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1.
Metarhizium anisopliae infects insects and ticks via a combination of specialized structures and cuticle degradation. Hydrolytic enzymes are accepted as key factors for the penetration step. The search for pathogenicity determinants has demonstrated that the process is multifactorial. Host specificity is an important factor to be addressed. The study of the enzymes produced during infection is important to discover those with a role in the process. To address some of the enzymes that take part during the infection of the tick, Boophilus microplus, we have analyzed the secretion of proteases and chitinases in single and combined carbon/nitrogen sources as compared with such complex substrates as chitin and B. microplus cuticles. Two chitinases, endo- and N-acetylglucosaminidases, and two proteases, subtilisin and trypsin-like proteases, were analyzed. Enzyme activities were detected in all carbon sources tested, but higher levels were found when combinations of carbon sources were used. A major 30-kDa protein apparently secreted during M. anisopliae growth on all carbon/nitrogen sources tested was demonstrated by SDS-PAGE.  相似文献   

2.
Beauveria bassiana is a well-known broad-range arthropod pathogen which has been used in biological control of several pest insects and ticks such as Boophilus microplus. Beauveria amorpha has both endophytic and entomopathogenic characteristics, but its capacity for biological control has still not been studied. During the processes of host infection, B. bassiana and B. amorpha produce several hydrolytic extracellular enzymes, including proteases and chitinases, which probably degrade the host cuticle and are suggested to be pathogenicity determinants. To access the role of these enzymes during infection in the tick B. microplus, we analyzed their secretion during fungus growth in single and combined carbon sources, compared to complex substrates such as chitin and B. microplus cuticle. Chitin and tick cuticle-induced chitinase in both fungus and protease was induced only by tick cuticle. SEM analysis of B. amorpha and B. bassiana infecting B. microplus showed apressorium formation during penetration on cattle tick cuticle.  相似文献   

3.
For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.  相似文献   

4.
The carbon and nitrogen source requirements of Aspergillus fumigatus NRRL 2436 for growth and production of the angiogenesis inhibitor fumagillin were studied in chemically defined media. Both carbon and nitrogen sources strongly influenced fumagillin formation. Two out of 29 carbon sources tested interfered with fumagillin biosynthesis. The best combination of two carbon sources was 30 g L−1 xylan and 50 g L−1 mannose. Of fifteen nitrogen sources tested, three ammonium salts (chloride, sulfate, and dibasic phosphate) failed to support fumagillin formation, presumably due to the low pH which developed. The dosage-response study of the best nitrogen source, L-glutamic acid, revealed that 9 g L−1 was optimal. Volumetric production of fumagillin was increased by 15-fold over that in the starting (Peterson-Goldstein) medium as a result of these findings. Received: 8 April 2002 / Accepted: 24 June 2002  相似文献   

5.
The acaricidal effects of 55 strains of Metarhizium anisopliae (Metschnikoff) Sorokin, 1883 (Hypocreales: Clavicipitaceae) isolated from paddocks of cattle farms were evaluated in two Rhipicephalus microplus (Canestrini 1887) (Ixodida: Ixodidae) populations, of which one was multi‐resistant and one was susceptible to chemical acaricides. Percentage mortality and reproductive efficiency indices in R. microplus were evaluated by adult immersion tests at a dose of 1 × 108 conidia/mL for each fungal strain. Some strains were selected to calculate lethal concentrations to kill 50% (LC50) and 99% (LC99) of engorged ticks. Strains MaV22, MaV26 and MaV55 induced 100% mortality in R. microplus on day 14. Strains MaV05, MaV09 and MaV22 caused mortality of >90% from day 12 onward in both tick populations. The most effective acaricidal fungal strain, MaV55, inhibited egg laying by 54.86 and 55.86% in acaricide‐resistant and ‐susceptible R. microplus populations, respectively. None of the fungal strains had statistically significant effects on larval hatching. In conclusion, nine strains of M. anisopliae demonstrated high acaricidal effects against R. microplus and reduced its egg laying. No differences in acaricidal effects were observed between the two populations of ticks tested.  相似文献   

6.
Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.  相似文献   

7.
The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection.  相似文献   

8.
《Experimental mycology》1991,15(4):310-315
The entomogenous fungusMetarhizium anisopliae attacks a broad range of insects, including the agricultural pestsGalleria mellonella (the Greater Wax Moth) andTrichoplusia ni (the Cabbage Looper). Five strains ofM. anisopliae from widely divergent isolation sources were culturedin vitro on media containing gelatin, glucose plus nitrate, or purified cuticle fromG. mellonella orT. ni larvae. The production of extracellular enzymes such as proteases, chitinases, and esterase was compared. A great deal of natural strain variability was found in enzyme patterns. The highest levels of proteases and endochitinase were produced in cuticle-grown cultures. Three of five strains produced exceptionally high levels of chymoelastase (47,000 to 98,000 IU/mg protein) on cuticle. Surprisingly, the highest levels ofN-acetyl glucosaminidase were produced in gelatin-grown cultures. Most strains produced esterase under all growth conditions. The source of insect cuticle did not strongly influence the production of enzymes.  相似文献   

9.
We determined the virulence of Beauveria bassiana, Metarhizium anisopliae, Isaria fumosoroseus and Lecanicilium lecanii against larvae of Chilo suppressalis Walker by bioassay and evaluated several enzymatic and non-enzymatic components. LC50 values of the entomopathogenic fungi revealed 90, 32, 45,000, 4600, 42,000 and 1,540,000 spores/larva for isolates BB1–BB3 of B. bassiana, I. fumosoroseus, M. anisopliae and L. lecanii, respectively. Isolate BB3 and I. fumosoroseus had the highest amounts of total protein and hydrophobin and isolates BB3 and M. anisopliae showed the highest activities of lipases and chitinases. In case of proteases, the highest activities were observed for Pr1 of BB1 and Pr2 of L. lecanii. The highest general esterase activities were obtained in I. fumosoroseus and BB1 when 1-naphtyl acetate and 2-naphtyl acetate were used as substrates. The highest activity of glutathione S-transferase (GST) was observed in I. fumosoroseus by using both reagents but BB1 demonstrated the highest activities of alkaline phosphatase and acid phosphatase. Clustering of the fungi using biochemical enzymes revealed BB2 and BB3 as a separate group of entomopathogenic fungi. In another group, I. fumosoroseus and L. lecanii had the most similarity and were separated from BB1 and M. anisopliae. The fungi exhibited different virulence on larvae of C. suppressalis by producing adhering protein and extracellular enzymes. Overall, results of the bioassays and clustering based on enzymatic activities revealed that isolate BB2 was the most effective fungus against larvae of C. suppressalis.  相似文献   

10.
11.
Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.  相似文献   

12.
Protease production by fourteen M. anisopliae isolates differing in geographical origin and host insect were investigated. Highest protease activity was observed during 4–8 days of culture incubation. Pr1 and Pr2 activity was investigated in various media containing different carbon and nitrogen source to evaluate the induction mechanism of these enzymes. Basal levels of Pr1 and Pr2 activity were observed in minimal medium suggesting constitutive production. Casein (1%) as an exogenous protein supplement was not able to induce significant release of Pr1 and Pr2 enzymes, whereas high levels of activity were observed in the medium containing colloidal chitin (2%) as sole carbon and nitrogen source. The pH, ammonia and oxalic acid production in in vitro conditions was also investigated and the alteration in pH for protease production was not significant in the different media used except for the medium containing casein (1%) as a supplement.  相似文献   

13.
Entomopathogenic fungi, such as Metarhizium anisopliae, for the control of arthropods, have been studied for more than 20 years. The aim of this study was to determine the best methodology to evaluate the in vitro effect of the fungus M. anisopliae on Rhipicephalus microplus tick larvae. We compared a modified Larval Packet Test (LPT) and a Larval Immersion Test (LIT). For the LPT filter papers were impregnated with 1 mL of M. anisopliae suspension in Triton X-100 at 0.02%, in concentrations of 106, 107 and 108 conidia/mL and subsequently folded to include the larval ticks. LIT was performed by immersing the larvae in M. anisopliae suspensions for 5 min using the same three concentrations, then the larvae were placed on filter paper clips. For LPT, the LT50 values obtained were 134.6, 27.2 and 24.8 days for concentrations of 106, 107 and 108 conidia/mL; and the mortality after 21 days was 17.3, 17.6 and 38%, respectively. The LT50 values of LIT were 24.5, 20 and 9.2 days with mortality after 21 days of 50.5, 64.7 and 98% for 106, 107 and 108 conidia/mL, respectively. For the same conidia concentration, LIT showed a higher mortality in a shorter time interval when compared with LPT. These differences between the methods tested must be taking into account in further screening and effect studies with M. anisopliae. The set of results shown here could optimize the protocol used to identify M. anisopliae strains pathogenic against R. microplus.  相似文献   

14.
The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO3. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.  相似文献   

15.
Polar P  Kairo MT  Moore D  Pegram R  John SA 《Mycopathologia》2005,160(2):151-157
Studies were conducted to identify oil-based formulating agents (paraffinic oil, palm oil and emulsifiable adjuvant oils (EAOs)) for Metarhizium anisopliae that were superior to water with simple surfactants using a germination test and a bioassay against Boophilus microplus. Germination of conidia in all formulations, except 10% coconut EAO, produced more than 68% germination at 24 h and nearly 100% at 48 h. Coconut oil (average survival time (AST)=4.6±0.28 days) and 10% liquid paraffin EAO (AST=4.4±0.15 days) enhanced the pathogenicity of M. anisopliae to B. microplus relative to water (AST=8.4±0.42 days). M. anisopliae in 10% liquid paraffin EAO was the most effective formulation having a moderately high germination after 24 h and a low AST as well as a high AST in the control. In the second experiment, germination of conidia in 2% liquid paraffin EAO and 2% Cropspray was higher than in 2% Codacide oil at 24 h, however, all treatments reached 100% germination after 48 h. The ASTs of the EAO based M. anisopliae formulations (Average AST=6.4±0.54 days) were similar but lower that the ASTs of the controls (Average AST=9.6±0.28 days).  相似文献   

16.
The high-conserved translation elongation factor 1 α (tef-1α) gene from the enthomopathogenic fungus Metarhizium anisopliae was characterized to select the promoter region. A 640-bp DNA fragment upstream to the start codon was employed to drive the expression of the reporter protein sGFP or a dominant selectable marker, the gene bar (resistance to ammonium glufosinate). Transformants carrying this homologous promoter system showed no difference in virulence bioassays against the cattle tick Boophilus microplus comparing to the M. anisopliae wild-type strain. Moreover, GFP fluorescence was detected during tick infection bioassay.  相似文献   

17.
There are no reports to date of entire gene sequences coding for chitinolytic enzymes from entomopathogenic fungi, even though these enzymes act synergistically with proteolytic enzymes to solubilize insect cuticle during the key step of host penetration, having considerable importance in the biological control of some insect pests. This paper reports the complete nucleotide sequence and analysis of the chromosomal and full-length cDNA copies of the regulated gene (chit1) coding one of the chitinases produced by the biocontrol agent Metarhizium anisopliae. Degenerated primers, encompassing conserved regions of other fungal chitinases, were used to amplify a 650-bp DNA fragment, which was used to isolate genomic and cDNA clones from M. anisopliae. Albeit at least two different chitinases are characterized in this fungus, only one chit gene was isolated. The chit1 gene is interrupted by three short typical fungal introns and has a 1,521-bp ORF, which encodes a protein of 423 amino acids with a stretch of 35 amino acid residues displaying characteristics of signal peptide. The deduced sequence of the mature protein predicts a 42-kDa protein with pI of 5.8. Southern analysis of genomic DNA indicates a single copy of chit1 in the M. anisopliae genome. Received: 13 March 1998 / Accepted: 14 April 1998  相似文献   

18.
The present study aimed to evaluate the in vitro pathogenicity of the Metarhizium anisopliae s.l. CG 112, CG 347, CG 32, CG 148 and CG 629 isolates formulated in vegetable or mineral oil against Rhipicephalus microplus. The bioassays were performed with R. microplus engorged females. The ticks were immersed for three minutes in oil-based formulations containing 10% oil (mineral or vegetable). The effects of the different fungal isolates were evaluated analyzing the females’ reproductive parameters (the total weight of the egg mass, hatching percentage, egg production index and nutritional index). The present study showed that all of the tested isolates and both oil formulations (vegetable or mineral oil) changed the biological parameters of the R. microplus females. However, the mineral oil formulation was more effective than the vegetable oil formulation, as the former showed a higher tick control percentage. It was concluded that there was variation in the virulence among the different M. anisopliae s.l. isolates and between the different types of oil. Moreover, that either of the isolates GC 148 and GC 629 formulated in oil confers the good potential for controlling R. microplus engorged females.  相似文献   

19.
Functional effects of ants in rainforest canopies depend on difficult to characterize ant diets. In Bornean dipterocarp forests, certain diurnal, arboreal, territorial, and ecologically dominant ‘COCY’ ant species (Colobopsis cylindrica clade) grazed epiphytic biofilms on adaxial leaf surfaces, as well as on tree trunks and branches. Microscopic examination of worker buccal pellets revealed numerous (mainly ascomycete) fungal spores, together with insect appendages and cuticle. Direct observations, video‐imaging, and δ15N isotope data rule out feeding by predation, but isotopes cannot separate fungi from plant and insect exudates as principal nitrogen sources. Lipid‐rich products, extracted from pellets in situ, are hypothesized sources of essential sterols. Also present in pellets were colorful mandibular gland (MG) compounds unique to this ant clade and deployed, as a derived character state, in suicidal defense of foraging territories. Mildly antimicrobial and highly adhesive MG products also occur basally in the clade and may have first evolved for roles in microbial sterilization and food‐gathering and processing. Proteomic studies of YG COCY ants detected 2% proteins in hypertrophied, product‐filled MG reservoirs, but SDS‐PAGE qualitative analysis revealed mostly low‐molecular mass proteins and peptides (8–15 kDa), too small for enzymes but consistent with membrane‐binding proteins and/or antimicrobial peptides. Breakdown of chitin and chitosan in pellets may occur with enzymes derived from molting fluids in insect cuticle (proteases and chitinases) and/or fungi and bacteria. To the extent that COCY workers collect and consume pathogenic and/or beneficial phyllosphere microbes, ant effects on plants may be mediated by these activities.  相似文献   

20.
Chitinases play an important role in the degradation of the cuticular chitin during the process of ecdysis. In this study, we compared the chitinases of two insect species, Bombyx mori (silkworm) and Helicoverpa armigera (bollworm), to assess the relation between characteristics and chitinase patterns. Differences between two chitinases were observed after purification using ammonium sulfate precipitation, affinity chromatography, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) assay. Although the specific activities of the purified enzymes were different, the purification yields were similar. One band of 88 kDa was observed for B. mori, and the other band of 75 kDa was detected for H. armigera. When a range of properties was tested, it was found that the optimum temperatures of B. mori and H. armigera chitinases were 45 and 50°C, respectively; the optimum pH value was 6.0 for both chitinases. Mn2+ played catalytic role while Cu2+ and SDS strongly inhibited activities of both enzymes. Between two chitinases, differences in K M were also observed. K M of chitinase from silkworm and bollworm was found to be 22.3 and 41.0 μmol/l, respectively. Both the chitinases significantly inhibited the spore germination of two fungal species, Saccharomyces cerevisiae and Penicillium.  相似文献   

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