Anisopliae Strain Research Articles

Identification of double-stranded RNA viruses in Brazilian strains of Metarhizium anisopliae and their effects on fungal biology and virulence

Brazil is the leading country in the use of the entomopathogenic fungus Metarhizium anisopliae against agricultural pests. Very little information has been documented about the diversity and the impact of mycoviruses on M. anisopliae compared to M. robertsii and M. brunneum. This study reports the discovery of mycoviruses associated with native Brazilian Metarhizium spp. strains from the entomopathogen collection of the University of São Paulo, including strains used for mass-production of aerial conidia in sugar-alcohol factories to meet their own demand for controlling sugarcane spittlebugs and used in commercial mycoinsecticides. We also investigated the potential effects of viral infection on the biological traits of some of these strains. Viral-like double-stranded RNA (dsRNA) was found in 61% of the 36 Metarhizium spp. strains analyzed and showed a large variation of different electrophoretic banding patterns consisting of 2 to 8 dsRNA bands in polyacrylamide gels (PAGE). No dsRNA was detected in any of the strains from commercial products used in this study. Efforts to “cure” strains with dsRNA revealed that the inhibitor of protein synthesis cycloheximide was not efficient in eradicating dsRNA viruses from Metarhizium spp. strains. Some dsRNA segments of M. anisopliae ESALQ 26 were lost following hyphal tip subculturing. The dsRNA-variants of M. anisopliae strains ESALQ 866, ESALQ 1256 and ESALQ 1595 were healed after either monoconidial or hyphal tip subculturing. Single spore cultures of M. anisopliae ESALQ 1256 exhibited significant differences in vegetative growth, conidia production, and virulence against Tenebrio molitor larvae, although these differences were not linked with dsRNA. Moreover, fungal variants free of or positive for dsRNA had similar heat and UV tolerance responses. Our results suggest that viral-like dsRNA are very common among Brazilian M. anisopliae strains, but there is sparse evidence that these mycoviruses are harmful to fungal biological traits.

Frozen section and electron microscopy studies of the infection of the red palm weevil, Rhynchophorus ferrugineus (coleoptera:curculionidae) by the entomopathogenic fungus Metarhizium anisopliae.

This study determined the pathogenicity of Metarhizium anisopliae strain SD-3 against invasive red palm weevil (RPW), Rhynchophorus ferrugineus Olivier (coleoptera:curculionidae) larvae in Hainan Province, China. Inoculation of 1 × 108 conidia/mL caused 100 % mortality of R. ferrugineus, indicating that the conidia of strain SD-3 were highly virulent. The process of invasion mechanism was showed by scanning electron microscopy (SEM) and frozen section as follows. Once R. ferrugineus was infected by strain SD-3, M. anisopliae hyphae first invaded the cuticular and body cavity of R. ferrugineus. Secondly, well-developed muscles, fat, tracheaes and digestive tube tissues in the abdomen of R. ferrugineus were then decomposed and absorbed by M. anisopliae hyphae, leading to the total destruction of the larvae. Finally, M. anisopliae hyphae reproduced, resulting in a large number of conidia in the body of RPW. The SEM and frozen section are convenient tools to observe the mode of action of entomopathogenic fungi and to observe how M. anisopliae is able to colonize and infect the host.

The production relationship of destruxins and blastospores of Metarhizium anisopliae with virulence against Plutella xylostella

Metarhizium anisopliae as an essential entomopathogenic fungus has been known to produce destruxins (a kind of cyclo-peptidic mycotoxins) and blastospores in submerged culture. Blastospores and destruxins are candidates for insecticides, but the relations of both productions and the impact factors are unclear yet. In this study, we investigated the effects of inoculums, rotation, dissolved oxygen (DO) on the productions of blastospores and destruxins A and B (DA and DB) in submerged culture of M. anisopliae strain MaQ01. The results indicated that DO levels were regulated by inoculum amounts and rotation speeds, meanwhile, the productions of DA, DB and blastospores were also closely influenced by those factors. Totally, when DO value was more than 40%, the higher productions of destruxins and blastospores were achieved, by contrast, lower than 40% of DO values resulted in lower productions. The regression analysis suggested that the productions of DA, DB and blastospores were positively correlated with the DO levels. Meanwhile, the positive correlations between the productions of DA or DB and blastospores were also found. Briefly, when the rotation is 150 r min−1 and the inoculum is 1.0×106 spore mL−1, the DA, DB and blastospores achieved the best production of 61.81 mg mL−1, 24.74 mg mL−1 and 5.73×106 spore mL−1, respectively. In addition, the pathogenicities of blastospores and conidia against Plutella xylostella were bioassayed. The higher mortalities of P. xylostella were totally recorded in blastospore treatments than in conidia treatments, especially in lower dosages and earlier periods. Our research will give some new insights to production of destruxins and blastospores by using M. anisopliae.

Different Effects of Metarhizium anisopliae Strains IMI330189 and IBC200614 on Enzymes Activities and Hemocytes of Locusta migratoria L.

BackgroundMetarhizium is an important class of entomopathogenic fungi in the biocontrol of insects, but its virulence is affected by insect immunity. To clarify the mechanism in virulence of Metarhizium, we compared the immunological differences in Locusta migratoria L. when exposed to two strains of Metarhizium anisopliae (Ma).ResultsThe virulence of Ma IMI330189 was significantly higher than that of Ma IBC200614 to locust, and IMI330189 overcame the hemocytes and began destroying the hemocytes of locust at 72 h after spray, while locust is immune to IBC200614. IMI330189 could overcome the humoral immunity of locust by inhibiting the activities of phenol oxidase (PO), esterases, multi-function oxidases (MFOs) and acetylcholinesterases in locust while increasing the activities of glutathione-S-transferases (GSTs), catalase and aryl-acylamidase (AA). However IBC200614 inhibit the activities of GSTs and AA in locust and increase the activities of MFOs, PO, superoxide dismutase, peroxidase and chitinase in locust. The changes of enzymes activities in period of infection showed that the time period between the 2nd and the 5th day after spray is critical in the pathogenic process.ConclusionThese results found the phenomenon that Ma initiatively broke host hemocytes, revealed the correlation between the virulence of Ma and the changes of enzymes activities in host induced by Ma, and clarified the critical period in the infection of Ma. So, these results should provide guidance for the construction of efficient biocontrol Ma strains.

English

Entomopathogenic fungi considerably vary in their action and virulence mode. The contamination mainly depends on the adhesion and penetration of the fungus in the host integument. Four isolates from Beauveria bassiana and Metarhizium anisopliae were molecularly characterized by rDNA-ITS sequencing. Their virulence against last instar larvae of Galleria mellonella and Tenebrio molitor at four conidia concentrations: 1×106, 1×107, 1×108 and 1×109 conidia.ml-1 were in vitro evaluated and the lethal concentrations (LC50 and LC90) as lethal time (LT50 and LT90) were determined. Sequencing of rDNA-ITS 500 bp fragments allowed the identification of Ma10MI, Ma12MI, Ma58MI and Ma11MI isolates as M. anisopliae and Bb11MI, Bb79MI, Bb53MI and Bb27MI isolates as B. bassiana by sequence comparison to GenBank. According to the pathogenicity test, B. bassiana strains with higher performance for G. mellonella were Bb53MI and Bb79MI with LC50= 3.98×107 and 1.04×107 conidia.ml-1 and LT50= 5.46 and 5.27 days, respectively, as well as to T. molitor was Bb79MI with LC50= 1.03×107 conidia.ml-1 and LT50= 5.57 days. The M. anisopliae strains with the best performance were Ma58MI and Ma10MI both T. molitor (LC50= 1.0×106 and 1.0×107 conidia.ml-1 and LT50= 4.06 and 5.22 days, respectively) and G. mellonella (LC50= 6.3×107 and 1.1×108 conidia.ml-1 and LT50= 5.18 and 6.37 days, respectively), these isolates might be considered as new promising candidates for the microbial pest control.   Key words: Pathogenicity, molecular taxonomy, entomopathogenic fungi, microbial control.

Differential susceptibility of Hyalomma excavatum adults and nymphs to the entomopathogens Metarhizium anisopliae ARSEF 4556 and Steinernema carpocapsae

ABSTRACTThis study investigated the virulence of the entomopathogenic fungus, Metarhizium anisopliae strain ARSEF 4556, and the entomopathogenic nematode Steinernema carpocapsae against adult and nymphal stages of the tick Hyalomma anatolicum excavatum. The fungus was tested at three different concentrations, 106, 107, and 108 conidia ml−1. Ticks were exposed to 4000 IJs of S. carpocapsae in 0.5 ml distilled water in Petri dishes lined with 2 layers of moist filter paper. Adult ticks were not susceptible to M. anisopliae, whereas the nymphs were highly susceptible. Nymph mortality was dose dependent with all ticks being killed at the highest dose 96 hours post-inoculation. In contrast, S. carpocapsae was much more effective against engorged adults than unfed adults or nymphs. Tick susceptibility was not influenced by gender. The mean mortalities of the unfed adult males, females, and nymphs were 30% ± 9.2%, 40% ± 7.5%, and 36% ± 4.7%, respectively. Nymphal mortality was 36% and there was a significant difference between nematode treatment and control group (P < 0.05).

Effect of Three Entomopathogenic Fungi on Three Species of Stingless Bees (Hymenoptera: Apidae) Under Laboratory Conditions.

Development of alternative strategies for pest control with reduced effect on beneficial organisms is a priority given the increasing global loss of biodiversity. Biological control with entomopathogenic fungi arises as a viable option to control insect pests. However, few studies have focused on the consequences of using these organisms on pollinators other than the honey bee ( Apis mellifera L.) or bumble bees ( Bombus spp). We evaluated the pathogenicity of commercial formulations of three widely used entomopathogenic fungi, Metarhizium anisopliae (Metschnikoff) Sorokin, Beauveria bassiana Vuillemin, and Isaria fumosorosea (Wize), to three species of stingless bees: Tetragonisca angustula Latreille, Scaptotrigona mexicana Guérin-Meneville, and Melipona beecheii Bennett. Bioassays consisted of exposing groups of bees to the recommended field concentration of each fungus using a microspray tower under laboratory conditions. Susceptibility to fungi varied greatly among species . Isaria fumosorosea (strain Ifu-lu 01) and the two formulations of B. bassiana (Bea-TNK and BotanicGard) caused <30.3% mortality in all bee species. Metarhizium anisopliae (Meta-TNK and strain Ma-lu 01) was highly active against T. angustula (94.2% mortality) and moderately active against M. beecheii (53.0% mortality) and S. mexicana (38.9% mortality). Though our laboratory-derived results suggest a moderate to high impact of these entomopathogenic fungi on stingless bees, further field studies are required to support this finding.

Agar plate analysis of chitinase expression by pathogens of the Formosan subterranean termite,Coptotermes formosanus(Isoptera: rhinotermitidae)

Strains of the entomopathogens Isaria fumosorosea and Metarhizium anisopliae were grown on PdA plates containing chitin azure to determine whether chitinase expression could be correlated with their previously measured pathogenic effects against Formosan subterranean termites. Isaria strains 3581 and 4491 formed chitin azure conversion zones that were significantly larger, 40.0 mm on day 12, than the zone formed by strain 92118, 36.8 mm on day 12. However in past studies, strain 4491 was less pathogenic than strain 92118. the zones formed by M. anisopliae strain 30905 were significantly smaller than those formed by the Isaria strains, 7.0 mm on day 12, but the Metarhizium strain is known to cause mortality of termites. to determine if the glucose in PdA inhibited chitinase expression, the experiment was repeated on A&M plates supplemented with fructose instead of glucose. More variation was seen between strains 3581 and 4491, but the expression of chitinase was not enhanced.

Tracking coformulated strains of the entomopathogenic fungi Beauveria bassianaand Metarhizium anisopliae using SCAR markers

This article examines the development of strain-specific sequence-characterized amplified region (SCAR) molecular markers in two strains of Beauveria bassiana and Metarhizium anisopliae, as well as their use for tracking pathogens in coinfected insect pests. The markers were designed based on the polymorphic introns of the large subunit region of the ribosomal DNA. These markers were used to distinguish between two strains of Beauveria (B55 and B51) and two strains of Metarhizium (M20 and M48). The entomopathogenic strains demonstrated synergistic increase in mortality against Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae when infected with the coformulated M. anisopliae strain M20 + B. bassiana strain B55, particularly at a 2:1 proportion of LC50 concentration. The study revealed a disparity between intergeneric and interstrain coformulations. In intergeneric coformulations, one strain appeared predominant over the other strain at 1:2 and 1:4 proportions, both under in vivo and in vitro conditions. On the other hand, in interstrain coformulations, both strains survived and formed heterokaryons. Molecular studies revealed that the heterokaryons were unstable and reverted back to any one of the parent strains after 3 or 4 generations.

Enhancing purification of chitosanase from Metarhizium anisopliae by expanded bed adsorption chromatography using Doehlert design

Abstract The use of expanded bed adsorption with three different adsorbents Streamline (SP XL, DEAE and PHENYL) to purify the chitosanase from Metarhizium anisopliae strain with and without clarification using Doehlert design for the optimization of the experiments has been investigated. Chitosanases are enzymes that hydrolyze chitosan, a carbohydrate, resulting oligosaccharides that have many biological remarkable activities, such as anti-cancer, anti-HIV and anti-oxidants activities. The superficial velocity was evaluated in five levels, from 40 to 260 cm h−1, while the type of adsorbent, a qualitative variable, was assessed at three levels, saving materials and time according to the Doehlert design. The results showed that it is possible to purify chitosanase from M. anisopliae by expanded bed adsorption. Chitosanase purification factor ranged from 2 to 3.1-fold and yield ranged from 27.7 to 45.3%. The highest enzyme activity in the elution was 0.06 U/mL values which were higher than or close to those reported. The molecular mass of chitosanase was about 45 kDa using SDS-PAGE.

Effect of Beauveria bassiana and Metarhizium anisopliae on the Trialeurodes vaporariorum-Encarsia formosa system

The virulence of three strains of Beauveria bassiana and one of Metarhizium anisopliae was tested against Trialeurodes vaporariorum and its parasitoid Encarsia formosa in laboratory assays. These strains were previously selected for their virulence against Galleria mellonella and Tenebrio molitor. The commercial B. bassiana strain ATCC 74040, both as pure fungal culture and formulated myco-insecticide (Naturalis), was included in the assays as positive control. First, the entomopathogenic fungal strains were tested for their virulence against T. vaporariorum nymphs on tomato leaf disks. Then, the E. formosa development was evaluated under treatment with the entomopathogenic fungal strains at five different time points from the parasitization of T. vaporariorum nymphs. The virulence of our entomopathogenic fungal strains was superior to that of ATCC 74040, although not significantly, resulting in a cumulative mortality (CM) of T. vaporariorum nymphs 7 days after inoculation (DAI) greater than 86 %. Our M. anisopliae strain CIST8 was the most effective (96.6 % CM 7 DAI), even superior to the myco-insecticide Naturalis (94.2 %), which was more effective than the ATCC 74040 pure strain (85.6 %). The entomopathogenic fungal strains, and especially Naturalis, negatively affected E. formosa development and its parasitization activity of T. vaporariorum nymphs. This effect was more pronounced when the fungal strains were applied before parasitization. Results suggest that the application of entomopathogenic fungi is incompatible with E. formosa release on crops.

Assessment of Metarhizium anisopliae (Clavicipitaceae) and its vector, Scleroderma guani (Hymenoptera: Bethylidae), for the control of Monochamus alternatus (Coleoptera: Cerambycidae)

AbstractThe pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae), is a major disease of pines forests in China. Its vector, Monochamus alternatus Hope (Coleoptera: Cerambycidae), has been the focus of an intensive study to improve the efficiency of the parasitoid Scleroderma guani Xiao and Wu (Hymenoptera: Bethylidae) by using the parasitoid both as a larval parasitoid in its own right and additionally as a vector for the entomophagous pathogen Metarhizium anisopliae (Metchnikoff) Sorokin (Clavicipitaceae). Twenty-one M. anisopliae strains were screened and the best four tested for sporulation capabilities at a range of temperatures. The best strain was then evaluated at several sporulation concentrations to define the lethal concentration 50 (LC50) against the larvae of M. alternatus. Scleroderma guani was inoculated with the best strain and tested against larvae of M. alternatus, which were constrained singly in glass vials. Three S. guani females per vial infected with M. anisopliae (strain 789) were shown to cause the death of 100% of the M. alternatus larvae they were exposed to as compared exposures to just one or two infected S. guani females per vial. This demonstrates a successful interaction of two biocontrol agents for the control of the pinewood nematode vector M. alternatus.

Integrated control of an acaricide-resistant strain of the cattle tick Rhipicephalus microplus by applying Metarhizium anisopliae associated with cypermethrin and chlorpyriphos under field conditions

The efficacy of the fungus Metarhizium anisopliae to control ticks has been shown in several in vitro experiments. However, few studies have been undertaken in field conditions in order to demonstrate the applicability of its use as a biological control of ticks and its combination with chemical acaricides. The aim of the present study was to evaluate the efficacy of M. anisopliae to control an acaricide-resistant strain of Rhipicephalus microplus under laboratory and field conditions. First, the compatibility of M. anisopliae strain (TIS-BR03) with commercial acaricides and its potential to control the cattle tick were evaluated in vitro. In general, acaricide treatments had mild effects on fungus viability. In the field experiment, the median of treatment efficacy with acaricide only, M. anisopliae only and combination of M. anisopliae with acaricide were 71.1%, 56.3% and 97.9%, respectively. There is no statistical difference between groups treated with M. anisopliae and acaricide alone. Thus, in this work we have demonstrated the applicability of M. anisopliae use associated or not with chemical acaricides on field conditions in order to control an acaricide-resistant strain of the cattle tick R. microplus.

Diversity of glycosil hydrolase family 18 genes in environment isolates of the entomopathogenic fungus Metarhizium anisopliae

Backgound Metarhizium anisopliae is a model for host-pathogen studies due to its ability to infect several different arthropods. The first barrier to accomplish successful host-infection is transverse the host cuticle, which is a rigid chitin-rich structure. To surpass this barrier, the fungus produces several hydrolytic enzymes, among which are chitinases and endo-b-N-acetylglucosaminidases, glycosil hydrolase 18 (GH18) members [1,2]. In fungi, GH18 enzymes have nutritional importance and exhibit morphogenic and autolytic functions, acting at different processes of fungal development and life cycle maintenance. Assigning functional role for these genes in each process is one of the goals in entomopathogenic fungi study [3]. A genomic analysis performed in our laboratory, in M. anisopliae E6 strain, identified twentythree GH18 putative genes [3-5]. Considering this variety, this study aims to evaluate the diversity of these genes amongst M. anisopliae strains, to access their distribution in environmental isolates of the fungus. DNA samples from 23 M. anisopliae strains (CG291, NORDESTE, CARO7, CG125, CG343, CG374, CG46, CARO12, CARO14, CARO19, CG30, CG97, CG320, AL, MT, M5, CARO11, CARO15, CARO16, CG47, PL57, CG87 e CG491) isolates from different arthropods and places were subjected to PCR analysis to evaluate the presence of each of the 23 GH18 putative genes found in the genome of strain E6. Methods All strains were grown on Cove’s Complete Medium (MCc) agar plates at 28°C until sporulation. Spores were harvested with 0.01% Tween solution and inoculated into liquid MCc cultured in a rotatory shaker at 28oC, 180 rpm for 48 hours. After growth, DNA was extracted using lysis solution and phenol/chloroform method. The presence of GH18 genes was detected by PCR, using primers designed for M. anisopliae strain E6 genes.

Management of Bactericera cockerelli (Sulc) (Hemiptera: Triozidae) in Greenhouses with Entomopathogenic Fungi (Hypocreales)

Research on entomopathogenic fungi has been conducted to control the potato psyllid, Bactericera cockerelli, to find new strategies and because of its mode of action, that, unlike other entomopathogenic microorganisms, does not require being ingested. Two Beauveria bassiana strains, a wild type (BB42) and a commercial (BB09), and two Metarhizium anisopliae strains (MA25 and MA28) were used at 1×107 spores ml-1, on B. cockerelli nymphs on chili peppers under greenhouse conditions. Three applications (at 1, 3, and 5 days) killed 36% (MA25) to 47% (BB09), achieving more control than imidacloprid.

Correlation between Pr1 and Pr2 gene content and virulence in Metarhizium anisopliae strains.

Metarhizium anisopliae is a widely studied model to understand the virulence factors that participate in pathogenicity. Proteases such as subtilisin-like enzymes (Pr1) and trypsin-like enzymes (Pr2) are considered important factors for insect cuticle degradation. In four M. anisopliae strains (798, 6342, 6345, and 6347), the presence of pr1 and pr2 genes, as well as the enzymatic activity of these genes, was correlated with their virulence against two different insect pests. The 11 pr1 genes (A, B, C, D, E, F, G, H, I, J, and K) and pr2 gene were found in all strains. The activity of individual Pr1 and Pr2 proteases exhibited variation in time (24, 48, 72, and 96 h) and in the presence or absence of chitin as the inductor. The highest Pr1 enzymatic activity was shown by strain 798 at 48 h with chitin. The highest Pr2 enzymatic activity was exhibited by the 6342 and 6347 strains, both grown with chitin at 24 and 48 h, respectively. Highest mortality on S. exigua was caused by strain 6342 at 48 h, and strains 6342, 6345, and 6347 caused the highest mortality 7 days later. Mortality on Prosapia reached 30% without variation. The presence of subtilisin and trypsin genes and the activity of these proteases in M. anisopliae strains cannot be associated with the virulence against the two insect pests. Probably, subtilisin and trypsin enzyme production is not a vital factor for pathogenicity, but its contribution is important to the pathogenicity process.

Growth and sporulation of entomopathogenic Beauveria bassiana, Metarhizium anisopliae, Isaria farinosa and Isaria fumosorosea strains in relation to water activity and temperature interactions

This study examined six strains of Beauveria bassiana s.l. and Isaria farinosa, one strain of Isaria fumosorosea and five strains of Metarhizium anisopliae s.l. to identify the ability for (1) growth and (2) sporulation under interacting environmental factors of water activity (aw) and temperature stress. Growth on Sabouraud Dextrose Agar (SDA; water activity, aw = 0.995) or SDA modified with glycerol to 0.98, 0.96 and 0.94 aw was measured at four different temperatures (25, 30, 35 and 37°C). All M. anisopliae strains grew at 25–35°C and 0.995 aw while only two strains tolerated extreme water stress at 0.94 aw.Three strains of B. bassiana were able to grow at 25–37°C and 0.995 aw. Only one strain of I. farinosa was able to grow at 25–37°C and 0.995 aw. Aw and temperature interactions resulted in different strain-dependent responses, in terms of growth and sporulation. Only one strain of I. farinosa and three of M. anisopliae grew at 0.94 aw and none of the B. bassiana strains tolerated such water stress. At 0.96 and 0.94 aw and 35–37°C, sporulation by all the strains of the three species were significantly affected. Under elevated temperatures and drought stress, very few of these strains of entomopathogenic fungi are able to grow and sporulate. Indeed, the B. bassiana strains were unable to tolerate the extreme conditions examined. Resilience to such abiotic interactions is critical for selecting strains for formulations. Tolerance to water and temperature stress could be good criteria for selection of strains with secondary spread potential for use as part of an integrated pest management system where secondary cycling may be important, especially in sub-tropical and tropical environments.

Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae

Conidial spores are often used as the infectious agent during insect biocontrol applications of entomopathogenic fungi. Here we show differential virulence of conidia derived from Metarhizium anisopliae strain EAMa 01/58-Su depending upon the solid substrata used for cultivation, where LC50 values differed by up to ~10-fold (5.3×106−4.5×105 conidia/ml) and LT50 values by ~40% (9.8−7.1 d). This fungal strain is also known to secrete proteins that are toxic towards adult Mediterranean fruit flies, Ceratitis capitata, and the Greater wax moth, Galleria mellonella, larvae. In vitro production and intrahemoceol injection using G. mellonella as the host was used to test fractions during purification of the protein toxins, demonstrating that they elicited defence-related responses including melanisation and tissue necrosis. Production of these proteins/peptides along with a number of potential cuticle degrading enzymes was confirmed both in vitro and during the infection process (in vivo). Two-dimensional gel electrophoresis, followed by gel elution and bioassay, was used to identify at least three proteins or peptides (molecular mass=11, 15 and 15 kDa) as mediating the observed insect toxicity. These data demonstrate that in vitro screening for insect toxins can mimic in vivo (i.e. during the infection process) secretion and applies the use of proteomics to invertebrate pathology.

Evaluation of Biocontrol Potential of Metarhizium anisopliae Strains Against Larvae and Adults of Aedes eegypti (l.)

In the present study, we exposed larvae and adult females of Aedes aegypti (L.) to fungal suspensions of three virulent strains of Metarhizium anisopliae (Metsch.), viz., M34412, M34311 and M81123. The strains resulted in 7 to 37% survival as a result of fungal infection over the 8 d test period in case of adults and 2 to 90% survival for larvae. Mean survival period varied between 3 and 4 d for treated adults, whilst control survival exceeded 28 d. Values of LC 50 varied from 5.92×10 3 conidia/ml for M34412, 3.49×10 4 conidia/ml for M34311 and 5.12×10 5 conidia/ml for M81123. Based on virulence and stability, the most promising strain, M. anisopliae M34412 was used for lethal exposure time determination. An exposure time of only 4 h was necessary to cause 50% mortality. Supplementation with M. verrucaria chitinase enzyme increased the larval mortality at lower conidial concentrations.

Proteins differentially expressed in conidia and mycelia of the entomopathogenic fungusMetarhizium anisopliaesensu stricto

Metarhizium anisopliae is a well-characterized entomopathogenic fungus that attacks a variety of insects. Its conidia are involved in its propagation and also in its infection of host insects. To investigate the protein expression profiles and to identify the proteins related to development and pathogenesis, we performed a comparative proteomic analysis of the conidia and mycelia of an M. anisopliae strain (Ma1291). The analysis used 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We detected 898 ± 37 protein spots in conidia and 1072 ± 24 in mycelia of strain Ma1291. A comparison of the 2 protein-expression profiles indicated that only 28% of protein spots were common to both developmental stages. Finally, we identified 30 proteins (19 from conidia and 11 from mycelia). The identified proteins exclusive to conidia were those involved in protective processes, appressorium formation, and degradation of the host cuticle (protease PR1H). The identified proteins exclusive to mycelia included major proteins participating in biosynthetic and energy metabolism, such as UTP-glucose-1-phosphate uridylyltransferase and heat shock protein 70. This research provides the first proteomic analysis of different developmental stages of M. anisopliae, and the results should facilitate clarification of the molecular basis of these epigenetic variations.