in plant dry biomass (ANOVA, P = 0.024). Plant developmental stage and number of squares were also significantly enhanced in the endophyte treated plants (ANOVA, P = 0.005 and P = 0.027, respectively). The survivorship of H. zea was significantly different among the endophyte treatment groups (Kaplan–Meier, P = 0.02), where insects feeding on control plants exhibited higher survival than insects on the endophyte treated plants. There were no significant endophyte treatment effects on larval or pupal weights of H. zea individuals. There was no endophyte effect on days to pupation among treatments, but there was a marginal effect on days to eclosion (Kaplan–Meier, P = 0.07). Overall, our results demonstrate (i) the positive plant growth enhancing effects of the target endophyes on cultivated cotton under greenhouse conditions and (ii) the negative effects of endophytic P. lilacinum and B. bassiana on H. zea survivorship and development using whole plant assays.     

Effectiveness of Beauveria bassiana sensu lato strains for biological control against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in China

Owing to the need to combat the spread of acaricide-resistant ticks, the development of long-term biological control has become a hot topic for tick control. In this study, we investigated the pathogenicity of three Beauveria bassiana isolates on the engorged female Rhipicephalus (Boophilus) microplus ticks using different conidial concentrations. The results showed that B. bassiana B.bAT17 was highly pathogenic against engorged R. (B.) microplus females, resulting in lethal time (LT₅₀ and LT₉₀) of 7.14 and 9.33 days at a concentration of 10⁹ conidia/ml. R. (B.) microplus females treated with B. bassiana B.bAT17 significantly reduced the amount of ovipositioning; and most ticks died before they could begin to oviposit. Proteases and chitinases were analyzed in order to establish a screening method for identification of high virulent strains. This study has confirmed the significant pathogenic effect of entomopathogenic fungi against engorged R. (B.) microplus females in China, and further studies on the efficiency of the fungus against ticks in the field are required.     

The antifungal activity of fatty acids of all stages of Sarcophaga carnaria L. (Diptera: Sarcophagidae)

Fatty acids as components of cuticular lipids of insects play a significant role in antifungal in protection against fungal infection. The chemical composition of cuticular and internal extracts obtained from all developmental stages of flesh flies Sarcophaga carnaria was identified. The fatty acids were detected using gas chromatography coupled with mass spectrometry and the most abundant for all examined stages were: 18:1 > 16:0 > 16:1 > 18:0 > 18:2. Polyunsaturated fatty acids (PUFA) C20 were found in both, cuticular and internal extracts. GC–MS analysis showed higher relative content of PUFA in adults than in preimaginal stages.Fatty acids alone as well as their cuticular and internal extracts obtained from larvae, pupae male and female of S. carnaria were tested according to their potential antimicrobial activity against entomopathogenic fungi: Paecilomyces lilacinus, Paecilomyces fumosoroseus, Lecanicillium lecanii, Metarhizium anisopliae, Beauveria bassiana (Tve-N39) and B. bassiana (Dv-1/07). FA presented diverse antimicrobial activity depending on the length of the chain and the presence of unsaturated bonds. Short chain and unsaturated FA (6:0, 11:0, 13:0) have shown significantly stronger activity against fungi but they were detected in lower concentrations. PUFA inhibit fungal growth more effectively than unsaturated long chain fatty acids. Cuticular and internal extracts of all living forms of S. carnaria exhibited approximately equal activity against tested entomopathogenic fungi. We presumed that the most abundant saturated long chain FA and additionally PUFA founded in our analysis are involved in protecting the flies against fungal infection.