Production in vitro of toxic macromolecules by strains of Beauveria bassiana, and purification of a chitosanase-like protein secreted by a melanizing isolate

The production of macromolecular insecticidal toxins in Adamek's medium by two selected strains of Beauveria bassiana was investigated. Filtrates and dialysates of the melanizing strain 618 were toxic when injected into the lepidopteran insect Galleria mellonella. Separation by DEAE chromatography revealed that peaks eluted respectively with 100 and 200 mM NaCl (P 100 and P 200) had an insecticidal activity and induced cuticular blackening. A hydrophilic protein, Bclp, which causes the formation of brownish spots of the integument, was purified from P 200 by means of chromatographic and electrophoretic methods. Bclp exhibited clear sequence homologies with fungal chitosanases of Fusarium solani. It has a molecular mass of 28 kDa, a pHI of 4 and is thermolabile. Injection of Bclp causes the same cytoxic effects and alterations of the cuticule as those observed during mycosis, and may contribute to the virulence of strain 618. Comparatively, the most obvious characteristic of the weakly melanizing strain 101 is the lack of significant toxic activity of its P 200, which does not contain Bclp. However, this strain secretes other insecticidal molecules active on lepidopterans, presently unidentified.     

Inhibition of Beauveria bassiana Proteases and Fungal Development by Inducible Protease Inhibitors in the Haemolymph of Galleria mellonella Larvae

Injection of zymosan or dead yeast cells enhanced the inhibitory activity against exocellular Beauveria bassiana proteases in the cell - free haemolymph of Galleria mellonella larvae . Pre - injected larvae exhibited no decreased mortality after subsequent injection with living B. bassiana blastospores but survived for a prolonged time before death . Increased levels of protease inhibitors in the haemolymph were also observed after injection of B. bassiana proteases . In contrast , no enhanced inhibitory activity against B. bassiana proteases was detected in infected larvae when mycosis was initiated with conidia which enabled the fungus to invade host larvae through the integument in a natural manner . B. bassiana proteases were not completely inhibited by the addition of cell - free haemolymph . Protease inhibitors obtained after heat and trichloroacetic acid precipitation of cell - free haemolymph were added to the protein medium of B. bassiana to study the effect on its growth in vitro. Enriched fractions from pre - injected larvae delayed fungal growth in comparison with fractions from untreated larvae , suggesting that delayed mortality of immunized G. mellonella larvae infected with B. bassiana is due to enhanced levels of protease inhibitors . A non - virulent form of the same strain exhibited reduced capacity to release proteases in vitro. The results strongly suggest that the capacity of insects to release inhibitors against fungal proteases influences their susceptibility against entomopathogenic fungi .     

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.     

Infection of the immature stages of Diadegma semiclausum, an endolarval parasitoid of the diamondback moth, by Beauveria bassiana

Interactions between the immature stages of Diadegma semiclausum, an endolarval parasitoid of Plutella xylostella, and the fungal entomopathogen Beauveria bassiana were investigated in the laboratory. Detrimental effects of B. bassiana on D. semiclausum cocoon production and adult parasitoid emergence increased with increasing pathogen concentration and some parasitoid larvae became infected by B. bassiana within hosts. The negative impact of B. bassiana on D. semiclausum cocoon production decreased as temporal separation between parasitism and pathogen exposure increased. Adult parasitoid emergence was significantly compromised by the highest rates of B. bassiana tested even when exposure of host larvae to the pathogen was delayed until one day before predicted parasitoid cocoon formation. Parasitoid pupae were infected by the pathogen in all B. bassiana treatments which did not preclude their development.     

Effect of exposure method to Beauveria bassiana and conidia concentration on mortality, mycosis, and sporulation in cadavers of Chilo partellus (Lepidoptera: Pyralidae)

The effects of exposure methods, conidial concentrations, and temperature on mortality, mycosis and sporulation in second instar Chilo partellus cadavers infected by Beauveria bassiana was investigated in laboratory studies. Larvae directly sprayed with conidia, exposed to conidia-treated leaves, and dipped into conidial suspension resulted in high mortality (98-100%). The longest LT50 (3.5 days) and days to mortality (2.6 days) were observed in the treated-leaves exposure method. The shortest LT50 (1 day) and days to mortality (1 day) were recorded for the dipping method. With increasing conidial concentrations, there were decreasing LT50 and days to mortality. Larvae exposed to treated leaves and larvae directly sprayed with conidial suspensions produced high mycoses in cadavers. Exposure of larvae to treated-leaves resulted in high sporulation. At lower concentrations of conidia, both mycosis and sporulation in cadavers were high. The optimum temperature for mycosis was 20 and 15 degrees C for sporulation.     

Selection of a Highly Virulent Fungal Isolate,Metarhizium anisopliae Ma TB 101, for Control of Taro Beetle,Papuana uninodis (Coleoptera: Scarabaeidae)

Fungal isolates (31 Metarhizium anisopliae var. anisopliae , five M. anisopliae var majus , three Beauveria bassiana and four B. brongniartii ) originating from a wide range of geographical locations, insect species and soil types were tested against Papuana uninodis (Coleoptera: Scarabaeidae). In a first test series, spores were applied topically to third-instar larvae and adults. The most effective strain against P. uninodis larvae and adults was Ma TB 101, a M. anisopliae isolate from adult P. woodlarkiana . For adults, strain Ma FI 384, a M. anisopliae from Popillia japonica , was almost equally effective. The 11 most effective isolates (nine M. anisopliae var. anisopliae , one B. brongniartii and one B. bassiana ) with LT values of less than 6 weeks in 50 adults and/or less than 4 weeks in larvae were tested for their efficacy against adults and larvae of P. uninodis by application of spores to soil (107 spores/g). Ma TB 101 was significantly more effective against both adults and larvae (LT ca. 10 days) than all other isolates (LT > 50 50 3 weeks). Two other M. anisopliae strains, Ma F 248 from soil and Ma FI 384 from P.japonica , were more effective than most isolates in adults. The latter three M. anisopliae isolates were tested in a concentration series against third-instar larvae and adults. Mortality was concentration related. Isolates Ma F 248 and Ma FI 384 did not achieve 50% mortality within the test period at concentrations lower than 107 spores/g of soil or feed. For concentrations of Ma TB 101 from 1 107 to 2 105 spores/g the LT ranged from 13 to 30 days in adults and 12 to 24 50 days in third-instar larvae. Accordingly, concentrations causing 50% mortality (LC ) for Ma 50 TB 101 were significantly lower than for the two other M. anisopliae isolates tested.     

Selection of entomopathogenic fungi for the control of the western corn rootworm Diabrotica virgifera virgifera

Abstract:  The western corn rootworm Diabrotica virgifera virgifera Le Conte (Col., Chrysomelidae), a serious pest of maize, has been recently introduced into Europe. Several approaches for its control are presently under investigation including microbial agents. During a field survey in Hungary in 2005, naturally occurring entomopathogenic fungi were found to attack this pest. These novel isolates together with standard isolates were tested for virulence against D. v. virgifera larvae and adults. Twenty strains of Metarhizium anisopliae , Beauveria bassiana and Beauveria brongniartii were used in bioassays in the laboratory. Larvae and adults were dipped into a spore suspension with a concentration of 1 × 10⁷ conidia (con.)/ml. They were kept for 14 days at 22°C (±2°C) and 70% relative humidity. The number of infected larvae and adults were counted and infection rates were calculated. Adults were significantly more susceptible to entomopathogenic fungi than larvae. The most virulent isolate infected about 47% of larvae ( M. anisopliae Ma2277), whereas the infection rate in adults was up to 97% ( M. anisopliae Ma2275). Isolates of M. anisopliae caused significantly higher mortalities than isolates of B. brongniartii and B. bassiana . Most of the adult beetles were killed within 12 days. Isolates from D. v. virgifera were more virulent than those from other hosts.     

Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker

Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice. To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B. bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B. bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations. With this highly efficient transformation method for B. bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence. Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B. bassiana.     

马尾松林中球孢白僵菌寄主转移和专化性的SSR标记分析

球孢白僵菌是一种最常见的虫生真菌,已被广泛应用于害虫生物防治。利用SSR简单序列重复(simple sequence repeat,SSR)分子标记对来自安徽省麻姑山马尾松林的102株球孢白僵菌进行基因分型、寄主转移和寄主专化性研究。9对微卫星引物将102株白僵菌分成31个微卫星基因型。在这31个微卫星基因型中,有5种基因型株系为相对优势菌株,这5种基因型株系通过侵染不同寄主昆虫即寄主转移延续自身在马尾松林生态系统中的传播和流行,从而证实寄主转移是白僵菌群体中的普遍现象。同时,这些相对优势基因型并不是在各个月份均匀分布,在大部分月份中,存在1–2种优势度高的基因型。相同基因型株系可侵染不同寄主的结果揭示出白僵菌不仅在种的水平,而且在菌株水平上寄主专化性也较弱。正是白僵菌较弱的寄主专化性特征促使部分基因型株系通过寄主转移,在马尾松林生态系统中得以宿存和延续。     

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strain's virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.     

Adult polyphagous coleopterans overwintering in cereal boundaries: winter mortality and susceptibility to the entomopathogenic fungus Beauveria bassiana

Autumn-collected Bembidion lampros and Agonum dorsale were kept outdoors under semi field conditions to determine winter mortality. On three occasions (autumn, mid-winter and late winter) sub-samples of the population were incubated in the laboratory at room temperature and mortality was recorded. Generally the mortality was low in autumn and mid-winter, not exceeding 5%. A more pronounced post-winter mortality of up to 30% was observed at the end of hibernation. Only a negligible number were infected by the entomopathogenic fungi Beauveria bassiana and Paecilomyces farinosus. Infection experiments with B. bassiana isolates from Sitona lineatus, Tachyporus hypnorum and three species of ground beetles showed a similarly low susceptibility of B. lampros and A. dorsale to B. bassiana and, although Tachyporus spp. showed a distinct mycophagy and a significantly higher mortality, this could not be related to mycosis.     

A Beauveria phylogeny inferred from nuclear ITS and EF1-alpha sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs

Beauveria is a globally distributed genus of soil-borne entomopathogenic hyphomycetes of interest as a model system for the study of entomopathogenesis and the biological control of pest insects. Species recognition in Beauveria is difficult due to a lack of taxonomically informative morphology. This has impeded assessment of species diversity in this genus and investigation of their natural history. A gene-genealogical approach was used to investigate molecular phylogenetic diversity of Beauveria and several presumptively related Cordyceps species. Analyses were based on nuclear ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-alpha) sequences for 86 exemplar isolates from diverse geographic origins, habitats and insect hosts. Phylogenetic trees were inferred using maximum parsimony and Bayesian likelihood methods. Six well supported clades within Beauveria, provisionally designated A-F, were resolved in the EF1-alpha and combined gene phylogenies. Beauveria bassiana, a ubiquitous species that is characterized morphologically by globose to subglobose conidia, was determined to be non-monophyletic and consists of two unrelated lineages, clades A and C. Clade A is globally distributed and includes the Asian teleomorph Cordyceps staphylinidaecola and its probable synonym C. bassiana. All isolates contained in Clade C are anamorphic and originate from Europe and North America. Clade B includes isolates of B. brongniartii, a Eurasian species complex characterized by ellipsoidal conidia. Clade D includes B. caledonica and B. vermiconia, which produce cylindrical and comma-shaped conidia, respectively. Clade E, from Asia, includes Beauveria anamorphs and a Cordyceps teleomorph that both produce ellipsoidal conidia. Clade F, the basal branch in the Beauveria phylogeny includes the South American species B. amorpha, which produces cylindrical conidia. Lineage diversity detected within clades A, B and C suggests that prevailing morphological species concepts underestimate species diversity within these groups. Continental endemism of lineages in B. bassiana s.l. (clades A and C) indicates that isolation by distance has been an important factor in the evolutionary diversification of these clades. Permutation tests indicate that host association is essentially random in both B. bassiana s.l. clades A and C, supporting past assumptions that this species is not host specific. In contrast, isolates in clades B and D occurred primarily on coleopteran hosts, although sampling in these clades was insufficient to assess host affliation at lower taxonomic ranks. The phylogenetic placement of Cordyceps staphylinidaecola/bassiana, and C. scarabaeicola within Beauveria corroborates prior reports of these anamorph-teleomorph connections. These results establish a phylogenetic framework for further taxonomic, phylogenetic and comparative biological investigations of Beauveria and their corresponding Cordyceps teleomorphs.     

Effect of Beauveria bassiana and Metarhizium anisopliae (Deuteromycetes) upon the coffee berry borer (Coleoptera: Scolytidae) under field conditions

The effect of three strains of the fungus Beauveria bassiana (Balsamo) Vuillemin and two strains of Metarhizium anisopliae (Metschnikoff) Sorokin upon the coffee berry borer, Hypothenemus hampei (Ferrari), was studied in three coffee farms at different altitudes (450-1,100 m above sea level) in Soconusco, Chiapas, Mexico. The maximum average percentage mycosis varied according to altitude. At 450 m asl (El Rincon) mycosis was 14.3% for B. bassiana and 6.3% for M. anisopliae; at 880 m asl (Santa Anita) mycosis was 40.6% for B. bassiana and 12.6% for M. anisopliae, and at 1,100 m asl (Alpujarras) 33.9% for B. bassiana and 22. 1% for M. anisopliae. The effect of fungal mycosis through time was not significant (P > 0.01) in any of the farms, but there was a significant difference between the strains of the fungus (P < 0.01); the best strains being Bb25 and Ma4 at the lower altitude, Bb26 and Ma4 for the middle altitude and Bb26 and Ma4 at the higher altitude. Environmental factors such as temperature, relative humidity and rain were not correlated with the percentage mycosis caused by B. bassiana and M. anisopliae. However, in the case of B. bassiana there was a significant, positive correlation (P < 0.01) between the infestation levels of the pest and the mycosis response of the entomopathogen.     

Effect of temperature and poultry litter in Beauveria bassiana (bals.) Vuill. and Metarhizium anisopliae (Metsch) virulence against the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

This study was carried out to evaluate the influence of temperature and poultry litter on germination vegetative growth virulence and conidial production of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates on larvae and adults of the lesser mealworm (Alphitobius diaperinus) (Panzer). The vegetative growth and conidial production were evaluated on culture media. Virulence was studied submerging larvae and adults in a conidial suspension (1 x 10(8) conidia/ml). All the experiments were carried out in growth chamber (26 degrees C and 32 degrees C and 14h photophase). Fungus-killed insects were daily collected and used for microscopic conidial counts. The poultry litter effect was evaluated by submerging the insects in a fungal suspension (10(8) conidia/ml) and then transferring them to cups containing poultry litter (new and used). B. bassiana isolates were more sensitive than M. anisopliae to high temperature because conidia viability, vegetative growth and virulence were negatively affected (P < 0.05). The conidial production was higher to B. bassiana in 26 degrees C (7 to 11 x 10(8) conidia/larval cadaver and 8 x 10(8) conidia/adult cadaver) (P < 0,05). Larval stage was about 10 times more sensitive to M. anisopliae at 26 degrees C than adults stage. Regarding B. bassiana, differences on sensitivity between larval stages and adults were not observed at this temperature. However, at 32 degrees C, larval stage was more sensitive for CB116 and UEL50 isolates. Mortality was higher when larvae and adults (15.7 and 66.7% respectively) were treated by B. bassiana and maintained on new poultry litter at 26 degrees C) (P < 0.05).     

Bursicon-mediated control of tanning in melanizing and non-melanizing first instar larvae of Schistocerca gregaria

Both melanization and sclerotization in first instar Schistocerca larvae are shown to have a common hormonal control mechanism. This hormone is shown to be analogous to bursicon, the tanning hormone of other insects, by the standard blowfly bioassay. The hormone is identical in both melanizing and non-melanizing first instar larvae of Schistocerca.     

Susceptibility of a native and an exotic lady beetle (Coleoptera: Coccinellidae) to Beauveria bassiana

The exotic multicolored Asian lady beetle, Harmonia axyridis, became established and recently spread across much of North America and southern Canada. In a habitat now used by both the invading H. axyridis and a native lady beetle, Olla v-nigrum, we discovered that the native lady beetle was commonly infected by Beauveria bassiana; whereas, the exotic H. axyridis, was not. Laboratory assays revealed that B. bassiana isolates collected from naturally infected O. v-nigrum were pathogenic to adult O. v-nigrum but not to adult H. axyridis. In contrast, the GHA strain of B. bassiana was not significantly pathogenic to O. v-nigrum nor H. axyridis. Late-season field collections revealed significantly higher B. bassiana infection of O. v-nigrum than H. axyridis. Our results lead us to hypothesize that low susceptibility of H. axyridis to B. bassiana (found to infect O. v-nigrum) may provide an intraguild advantage to H. axyridis over O. v-nigrum; this may also occur with other species of native lady beetles and other endemic entomopathogens in different habitats and regions.     

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.     

Differential Susceptibility of Two Melolontha Populations to Infections by the Fungus Beauveria brongniartii

The susceptibility of second-instar larvae of the European cockchafer, Melolontha melolontha L., originating from two different geographical locations was investigated with eight isolates of the insect pathogenic fungus Beauveria brongniartii (Sacc.) Petch. 77-94% of larvae from northern Italy (Auer, South Tyrolia) succumbed to mycosis with an average survival time (AST) of 22.9-40.1 days. Infections in larvae from south-western Switzerland (Bramois, Valais) were 28-72% and the ASTs varied between 34.7-56.4 days. Differences in susceptibility between the two host populations may be explained by the historical ages of the two populations and the presence of B. brongniartii resulting in a coevolution of tolerance between the host and pathogen. The Italian population is occasionally infected by B. brongniartii; in comparison, the Swiss population has existed for at least 50 years and regular infections by the pathogen are observed. Coevolution between B. brongniartii and M. melolontha from Switzerland may explain the apparent resistance of the host towards this pathogen in laboratory assays.     

Avoidance of a generalist entomopathogenic fungus by the ladybird, Coccinella septempunctata

Fungal entomopathogens are ubiquitous within the environment and susceptible insects are predicted to be under strong selection pressure to detect and avoid virulent isolates. Beauveria bassiana is an entomopathogenic fungus with a wide host range including coccinellids. Seven-spot ladybirds, Coccinella septempunctata, overwinter predominantly in leaf litter and B. bassiana is one of their major mortality factors during winter in temperate regions. Behavioural assays were conducted to assess the ability of adult C. septempunctata to avoid lethal densities of B. bassiana conidia in soil or on leaves. Further assays considered avoidance by C. septempunctata of mycosed (B. bassiana) C. septempunctata cadavers compared with uninfected C. septempunctata cadavers or in vitro B. bassiana. Treatments in any bioassays entirely avoided by C. septempunctata were regarded as censored data, to overcome the difficulties associated with zeros in log-ratio analyses. Male and female C. septempunctata avoided contact with leaf surfaces and soil inoculated with B. bassiana and mycosed cadavers. The ability of C. septempunctata to detect and avoid B. bassiana conidia is an adaptation that undoubtedly increases survival and ultimately fitness. We predict that such behavioural responses are widespread and driven by the high cost of fungal infection to a host.