球孢白僵菌和金龟子绿僵菌不同菌株对黑足角胸叶甲成虫的致病力评价

本研究在测定不同球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae菌株的生长速率与产孢量的基础上, 采用孢悬液浸渍法进行了对油茶新害虫--黑足角胸叶甲Basilepta melanopus成虫的生物测定, 旨在筛选出感染该成虫的高致病力菌株, 为防治该虫提供新的生物资源。结果表明： 不同菌株生长速率和产孢量间存在显著差异, MaYTTR-04, BbFZ-17, MaZPTR-01和BbTK-01生长速率和产孢量均显著高于其他菌株。接种后, 叶甲成虫累积死亡率随时间的增加而逐渐增高, 接种白僵菌7 d后, 成虫校正死亡率全部达到100%; 接种MaZPTR-01和MaYTTR-04两个绿僵菌菌株14 d后, 成虫死亡率分别为80.3%和78.8%。而且接种白僵菌后, 叶甲成虫的僵虫率显著较绿僵菌高, 尤其以BbTK-01和BbFZ-17两个菌株较好, 分别达到85.7%和75.8%。BbXJ-01, BbFZ-17和BbTK-01 3个白僵菌菌株的LT₅₀最小, 分别为3.0, 3.3和3.4 d; MaYTTR-04和MaZPTR-01两个绿僵菌的LT₅₀分别为6.0和6.2 d。结果说明, 白僵菌对叶甲成虫的致病力较强, 尤其是BbTK-01和BbFZ-17两个菌株, 不仅致死率高, 且致死速度快, 僵虫率高, 同时这2个菌株生长速度快、 产孢量大, 具有优良的生产特性, 在黑足角胸叶甲的生物防治中将有重要的应用价值。     

Isolation and characterization of nucleotide excision repair deficient mutants of the entomopathogenic fungus, Beauveria bassiana

To better understand DNA repair in the entomopathogenic fungus Beauveria bassiana, three ultraviolet (UV) light sensitive mutants were isolated and characterized to be deficient in nucleotide excision repair (NER). The UV sensitive mutants were scored by comparison to survival of the parental isolate, GK2016, after 36 J/m² UV-C irradiation. At this dose, conidial survival of GK2016 was 98% and the mutants LC75, LC194, and LC85 had survival values of 63%, 45%, and 31%, respectively. An immunological method which measured the removal of pyrimidine-(6-4)-pyrimidone photoproducts during repair confirmed the decreased ability of LC75, LC194, and LC85 to remove these UV-induced dimers by NER. The mutants were also found to be deficient in NER at swollen/ germinating conidia and blastospore life cycle stages. The germination of the moderately UV sensitive mutant, LC75, was similar to that of the parental isolate, GK2016, after UV irradiation and incubation to enhance NER. The more sensitive mutants, LC194 and LC85 were 2.1- or 2.7-fold, respectively, less likely to germinate after UV irradiation based on their ability to carry out NER. These NER deficient mutants, the first to be derived from B. bassiana, reveal the importance of NER in spore survival post-UV irradiation.     

Transformation system of Beauveria bassiana and Metarhizium anisopliae using nitrate reductase gene of Aspergillus nidulans

An heterologous transformation system for entomopathogenic fungi B. bassiana and M. anisopliae was developed based on the use of A. nidulans nitrate reductase gene (niaD). B. bassiana and M. anisopliae niaD stable mutants were selected by treatment of protoplast with ethane methane sulphonate (EMS) and regenerated on chlorate medium. The cloned gene was capable of transforming B. bassiana and M. anisopliae at a frequency of 5.8 to 20 transformants per microg of DNA. Most of them were mitotically stable.     

Susceptibility of Agrilus planipennis (Coleoptera: Buprestidae) to Beauveria bassiana and Metarhizium anisopliae

The susceptibility of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae) to selected strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin was evaluated through bioassays with direct immersion or foliar exposure under laboratory conditions. Results showed that A. planipennis adults were susceptible to B. bassiana and M. anisoplae. Significant time-mortality response was found for each isolates. Isolate B. bassiana GHA killed A. planipennis adults at a faster rate compared with other isolates tested, with the lowest average time-to-death values. The LC50 values estimated under direct immersion method ranged from 1.7 x 10(5) to 1.9 x 10(7), 3.5 x 10(4) to 5.3 x 10(5), and 4.1 x 10(3) to 2.9 x 10(5) conidia/ml for B. basissiana and from 3.2 x 10(6) to 1.1 x 10(7), 4.5 x 10(3) to 4.5 x 10(5), and 1.4 x 10(2) to 1.2 x 10(5) conidia/ml for M. anisopliae at 4, 5, and 6 d after treatment, respectively. By days 5 and 6, B. bassiana GHA outperformed all other isolates tested except ARSEF 7234, followed by ARSEF 7152, 6393, and 7180. Significant concentration-mortality response was also observed for two B. bassiana GHA formulations, BotaniGard ES and Mycotrol O, and M. anisopliae F52 when insects were treated through foliar exposure. The LC50 values ranged from 114.5 to 309.6, 18.4 to 797.3, and 345.3 to 362.0 conidia/cm2 for BotaniGard, Mycotrol, and M. anisopliae F52, respectively. Based on the results of these bioassays, the efficacy of both B. bassiana GHA formulations and M. anisopliae F52 were similar against adult A. planipennis. The potential use of entomopathogenic fungi for management of A. planipennis in North America is discussed.     

Conidium production by insect pathogenic fungi on commercially available agars

AIMS: Conidium production by three species of insect pathogenic fungi, Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii, was assessed on various depths and types of commercially available agars. METHODS: Conidium production was assessed after 14 d of growth on commercially available media as well as at three different agar depths. RESULTS: Metarhizium anisopliae and B. bassiana isolates showed greatest conidium production on potato dextrose agar (PDA) at a depth of 2 mm, whereas V. lecanii showed greatest conidium production on yeast extract-peptone-dextrose agar (YPDA) regardless of agar depth. Optimum conidium production for M. anisopliae and B. bassiana was not only dependent upon the isolate used but also on the medium type and agar depth. SIGNIFICANCE AND IMPACT OF THE STUDY: Conidia are the infective structures for insect pathogenic fungi and this study suggests a rationale basis for consistent conidium production for laboratory and commercial practices.     

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidine dimers and their removal in Beauveria bassiana conidiospore DNA

Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

Effects of Metarhizium anisopliae (Metsch.) Sorok. and Beauveria bassiana (Bals.) Vuill. on the predatory stinkbug Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae)

Entomopathogenic fungi and the predatory stinkbug Podisus nigrispinus (Dallas) can target simultaneously the same or different pests in agroecosystems. Topical contact during fungal dispersion or spraying, walking on plant surfaces and ingestion of contaminated prey are some of possible ways of interactions between fungi and the predatory stinkbug. The impact of three isolates of Metarhizium anisopliae (Metsch.) Sorok. (866, 1022 and 1189) and Beauveria bassiana (Bals.) Vuill. (604, 634 and 561) against nymphs and adults of P. nigrispinus was investigated under laboratory conditions. M. anisopliae caused higher mortality compared to B. bassiana either by topical application or by dry residue on cotton leaves. Topic contact with both fungi caused higher mortality to the predator. No mortality confirmation was reported for the adults. Nymphs fed with cotton leafworm larvae Alabama argillacea (Hübner) (Lepidoptera: Noctuidae) contaminated by the fungi had their reproduction affected, but this was not observed when adult predators fed on contaminated larvae. These findings suggest that isolates of M. anisopliae can cause mortality of P. nigrispinus nymphs by topical contact, while isolates of B. bassiana were less harmful by all ways of infection as compared to M. anisopliae.     

Characterization and virulence of Beauveria spp. recovered from emerald ash borer in southwestern Ontario, Canada

The emerald ash borer (EAB), Agrilus planipennis (Coleoptera: Buprestidae), is an invasive wood boring beetle that is decimating North America's ash trees (Fraxinus spp.). To find effective and safe indigenous biocontrol agents to manage EAB, we conducted a survey in 2008-2009 of entomopathogenic fungi (EPF) infecting EAB in five outbreak sites in southwestern Ontario, Canada. A total of 78 Beauveria spp. isolates were retrieved from dead and mycosed EAB cadavers residing in the phloem tissues of dead ash barks, larval frass extracted from feeding galleries under the bark of dead trees. Molecular characterization using sequences of the ITS, 5' end of EF1-α and intergenic Bloc region fragments revealed that Beauveria bassiana and Beauveria pseudobassiana were commonly associated with EAB in the sampled sites. Based on phylogenetic analysis inferred from ITS sequences, 17 of these isolates clustered with B. bassiana, which further grouped into three different sub-clades. However, the combined EF1-α and Bloc sequences detected five genotypes among the three sub-clades. The remaining 61 isolates clustered with B. pseudobassiana, which had identical ITS sequences but were further subdivided into two genotypes by variation in the EF1-α and Bloc regions. Initial virulence screening against EAB adults of 23 isolates representing the different clades yielded 8 that produced more than 90% mortality in a single concentration assay. These isolates differed in virulence based on LC(50) values estimated from multiple concentration bioassay and based on mean survival times at a conidia concentration of 2×10(6) conidia/ml. B. bassiana isolate L49-1AA was significantly more virulent and produced more conidia on EAB cadavers compared to the other indigenous isolates and the commercial strain B. bassiana GHA suggesting that L49-1AA may have potential as a microbiological control agent against EAB.     

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.     

Pathogenicity of three formulations of entomopathogenic fungi for control of adult Haematobia irritans (Diptera: Muscidae)

Powder formulations of three species of entomopathogenic fungi were evaluated for their pathogenic effect upon adult horn flies, Hematobia irritans (L.) (Diptera: Muscidae). Flies were treated with conidia and blastospores of the entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. (strain GHA), Metarhizium anisopliae (Metschnikoff) Sorokin (strain ESCI), and Paecilomyces fimosoroseus (Wize) Brown & Smith (strain ARSEF 3581) in the laboratory. At 4 d postexposure, flies treated with B. bassiana had an average of 98.4% mortality versus 43.5% from treatment with M. anisopliae and 13.0% from treatment with P. fiimosoroseus. At 7 d postexposure, flies treated with B. bassiana had an average of 100.0% mortality compared with 73.0% from treatment with M. anisopliae and 33.3% from treatment with P.fumosoroseus. Mean lethal time (LT50) was 2.70, 4.98, and 7.97 d for B. bassiana, M. anisopliae, and P. fiumosoroseus, respectively. Entomopathogenic fungi such as B. bassiana and M. anisopliae may have the potential for controlling populations of horn flies. These studies indicate that B. bassiana and M. anisopliae were not only pathogenic to adult horn flies, but they caused mortality in a short time.     

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.     

Interaction of Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill. and the parasitoid Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) with larvae of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Chemical insecticides are broadly applied to control diamondback moth, Plutella xylostella (L.). Diamondback moth is a major pest of cruciferous worldwide, and resistance of this pest to insecticide has been often reported. Thus, this research investigated the interactions among the fungi Metarhizium anisopliae (Metsch.) Sorok., Beauveria bassiana (Bals.) Vuill., and the larval-pupal parasitoid Oomyzus sokolowskii (Kurdjumov) before and after application of the fungi on DBM larvae offered to the parasitoid. The experiment was carried out at 26+/-l degreeC, 75+/- 5% RH and 12h photophase using a completely randomized design, with eight treatments with six replications each. The isolates E9 of M. anisopliae and ESALQ 447 of B. bassiana, were used at the concentration of 10(7) conidia ml(-1). The results showed that M. anisopliae and B. bassiana reduced the parasitism of P. xylostella by O. sokolowskii. Additive effects were found on the mortality of P. xylostella with the different combinations among the fungi and parasitoid, except for the treatment B. bassiana inoculated 24h before exposition of the larvae to O. sokolowskii. The isolates were more efficacious when applied after exposition of the larvae to the parasitoid. The efficiency of O. sokolowskii was negatively influenced by the presence of the fungi, mainly when the fungi were applied 24h before diamondback's larvae were exposed to the parasitoid. The association of the fungi with the parasitoid presents potential to be tested in field. The use of these natural enemies in the integrated management of P. xylostella may economically improve the cabbage productive system, especially for organic farming.     

球孢白僵菌和绿僵菌对日本双棘长蠹林间防治效果的研究

日本双棘长蠹（Sinoxylon japonicum）是危害国槐和刺槐等园林树木重要的蛀干害虫。本研究采用喷雾法,用含0.1%（体积分数）吐温-80的1.0×109分生孢子/mL的球孢白僵菌（Beauveria bassiana）和绿僵菌（Metarhizium anisopliae）悬液,分别于日本双棘长蠹生活史各阶段开始时在林间进行喷雾实验。实验结果显示,球孢白僵菌的致死效果比绿僵菌好,前者喷药效果最佳的时期为成虫越冬前期;这个时期折枝内虫体校正死亡率为94.1%,僵虫率为75.0%,相对折枝率为48.9%,相对蛀孔率为49.2%。绿僵菌防治效果最佳时期是成虫羽化期;这个时期折枝内虫体校正死亡率为79.9%,僵虫率为83.3%,相对折枝率为71.0%,相对蛀孔率为54.2%。     

Variability in tolerance to UV-B radiation among Beauveria spp. isolates

Solar radiation, particularly the UV-B component, negatively affects survival of entomopathogenic fungi in the field. In an effort to identify Beauveria spp. isolates with promise for use in biological control settings with high insolation, we examined 53 Beauveria bassiana isolates, 7 isolates of 4 other Beauveria spp. and Engyodontium albus (=Beauveria alba). The origins of these fungi varied widely as to host/substrate and country, but approximately 30% of these isolates were B. bassiana from ticks in Brazil. A preliminary trial with three B. bassiana isolates (Bb 19, CG 310 and CG 481) at several UV-B dosages indicated that 2h of weighted UV-B irradiance at 978mWm(-2) (providing a total dose of 7.04kJm(-2)) allowed separation of isolates into low, medium or high UV-B tolerance. This dose, therefore, was selected as a single dose to compare UV-B tolerances of all 60 Beauveria spp. isolates. There was high variability in tolerance to UV-B radiation among the B. bassiana isolates, ranging from virtually zero tolerance (e.g., Bb 03) to almost 80% tolerance (e.g., CG 228). In addition, surviving B. bassiana conidia demonstrated delayed germination; and this is likely to reduce virulence. Conidia of the other species were markedly more sensitive to UV-B, with E. albus (UFPE 3138) being the least UV-B tolerant. Among B. bassiana isolates originating from 0 degrees to 22 degrees latitudes, those from lower latitudes demonstrated statistically significant greater UV-B tolerances than those isolates from higher latitudes. Isolates from above 22 degrees , however, were unaffected by latitude of origin. A similar analysis based on host type did not indicate a correlation between original host and UV-B tolerance. The identification in this study of several B. bassiana isolates with relatively high UV-B tolerance will guide the selection of isolates for future arthropod microbial control experiments.     

Effects of virulence,sporulation, and temperature on Metarhizium anisopliae and Beauveria bassiana laboratory transmission in Coptotermes formosanus

Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.     

Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana

Abstract A Beauveria bassiana extracellular subtilisin-like serine endoprotease is a potential virulence factor by virtue of its activity against insect cuticles. A cDNA clone of the protease was isolated from mycelia of B. bassiana grown on cuticle/chitin cultures. The amino acid sequence of this gene was compared to that of Metarhizium anisopliae Pr1, the only pathogenicity determinant so far described from an entomopathogenic fungus, and proteinase K, isolated from Tritirachium album , a saprophytic fungus. The cDNA sequence revealed that B. bassiana Prl is synthesized as a large precursor ( M _r 37 460) containing a signal peptide, a propeptide and the mature protein predicted to have an M _r of 26 832.     

Potential capacity of Beauveria bassiana and Metarhizium anisopliae in the biosorption of Cd2+ and Pb2+

In this study Beauveria bassiana and Metarhizium anisopliae were used as inexpensive and efficient biosorbents for Pb(II) and Cd(II) from aqueous metal solutions. The effects of various physicochemical factors on Pb(II) and Cd(II) biosorption by B. bassiana and M. anisopliae were studied. The optimum pH for Cd(II) and Pb(II) biosorption by two fungal species was achieved at pH 6.0 for Pb(II) and 5.0 Cd(II) at a constant time of 30 min. The nature of fungal biomass and metal ion interactions was evaluated by Fourier transform infrared. The maximum adsorption capacities (q(max)) calculated from Langmuir isotherms for Pb(II), and Cd(II) uptake by B. bassiana were 83.33±0.85, and 46.27±0.12 mg/g, respectively. However, the q(max) obtained for Pb(II) uptake by M. anisopliae was 66.66±0.28 mg/g, and 44.22±0.13 mg/g for Cd(II). B. bassiana showed higher adsorption capacity compared to M. anisopliae. The data obtained imply the potential role of B. bassiana and M. anisopliae for heavy metal removal from aqueous solutions.     

Improving UV resistance and virulence of Beauveria bassiana by genetic engineering with an exogenous tyrosinase gene

Insect pathogenic fungi like Beauveria bassiana have been developed as environmentally friendly biocontrol agents against arthropod pests. However, restrictive environmental factors, including solar ultraviolet (UV) radiation frequently lead to inconsistent field performance. To improve resistance to UV damage, we used Agrobacterium-mediated transformation to engineer B. bassiana with an exogenous tyrosinase gene. The results showed that the mitotically stable transformants produced larger amounts of yellowish pigments than the wild-type strain, and these imparted significantly increased UV-resistance. The virulence of the transgenic isolate was also significantly increased against the silkworm Bombyx mori and the mealworm Tenebrio molitor. This study demonstrated that genetic engineering of B. bassiana with a tyrosinase gene is an effective way to improve fungal tolerance against UV damage.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO4, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20-35 degrees C in both fungal species.