Production of cuticle-degrading proteases by Nomuraea rileyi and its virulence against Anticarsia gemmatalis/Producao de proteases degradadoras de cuticula por Nomuraea rileyi e sua virulencia contra Anticarsia gemmatalis.
The entomopathogenic fungus Nomuraea rileyi (Farlow) Samson has the potential to control caterpillars of 30 different species of lepidopteran larvae, and the caterpillars of the Noctuidae family are among the most sensitive to this pathogen (DEVI et al., 2003; SRISUKCHAYAKUL et al., 2005). N. rileyi are an important natural control agent of Anticarsia gemmatalis (Hubner) (Lepidoptera: Noctuidae), one of the most damaging soybean pests in the American continent (IGNOFFO, 1981). In Brazil, N. rileyi often prevents populations from reaching economic threshold levels in soybean, avoiding the need for insecticide applications against this pest (CORREA AND SMITH, 1975; FARIA et al., 1993).
During the infection process, the integument is a barrier that prevents fungus penetration. Proteases are enzymes that are often considered critical in facilitating the host infection process (CLARKSON & CHANRLEY, 1996). The best known determinant of fungal entomopathogenicity is based on subtilisin-like serine protease (designated Pr1) of Metarhizium anisopliae, whose role in host invasion has been clearly demonstrated (St. LEGER et al., 1988). This enzyme is adapted to extensively degrade the insect cuticular protein (St. LEGER et al., 1987b) and has been ultrastructurally located in the host cuticle during the early stages of penetration (GOETTEL et al., 1989). Recent studies based on the analysis of expressed sequence tag (EST) revealed the expression of several subtilisin-like proteases by M. anisopliae during growth on insect cuticle (FREIMOSER et al., 2003). These different subtilisins may play different roles in pathogenicity increasing the scale of adaptability to the host, or having different survival functions in several habitats under extreme ecological conditions (BAGGA et al., 2004).
A trypsin-like enzyme (Pr2), which belongsto the serine protease group, also occurs during the early stages of cuticle colonisation suggesting that it has some role in degrading extracellular proteins complementary to that of Pr1 (St. LEGER et al., 1996). PATERSON et al., (1994) reported that this enzyme could be involved in Pr1 activation or induction in M. anisopliae.
Both Pr1 and Pr2-like proteases are also produced by other entomopathogenic fungi, including N. rileyi (ST. LEGER et al., 1987a). This fungus may form a mucillaginous mass around the germ tube and does not usuallly produce appressoria (SRISUKCHAYAKUL et al. (2005), suggesting that its penetration through the larval integument occurs enzymatically, particularly during the germination of conidia. ELSAYED et al. (1993a,b) showed that this fungus produces proteases and chitinases in the presence of larval cuticle from Trichoplusia ni, Helicoverpa (Heliothis) zea, and Heliothis virescens, but not in minimal medium or on non-host substrate, suggesting that their expression is a specific response to cuticle components. Furthermore, a correlation between the level of proteases produced and pathogenicity has been reported (EL-SAYED, et al., 1993b; SRISUKCHAYAKUL et al., 2005).
The entomopathogenic fungus B. bassiana showed high PR1 and PR2 enzyme activities when cultured in medium containing Rhammatocerus schistocercoides grasshopper cuticle (DONATTI et al., 2008) and in medium containing Hypothenemus hampei coffee berry borer cuticle (DIAS et al., 2008).
In this study, it was examined for the first time the production of Pr1 and Pr2 proteases by N. rileyi in the presence of A. gemmatalis cuticle, and its possible role in the process of pathogenicity and virulence to this caterpillar, extending knowledge about protease production by this fungus.
MATERIALS AND METHODS
Microorganism and culture conditions
N. rileyi strain CG434 (isolated from A. gemmatalis, collected at Patos de Minas/MG, from the Entomopathogenic Fungi Collection of CENARGEN at EMBRAPA Brasilia/DF) and strain NR458 (isolated from A. gemmatalis, collected at Londrina/PR, from the Entomopathogenic Fungi Collection of CNPSo EMBRAPA Londrina/PR) were reinvigorated in bioassays using A. gemmatalis larvae.
Conidia were obtained by harvesting sporulated cultures grown on agar plates composed of minimal medium (MM) (PONTECORVO et al., 1953) containing nitrate as nitrogen source.
For enzyme production, conidia were added to 25ml of liquid MM, MMI (MM lacking nitrogen source) to which a 1% p/v casein solution was added (MMI+CAS), MMI to which a 0.5% p/v cuticle solution was added (MMI+CUT), MMI to which a solution of exuvia was added (MMI+EXU), to a final concentration of 5x[10.sup.6] [ml.sup.-1] and grown in submerged culture (180rpm) at 28[degrees]C for up to 10 days.
Cuticle and exuvia were prepared using an aqueous solution of 1% (w/v) potassium tetraborate. The samples were taken at 0, 168, 192 and 216h of growth. Mycelium was harvested by centrifugation at 8000g for 15min and culture filtrates were stored at -20[degrees]C and used as the extracellular secreted fraction. All experiments were repeated three times, and the results represent mean values [+ or -] SD.
Subtilisin-like (Pr1) and trypsin-like (Pr2) activities were determined using specific synthetic substrates as Suc-Ala-Ala-Pro-Phe-p-nitroanilide for Pr1 and N-Benzoyl-Phe-Val-Arg-p-nitroanilide for Pr2, according to GUPTA et al. (1992). In the reaction 50[micro]L of substrate (1mM), 100[micro]L of sample and 850pL of Tris-HCl (15mM) pH 7.0, 8.0 and 8.5 buffer were mixed. The mixture was incubated for 1h at 28[degrees]C and the reaction was terminated by adding 0.25ml of 30% acetic acid and left to stand for 15min in ice, after which samples were centrifuged at 1250g for 5min at 4[degrees]C. The supernatants were read at 410nm. Activities were expressed as nmol p-nitroanilide [ml.sup.-1] [h.sup.-1]. Assays were performed in duplicate for each sample.
The insects were placed on Petri plates with filter paper containing 3mL of fungal suspensions ([10.sup.9] conidia [ml.sup.-1]) where they stayed for approximately 24 hours. After this time they were transferred to an artificial diet. Fifty insects were used for each strain tested, and an equal number for the control with Tween 80 (0,1% v/ v). Three repetitions were performed per treatment, and in all assays 3rd instar caterpillars were used. The mortality rate was assessed daily until the caterpillars reached the pupal stage.
Statistical analysis of enzymatic assays was performed using parametric tests with the help of the computer program SPSS for Windows version 10.0. The data were submitted to an Analysis of variance by the ANOVA tests with media comparison using the Tukey test (P<0.05).
Virulence was assessed using the PROBIT method, and the TL50 was determined. The confidence interval calculated was P=0.011 (Pearson). The Pearson Correlation Coefficient test was used to verify the level of correlation between virulence and proteolytic subtilisin-like activity (Pr1).
RESULTS AND DISCUSSION
Entomopathogenic fungi exhibit many attributes which determine virulence towards their hosts. Proteases are enzymes that are often considered critical in facilitating the host infection process (CLARKSON & CHANRLEY, 1996).
As it is well known, growth conditions such as composition of culture medium and time could modulate the synthesis of bioactive molecules in microorganisms (RAO et al., 1998). In this study, it was determined the production of Pr1 and Pr2-like proteases by N. rileyi in liquid culture, either in the presence or absence of cuticular substrate at different incubation periods.
In medium containing nitrate as sole nitrogen source (MM) no detectable Pr1-like activity occurred in the culture supernatants of the two fungal strains analysed. However, both strains of N. rileyi produced Pr1-like protease in all medium amended with exogenous nitrogen source, and it was highly expressed in the presence of insect cuticle (Tables 1 and 2).
In medium containing casein as a source of carbon, the Pr1-like activity in the culture supernatant of both strains analysed was higher in alkaline pH. In this culture condition, strain CG434 produced higher amounts of enzyme compared to that of strain NR458.
In the presence of cuticle substrate, strain NR458 presented greater enzyme activity, being up to 5.21U after 216h of incubation. For strain CG434, similar values of Pr1 activity were observed in all incubation period analysed. The Pr1 activity of strain NR458 in a culture amended with pupal exuvia was similar to the activity detected in a medium containing casein, and it was superior to that of strain CG434. Protease activity in the culture supernatant of both strains analysed was also higher in alkaline pH (Tables 1 and 2).
In this study, the highest expression of Pr1 was found in a medium containing a cuticle substrate compared with the other substrates, since no activity was detected in absence of exogenous protein, and low levels were observed in the casein and pupal exuvia substrates. These data suggest that components of the A. gemmatalis cuticle induce the production of Pr1-like enzymes. Similarly, in previous studies, N. rileyi demonstrated increased proteolytic activity in the presence of Manduca sexta, Trichoplusia ni and Helicoverpa zea cuticles (ST. LEGER et al., 1987a; EL SAYED et al., 1993a). Similar results were observed for other entomopathogenic fungi (ST. LEGER et al., 1989; GUPTA et al, 1992; PATERSON et al., 1994; GILLESPIE et al., 1998; TIAGO et al., 2002; PINTO et al., 2002; CAMPOS et al., 2005; DONATTI et al., 2008; DIAS et al., 2008). Analyses of the proteolytic and chitinolytic activities of M. anisopliae and B. bassiana, performed in media containing different sources of carbon/ nitrogen and complex substrates such as colloidal chitin and cuticle of different insects showed more activities of these enzymes in the media containing complex substrates, suggesting that the expression of the genes involved in the synthesis of Pr1 and Pr2, as well as that for chitinases, depends on the level of carbon and nitrogen sources in the culture medium (MORAES, et al., 2003; CAMPOS, et al., 2005, TIAGO et al., 2007; DONATTI et al., 2008).
It was also determined the production of Pr2-like protease by A. gemmatalis in liquid culture, either in the presence or absence of cuticular substrate. This activity was significantly inferior to Pr1-like activity, and it was detected only in some of the culture media and incubation periods tested (data not shown). In the culture supernatant of strain NR458, in a medium containing cuticle substrate, Pr2-like activity was found in all three periods evaluated at pH 8.5. In the other culture media tested, activity was found only at pH 4.0. The highest activity was observed (0.370U [+ or -] 0.224) in medium containing nitrate as nitrogen source; in a medium containing pupal exuvia and casein, activites were lower (up to 0.175U [+ or -] 0.05 and 0.146U [+ or -] 0.043 at 216h incubation, respectively). For this fungal strain, A. gemmatalis cuticle did not have a positive effect on trypsin-like protease (Pr2), since it was produced in substantive amounts in defined growth substrate (nitrate medium). Similar result was observed for Metarhizium anisopliae var. acridum (TIAGO et al., 2002).
Strain CG434 presented lower Pr2-like activity compared to strain NR458. In a medium to which casein was added (MMI+CAS) activity (0.162U [+ or -] 0.37) was detected only at pH 7.0 and after 168h of culture. In MM and in medium supplemented with cuticle Pr2-like activity was detected only at pH 8.0 (0.211U [+ or -] 0.128 and 0.188U [+ or -] 0.085, respectively). No Pr2 activity was detected at pH 8.5. Different results were described by GUPTA et al. (1992) who observed that the optimum pH for Pr2 production by N. rileyi was 8.5.
Studying the B. bassiana PR1 and PR2, DIAS et al. (2008) reported the effect of nitrogen sources and pH variation in enzyme regulation and the induction of the aforementioned enzymes in alcaline pH.
According to ST. LEGER et al. (1996) Pr2-like protease was secreted by M. anisopliae, by the infection structures (appresoria) on the surface of M. sexta cuticle and by the penetrating hypha, suggesting a complementary role of this enzyme to that of Pr1 in degrading cuticle proteins. However, N. rileyi does not produce appressoria, but rather a mucillaginous mass around the germinal tube, which would also function as an adhesive and facilitate enzyme production (ALVES, 1998a).
The virulence of a fungal strain can be measured in bioassays, considering the mean time needed to kill 50% ([TL.sub.50]) of a population of insects applying a single concentration of conidia (ALVES, 1998b) In this study it was found that strain NR458 induced a higher percentage of mortality (64.4%) with a 223-hour [TL.sub.50] and strain CG434 induced 35% mortality with a 253-hour [TL.sub.50].
Correlation analysis between the percentage of mortality and Pr1-like protease activity of strain NR458 (Table 3) suggests a positive correlation of these variables in the medium containing casein and cuticle at the three pHs evaluated. As to strain CG434, the correlation analysis of these variables was low compared with that observed for strain NR458. Strain CG434 also showed a positive correlation in the medium containing casein, at the three pHs, and in the medium containing cuticle at pH 8.5 (Table 3). This correlation may indicate the importance of this enzyme in the pathogenicity process of some strains of fungus N. rileyi to the A. gemmatalis caterpillar, and also indicates the occurrence of genetic variability among the different strains. Controversially, VARGAS et al. (2003) did not observe a correlation between enzyme activity and the virulence of fungus N. rileyi, suggesting that other factors may interfere in this process. Differences in pathogenicity indicate the natural occurrence of genetic variation (ST. LEGER et al., 1992).
According to the data obtained throughout this research, following the previous detailed experimental conditions, it was detected Pr1-like protease activity in the tested N. rileyi strains culture supernatant. No Pr1-like activity was detected in minimum media (MM); highest activity was detected in cuticle-containing media under the three pH values evaluated, and the most appropriate range was between 8.0 and 8.5 for the referred enzyme activity. The Pr2-like protease activity was lower when compared to Pr1-like protease activity and it was not constantly observed in all media, with most appropriate range of pH values between 7.0 and 8.0. The strains NR458 and CG434 induced 64.4% and 35% mortality, with LT50 of223 and 253 hours respectively. It was a positive correlation between the Pr1-like protease activity and virulence when the NR458 strain was tested and cultured in media with casein and host cuticle in three pH ranges. This positive correlation was also observed for CG434 strain when cultured in media containing casein in the three pH ranges and in cuticle-containing media under pH 8.5.
The results presented in this study increase knowledge about protease production in N. rileyi, opening new avenues for the study of the role of secreted proteases in virulence against A. gemmatalis larvae during the infection process.
This work was supported by grants and fellowships from UCS, UEL, CAPES, and FAPERGS.
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Ana Rita Fonseca Nunes (I,II) Juliana Nascimento Martins (I) Marcia Cristina Furlaneto (III) Neiva Monteiro de Barros (I) *
(I) Instituto de Biotecnologia, Centro de Ciencias Agrarias e Biologicas, Universidade de Caxias do Sul (UCS), CP 1352, 95001-970, Caxias do Sul, RS, Brasil. E-mail: firstname.lastname@example.org. * Autor para correspondencia
(II) Faculdade da Serra Gaucha, Os Dezoito do Forte, 2366, 95020-472, Caxias do Sul, RS, Brasil.
(III) Centro de Ciencias Biologicas, Universidade Estadual de Londrina (UEL), Londrina, PR, Brasil.
Received 07.29.09 Approved 07.30.10 Returned from the author 08.27.10 CR-2256
Table 1--Subtilisin-like (Pr1) activity of Nomuraea rileyi NR458 after growth in different carbon sources. Strain NR458 Time(h) MMI+CAS pH 7.0 pH 8.0 168 Zero 0.263 [+ or -] 0.14 (a) 192 0.376 [+ or -] 0.32 (b) 0.420 [+ or -] 0.17 (c) 216 0.370 [+ or -] 0.05 (d) 0.694 [+ or -] 0.07 (e) MMI+CUT 168 1.694 [+ or -] 0.64 (a) 3.707 [+ or -] 1.98 (b) 192 3.446 [+ or -] 0.08 (c) 5.031 [+ or -] 0.03 (d) 216 4.612 [+ or -] 0.79 (e) 5.210 [+ or -] 0.11 (f) MMI+EXU 168 0.246 [+ or -] 0.03 (a) 0.339 [+ or -] 0.09 (a) 192 0.159 [+ or -] 0.05 (b) 0.258 [+ or -] 0.01 (c) 216 0.58 [+ or -] 0.12 (d) 0.302 [+ or -] 0.02 (e) Time(h) MMI+CAS pH 8.5 168 0.379 [+ or -] 0.22 (a) 192 0.474 [+ or -] 0.23 (c) 216 0.628 [+ or -] 0.39 (e) MMI+CUT 168 3.321 [+ or -] 1.90 (b) 192 4.604 [+ or -] 0.04 (d) 216 4.742 [+ or -] 0.62 (f) MMI+EXU 168 0.420 [+ or -] 0.10 (a) 192 0.395 [+ or -] 0.07 (c) 216 0.327 [+ or -] 0 (e) MMI+CAS (Minimal medium lacking nitrogen source to which a 1% p/v casein solution. MMI+CUT (Minimal medium to which a 0.5% p/v cuticle solution). MMI+EXU (Minimal medium to which a solution of exuvia). The values are expressed in U (nmol of r-nitroanilide.[mL.sup.-1] [min.sup.-1]). Means followed by the same letter do not differ from each other, at each culture time evaluated by the Tukey test (P<0,05). Table 2--Subtilisin-like (Pr1) activity of Nomuraea rileyi CG434 after growth in different carbon sources. Strain CG434 Time(h) MMI+CAS pH 7.0 pH 8.0 168 0.632 [+ or -] 0.57 (a) 0.512 [+ or -] 0.08 (a) 192 1.4 [+ or -] 0.5 (b) 0.778 [+ or -] 0.22 (c) 216 0.943 [+ or -] 0.11 (d) 0.782 [+ or -] 0.32 (e) MMI+CUT 168 3.644 [+ or -] 0.37 (a) 3.396 [+ or -] 0.57 (b) 192 3.427 [+ or -] 0.81 (c) 3.048 [+ or -] 0.57 (d) 216 3.571 [+ or -] 0.6 (e) 3.633 [+ or -] 1.51f MMI+EXU 168 0.213 [+ or -] 9 (a) 0.18 [+ or -] 0.07 (a) 192 0.362 [+ or -] 0.22 (b) 0.142 [+ or -] 0.02 (c) 216 0.138 [+ or -] 0.03 (d) 0.188 [+ or -] 0.11 (e) Time(h) MMI+CAS pH 8.5 168 0.59 [+ or -] 0.09 (a) 192 0.728 [+ or -] 0.18 (c) 216 1.35 [+ or -] 1.00 (e) MMI+CUT 168 4.876 [+ or -] 0.56 (b) 192 4.782 [+ or -] 0.55 (d) 216 4.857 [+ or -] 0.25f MMI+EXU 168 0.175 [+ or -] 0.05 192 0.134 [+ or -] 0.02 (c) 216 0.134 [+ or -] 0.02 (e) MMI+CAS (Minimal medium lacking nitrogen source to which a 1% p/v casein solution). MMI+CUT (Minimal medium to which a 0.5% p/v cuticle solution). MMI+EXU (Minimal medium to which a solution of exuvia). The values are expressed in U (nmol of r-nitroanilide [mL.sup.-1] [min.sup.-1]). Means followed by the same letter do not differ from each other, at each culture time evaluated by the Tukey test (P<0,05). Table 3--Correlation analysis (Pearson) between the percentage of mortality and the Pr1 activity of strains NR458 and CG434. Medium/pH Strain NR458 Strain CG434 MMI+Cas pH 7.0 0.769 ** 0.577 * MMI+Cut pH 7.0 0.972 ** 0.555 (n.s.) MMI+Exu pH 7.0 0.573 (n.s.) 0.243 (n.s.) MMI+Cas pH 8.0 0.864 ** 0.679 * MMI+Cut pH 8.0 A % 0.863 0.57 (n.s.) MMI+Exu pH 8.0 0.407 (n.s.) 0.49 (n.s.) MMI+Cas pH 8.5 0.718 ** 0.698 * MMI+Cut pH 8.5 0.85 ** 0.582 * MMI+Exu pH 8.5 0.519 (n.s.) 0.373 (n.s.) MMI+CAS (Minimal medium lacking nitrogen source to which a 1% p/v casein solution). MMI+CUT (Minimal medium to which a 0.5% p/v cuticle solution). MMI+EXU (Minimal medium to which a solution of exuvia). (n.s.)--not significant; *--significant (5%); **--significant (1%).