Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

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.     

Production and Regulation of Cuticle-Degrading Proteases from <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in the Presence of <Emphasis Type="Italic">Rhammatocerus schistocercoides</Emphasis> Cuticle

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.     

Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus,Beauveria bassiana in the presence of Sunn pest,Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca^{2 +} and Mg^{2 +}, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes.     

Regulation of production of a trypsin-like protease by the insect pathogenic fungus Metarhizium anisopliae

Abstract The entomopathogenic fungus metarhizium anisopliae produces several cuticle-degrading proteases which may play a role in pathogenesis. The regulation of one of these, a trypsin-like protease PR2, has been investigated using depressed mycelia. Three insoluble protein sources, insect cuticle, elastin and collagen, as well as two soluble proteins, BSA and gelatin, induced PR2. The polymeric carbon sources cellulose and xylan resulted in depressed basal levels but not induced production of PR2. An approximately 15-fold increase in PR2 activity per mg dry weight of mycelium was observed when the fungus was grown in the presence of bovine serum albumin (BSA), as compared with conditions of depression alone. This indicates that PR2 is induced by BSA, and probably by other proteins. Basal levels of PR2 were detected after 8 h when mycelium was starved for both carbon and nitrogen but only after 16 h when starved for either nitrogen or carbon. In the presence of a protein source, nitrogen strongly repressed PR2 whereas carbon had little effect. There was no effect of sulphur on PR2 production.     

Cuticle-degrading proteases from the entomopathogen Metarhizium flavoviride and their distribution in secreted and intracellular fractions

AIMS: The aim of this study was to analyse a native isolate of Metarhizium flavoviride (CG423) which is being developed as a myco-insecticide against grasshoppers in Brazil for the production of the cuticle-degrading subtilisin-like (Pr1), and trypsin-like (Pr2) proteases. METHODS AND RESULTS: The results show that Pr1 activity occurred only in medium supplemented with grasshopper cuticle (Schistocerca pallens). In contrast, Pr2 was detected in higher amounts on defined growth substrate than on cuticle-supplemented medium. Both activities were detected after 48 h of growth, suggesting that in S. pallens cuticle-containing medium these protease types are not co-ordinately expressed. Low levels of enzyme activity were detected when pre-grown mycelium was used to investigate the induction of Pr1 proteases. CONCLUSIONS: The analysis of Pr1 and Pr2 distribution in both secreted and intracellular fractions revealed high percentage of extracellular activity, which may suggest the occurrence of an efficient mechanism of protein secretion by this fungus, probably related to substrate degradation which provides nutrients for fungal growth.     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles.

Proteinase 2 (Pr2) is a fungal (Metarhizium anisopliae) serine proteinase which has a tryptic specificity for basic residues and which may be involved in entomopathogenicity. Analytical and preparative isoelectric focusing methods were used to separate two trypsin components, produced during growth on cockroach cuticle, with isoelectric points of 4.4 (molecular mass, 30 kDa) and 4.9 (27 kDa). The catalytic properties of the proteases were analyzed by their kinetic constants and by a combination of two-dimensional gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme overlay membranes. Both Pr2 isoforms preferentially cleave at the carboxyl sides of positively charged amino acids, preferring arginine; the pI 4.4 Pr2 isoform also possessed significant activity against lysine. Compared with the pathogen's subtilisin-like enzyme (Pr1), the pI 4.4 Pr2 isoform shows low activity against insoluble proteins in a host (Manduca sexta) cuticle. However, it degrades most cuticle proteins when they are solubilized, with high-molecular-weight basic proteins being preferentially hydrolyzed. Polyclonal antibodies raised against each Pr2 isoform were isotype specific. This allowed us to use ultrastructural immunocytochemistry to independently visualize each isoform during penetration of the host (M. sexta) cuticle. Both isoforms were secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. The extracellular sheath, which is commonly observed around fungal cells, often contained Pr2 molecules. Intracellular labelling was sparse.     

Properties of Extracellular Enzymes from Aphanomyces astaci and Their Relevance in the Penetration Process of Crayfish Cuticle

In culture filtrates from the crayfish plague parasite, Aphanomyces astaci, protease and a low level of hyaluronidase activity were found. The hyaluronidase activity was highest at pH 6.5 or above and at about 23°C. The protease activity had a broad pH-optimum, between pH 7 and at least pH 10, and was partially denatured at 30°C. However, when incubated for 30 min with the substrate, casein, the activity increased logarithmically up to about 35–40°C and had an apparent optimum at 45–50°C. The proteases from the parasitic as well as from two less proteolytic, saprophytic Aphanomyces species were predominantly constitutive and were excreted mainly by the older mycelia. Proteases from the parasite and a saprophyte did not reach full activity until 10–30 min after substrate addition. No lipase activity was found in the case of the mycelium of the parasitic species. However, esterase was apparently present inside germinating zoospores. The native enzymes of A. astaci could degrade freeze-dried soft cuticle from crayfish. The relevance of the different enzymes of A. astaci for the penetration process within the cuticle of crayfish is discussed.     

Host-plant mediated interactions between two aphid species

Three isolates of Isaria fumosoroseus Wize (Hyphomycetes) were cultured on six media composed of differing amounts of chitin, carbon, and nitrogen. The effect of nutrition on growth and virulence was studied by measuring colony growth, spore yield, germination rate, spore-bound protease (Pr₁) and lipase activity, and virulence of inoculum produced by different media against second instars of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Chitin peptone nutrition media resulted in the highest colony growth but spore yields were lowest for this medium, whereas the osmotic stress medium resulted in the lowest colony growth with fewer spores. Highest lipase activity (4.14 µmol/ml/min) was observed for spores produced on high C/N medium for isolate IF28.2, whereas highest Pr1 activity (2.64 µmol/ml/min) was also observed on high C/N medium for isolate IF28.2. A higher rate of Pr1 and lipase activity for all three isolates was observed on low C/N, 2% peptone, and the osmotic stress media. Conidia from nutrient-poor media (2% peptone) proved to be the least virulent for all three isolates with median survival time values of 2.23, 2.05, and 1.79 days for IF32, IF28.2, and IF49, respectively. Median survival time values for the various nutrient media proved to be positively correlated with spore-bound Pr1 and lipase activity, having correlation coefficient values of 0.115 and 0.538, respectively.     

Evaluation of insecticidal activity of a bacterial strain, <Emphasis Type="Italic">Serratia</Emphasis> sp. EML-SE1 against diamondback moth

To identify novel bioinsecticidal agents, a bacterial strain, Serratia sp. EML-SE1, was isolated from a dead larva of the lepidopteran diamondback moth (Plutella xylostella) collected from a cabbage field in Korea. In this study, the insecticidal activity of liquid cultures in Luria-Bertani broth (LBB) and nutrient broth (NB) of a bacterial strain, Serratia sp. EML-SE1 against thirty 3rd and 4th instar larvae of the diamondback moth was investigated on a Chinese cabbage leaf housed in a round plastic cage (Ø 10×6 cm). 72 h after spraying the cabbage leaf with LBB and NB cultures containing the bacterial strain, the mortalities of the larvae were determined to be 91.7% and 88.3%, respectively. In addition, the insecticidal activity on potted cabbage containing 14 leaves in a growth cage (165×83×124 cm) was found to be similar to that of the plastic cage experiment. The results of this study provided valuable information on the insecticidal activity of the liquid culture of a Serratia species against the diamondback moth.     

Cuticle-Degrading Proteases Produced by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Their Induction in Different Media

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.     

The effect of microbial mycolytic agents on Trichophyton rubrum

Chitinolytic microorganisms isolated from forest soil and from healthy gypsy moth larvae (Porthetria dispar (L.) were screened for their ability to lyse Trichophyton rubrum mycelia. A few of these isolates were mycolytic on both autoclaved and on actively growing, intact, T. rubrum mycelia. Supernatants from these isolates, utilizing live T. rubrum as the sole carbon source, showed the same mycolytic ability. Assays of the supernatants for enzymatic activity revealed exocellular, stable enzymes that releases reducing substances including N-acetylglucosamine from the mycelia.     

Purification and characterization of a neutral serine protease with nematicidal activity from <Emphasis Type="Italic">Hirsutella rhossiliensis</Emphasis>

Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40°C, respectively. The Hnsp activity was stable at pH 6–8 and decreased radically at 50°C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.     

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.     

Addition of exogenous carbon and nitrogen sources to aphid exuviae modulates synthesis of proteases and chitinase by germinating conidia of Beauveria bassiana

Secretion of catabolic extracellular enzymes (ECE) is the hallmark of the infection of insects through the cuticle by entomopathogenic fungi (EPF). In this paper, we show that germinating conidia of Beauveria bassiana (Bb) regulate the synthesis of ECE through a multiple control mode during the initial stages of germination. We tested Bb conidial growth on aphid exuviae with or without supplementation of additional carbon and/or nitrogen (C/N) compounds. To understand the interrelation between conidial germination during growth, the synthesis of ECE activity, free amino nitrogen (FAN), glucose and fungal dry weight biomass were measured. Immediately (0.25 h) upon incubation of conidia, activity of subtilisin-like Pr1 and trypsin-like Pr2 enzymes and chitinase (NAGase) was observed in the culture filtrates. At 0.25 h, addition of exogenous C-source resulted in higher activities of Pr1 and Pr2, respectively. Conversely at 0.25 h, addition of N-sources repressed the synthesis of Pr2, but that of Pr1. C/N repression was observed only for exponentially growing mycelia. NAGase activity remained at basal level and unaffected by added C/N. We conclude that C/N repression occurs only when it is necessary for the Bb infective structures to establish a nutritional relationship with the host structures.     

绿僵菌素对爪哇棒孢霉SP053菌株的影响及其混用对小菜蛾的联合毒力

为了寻找更有效的利用虫生真菌爪哇棒孢霉Isaria javanicus防治小菜蛾Plutella xylostella的新方法, 测定了绿僵菌素（destruxin）对SP053菌株生长发育与产孢量与萌发率的影响, 利用协同毒力指数法和毒力回归分析法评价了两者混用对小菜蛾2龄幼虫的联合毒力。结果表明: 绿僵菌素对SP053菌株菌丝生长、孢子萌发率和孢子产量均无显著影响（P>0.05）。而一定浓度的绿僵菌素与SP053菌株分生孢子混用具有增效作用, S50-CD100（指爪哇棒孢霉SP053菌株的分生孢子浓度为50×10⁵个/mL和绿僵菌素粗毒素的浓度为100 mg/L的组合; 依此类推）、S25-CD100、S25-CD50及S12.5-CD100等混配组合都有显著的增效效果, 尤其以S25-CD100组合增效效果最好, 其48 h和72 h的协同毒力指数（c.f.）分别达到52.31和31.07。毒力回归分析结果也显示, 添加绿僵菌素粗毒素25~100 mg/L的混剂的LC50值明显降低, 比如添加100 mg/L绿僵菌素粗毒素的混剂（SP053-CD100）, 其48和72 h的LC50分别为17.45和10.55 （×10⁵个孢子/mL）, 而SP053菌株分生孢子单剂的LC50相应值为>50和35.85（×10⁵个孢子/mL）。本研究证明绿僵菌素与爪哇棒孢霉SP053菌株混用有增效作用, 对改进小菜蛾的生物防治方法有一定指导意义。     

湖北高海拔地区性信息素对小菜蛾的诱捕和防治效果

2002年应用性诱剂对海拔1 200 m山区甘蓝田小菜蛾的发生及防治进行了研究。在第一茬蔬菜生长期有2个诱蛾高峰,诱蛾量(头/盆)分别为11.7±2.4和9.2±1.0;第二茬蔬菜生长期有3个诱蛾高峰,诱蛾量(头/盆)分别为70.9±8.0、16.1±2.5和11.1±1.9。应用性诱剂诱捕山区甘蓝田小菜蛾时,第一茬田间蛾密度与单盆诱捕量相关性不显著,而第二茬菜生长期田间蛾密度与单盆诱蛾量相关性显著(y=0.0116x+0.1614, r=0.9213, P=0.0011)。性诱剂在光期与暗期都可诱到小菜蛾雄虫, 没有明显的诱蛾高峰。应用性诱剂可以使菜田的农药使用减少3～5次,降低田间子代幼虫密度。     

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, <Emphasis Type="Italic">Lecanicillium</Emphasis> spp: implications for host specificity

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).     

球孢白僵菌凝乳弹性蛋白酶(BbPr1)的纯化与特性

以几丁质为底物,加入基本盐培养基中,诱导球孢白僵菌(Beauveria