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Revista mexicana de micología
versão impressa ISSN 0187-3180
Rev. Mex. Mic vol.28 spe Xalapa Dez. 2008
Contribuciones
Relationship between protease and chitinase activity and the virulence of Paecilomyces fumosoroseus in Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)
Relación de la actividad de proteasa y quitinasa con la virulencia de Paecilomyces fumosoroseus en Trialeurodes vaporariorum (Hemiptera: Aleyrodidae)
Judith CastellanosMoguel1, 3, Ramón CruzCamarillo1, Eduardo Aranda2†, Teresa Mier3, Conchita Toriello4 *
1 Laboratorio de Enzimas Microbianas, Escuela Nacional de Ciencias Biológicas, IPN, México D.F 11340, México.
2 Centro de Investigación en Biotecnología, UAEM, Cuernavaca 62250, México.
3 Departamento El Hombre y su Ambiente, UAMXochimilco, México D.F. 04960, México.
4 Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM, México D.F. 04510, México.
*Autor para correspondencia:
toriello@servidor.unam.mx
Recibido 20 de agosto 2008
Aceptado 8 de diciembre 2008
Resumen
Se evaluó la actividad de proteasa y quitinasa y la virulencia en ninfas de mosquita blanca de aislados del hongo entomopatógeno Paecilomyces fumosoroseus. La producción de enzimas se ensayó con l8 aislados fúngicos, en un medio sintético líquido adicionado de cutícula de camarón coloidal y de quitina coloidal teñida con azul brillante de remazol como substratos para proteasa y quitinasa, respectivamente. Los bioensayos de virulencia se realizaron en ninfas de mosquita blanca (Trialeurodes vaporariorum) de segundo estadio, con 9 cultivos monospóricos. La virulencia fue determinada a partir de la concentración letal media (CL50) en mosquita blanca. Los aislados EH506/3 y EH511/3 fueron los más virulentos con una CL50 de 1.1 x 103 y 6.9 x 103 conidios/mL, respectivamente, con la más alta actividad de proteasa (39.8y 14.9 PU/mL, respectivamente) a las 120 h del experimento. Las actividades de quitinasa alcanzaron sus valores más altos al final (264 y 312 h) del experimento. Se presenta por primera vez, en una muestra representativa de cepas de P. fumosoroseus aisladas en México, una gran variabilidad intraespecífica en las actividades de proteasas y quitinasas y en la virulencia. Se sugiere que la actividad de proteasas podría ser utilizada en la selección de cepas de este hongo para el control biológico de mosquita blanca.
Palabras clave: control biológico, factores de virulencia, hongos entomopatógenos.
Abstract
The production of protease and chitinase activities of isolates of Paecilomyces fumosoroseus and their virulence in whitefly nymphs were evaluated. Enzyme production was tested in l8 fungal isolates, on synthetic liquid medium with colloidal shrimp cuticle and remazol bright blue stained colloidal chitin as substrates for protease and chitinase activity, respectively. The virulence bioassay was carried out in second stage whitefly (Trialeurodes vaporariorum) nymphs in 9 monospore cultures of selected isolates. The virulence was determined by the median lethal concentration (LC50) in whiteflies. Isolates EH506/3 and EH511/3 with the highest virulence (LC50 of 1.1 x 103 and 6.9 x 103, respectively) also showed the highest protease activity (PU/mL of 39.8 and 14.9, respectively) at 120 h. Chitinases reached maximum activities at the end (264 and 312 h) of thein vitro enzymatic assay. This is the first time that an ample intraspecific variability in protease and chitinase activities as well as in virulence has been reported in a representative sample of P. fumosoroseus strains from Mexico, which suggest that protease activities could be used in the selection of candidates for whiteflies biological control.
Key words: biological control, virulence factors, entomopathogenic fungus.
Introduction
Unlike most other pathogens that need to be ingested, fungi usually infect insects by active penetration through the cuticle, being able to control insects independent of their feeding habits (Smith and Grula, 1983). Insect cuticle is the first barrier that fungi encounter, therefore a wide variety of extracellular enzymes are synthesized for the enzymatic processes involved in the degradation of protein, chitin and lipids, principal cuticle components (Charnley and St. Leger, 1991; St. Legeretal., 1986a; St. Legeretal., 1986b; St. Leger et al., 1996). Different studies suggest proteases and chitinases as major determinants of fungal virulence in the complex and multifactorial phenomenon insect host/pathogen relationship (Fang et al., 2007; Gillespie et al., 1998; Gillespie et al., 2000; Gupta et al., 1994; Paterson, 1994; St. Leger, 1995; Yang et al., 2007). Recently CastellanosMoguel et al. (2007) have pointed subtilisinelike (Pr1) protease as a phenotypic marker of virulence for Paecilomyces fumosoroseus (Wize) Brown & Smith.
Paecilomyces fumosoroseus, a mitosporic ascomycete, has been successfully used as a biocontrol agent of whiteflies (Hemiptera: Aleyrodidae) and other insect pests (Butt et al., 2001; Shah and Pell, 2003). In Mexico, the whitefly is considered an important pest of vegetable crops, and of ornamental plants (Mier et al., 1991; RamírezVillapudua, 1996). Due to the relevance of enzymatic activities for fungal penetration in the pathogenic mechanism to attack the host and the importance of virulent strains for an efficient biological control, in this study, protease and chitinase activities was measured in 18 isolates of P. fumosoroseus from whiteflies in Mexico, as well as their relationship in fungal virulence, to be able to select the most suitable strains for biological control of whiteflies adapted to Mexican environmental conditions.
Materials and methods
Fungal isolates
All Paecilomyces fumosoroseus strains were isolated from whiteflies and obtained from the National Center for Biological Control, Mexico (Centro Nacional de Referencia de Control BiológicoCNRCB, SAGARPA, Tecomán, Colima) (Table 1). Single spore cultures were prepared as initially described by Goettel and Inglis (1997) and later modified by Cavallazzi et al. (2001). Monospore cultures (MCs) were preserved in sterile water, mineral oil, and in liquid nitrogen cryopreservation at 196 °C and deposited in the fungal collection of the Basic Mycology Laboratory, Microbiology and Parasitology Department, Faculty of Medicine, National Autonomous University of Mexico (UNAM) and in the Mycology Laboratory, Depto. El Hombre y su Ambiente, División de Ciencias Biológicas y de la Salud, Metropolitan Autonomous UniversityXochimilco (UAMX). Isolates were maintained on Sabouraud dextrose (SAB, Bioxon, Mexico) slants until used.
Polyspore original cultures were used for the determination of enzyme activities. Nine MC were selected for whitefly bioassays based on high, medium and low protease and chitinase activity (CastellanosMoguel et al., 2001) to investigate their relationship with virulence. Lethal median concentration (LC50) of three of these nine MC were previously reported (CastellanosMoguel et al., 2007).
Shrimp cuticle and chitin substrates
Colloidal shrimp cuticle and remazol bright blue stained colloidal chitin were used as substrates for protease and chitinase activity production, respectively. Both, shrimp cuticle and commercial chitin were treated with phosphoric acid (10 g/100 mL) overnight. Then, several water washings and filtrations were performed until a pH 7 was reached. Substrates were then sterilized and kept at 4 °C (ChávezCamarillo y CruzCamarillo, 1984). Remazol bright blue stained chitin was prepared by the addition of 0.23 g of bright blue remazol diluted in water to 7 g of colloidal chitin, mixed and heated for 1 h, then filtrated and washed. After, 5 g of stained colloidal chitin with 0.075 g of sodium dichromate and 0.075 g of sodiumpotassium tartrate was boiled (10 min), washed again, and sterilized by autoclave at 121 °C for 15 min (GómezRamírez, 2000).
Enzyme activity assays
For all shakeflask enzyme activity assays, cultures of P. fumosoroseus were grown in 125mL Erlenmeyer flasks containing 25 mL of synthetic liquid medium (per liter: 0.375 g of KH2PO4; 0.375 g of Na2CO3; 0.250 g of NaCl; 0.275 g of MgSO4) supplemented with 14% colloidal shrimp cuticle or 1% of remazol bright blue stained colloidal chitin as substrates, for protease or chitinase production, respectively. Inocula for both enzyme activities consisted of conidia from 10day old slant SAB medium cultures with a final density of 1 x 106 conidia/mL. Flasks were incubated with orbital shaking (150 rpm) at 28 °C, for 312 h (13 days). Control flasks of liquid medium without inocula were maintained under the same experimental conditions. All experiments were reproduced at least three times.
At 24, 72, 120, 168, 216, 264 and 312 h, 3ml aliquots of cultures for protease and chitinase activities and controls were collected. Samples were centrifuged (5,000 g, 10 min) and cellfree supernatants used immediately for chitinase activity determination or maintained at 20 °C until protease activity was determined.
A nonspecific proteolytic assay with azocasein was performed for cellfree supernatants and controls of synthetic liquid medium supplemented with colloidal shrimp cuticle, according to Sarath et al. (1989), and described in Castellanos et al. (2007). Briefly, 2% azocasein was dissolved in phosphate buffer pH 7.0. 150 µL of cellfree supernatant (enzyme) and 250 µL of substrate were incubated at 25°C for 1 h. Reaction was stopped with 1.2 mL of 10% trichloroacetic acid for 15 min. The reaction mixture was centrifuged at 10,000 rpm for 5 min, NaOH was added to develop color and absorbance read at 440 nm. One unit of protease activity (PU) was defined as the amount of enzyme that produced a change of 0.010 in optical density at 440 nm.
Chitinase activity was determined for cellfree supernatants and controls of synthetic liquid medium supplemented with remazol bright blue stained colloidal chitin, by the color development from the dye liberation by chitinase, and absorbance was read at 595 nm in a Beckman DU650 spectrophotometer (Beckman Instruments, CA, USA). One unit of chitinase activity (CU) was defined as the amount of enzyme that produced a change of 0.010 in optical density at 595 nm.
Insects
The whitefly nymphs (Trialeurodes vaporariorum) used for bioassays originated from colonies maintained at the greenhouse and experimental field of the Center for Biotechnology Research, Autonomous University of the State of Morelos (CEIBUAEM), Mexico. Whiteflies were reared on "flor de mayo" bean (Phaseolus vulgaris) leaves.
Bioassay procedure
The method used was according to Vidal et al. (1997), with minor modifications as described in Castellanos et al. (2007). Fungal conidia were produced in SAB medium cultures, and five doses ranging from 4.7 x 102 to 4.7 x 106 conidia/mL in 0.05% sterile Tween 80 (v/v) were used and only 0.05% sterile Tween 80 for controls.
Experiments were conducted during a total of 10 days. Individual selected nymphs were monitored for mortality at day 10. Emerged whiteflies (empty pupal cases) were considered noninfected. A total of 75 nymphs were used, 25 for each replicate bioassay, and another 25 nymphs as control, for each conidial concentration tested. The number of nymphs of second instar that did not change to the next developmental stage was recorded (dead nymphs). All nymphs were then removed from the leaf surface, placed on sterile water agar dishes, and incubated at 24 °C for 57 days to determine the percentage of mortality caused by mycosis, based on fungal sporulation on the insects. Nymphs that remained in second instar, were considered dead, and lethal median concentration (LC50) was calculated using second instar data.
Chemicals
Unless otherwise stated, all chemicals used were obtained from SigmaAldrichQuímica (Toluca, Mexico).
Statistical analysis
Mortality of second stage nymphs was subjected to Probit analysis (Finney, 1971), generating a concentrationmortality relationship for the estimates of LC50 and its 95% confidence intervals for each of the MC tested, using POLOPC program (1996). The data of protease and chitinase activities were analyzed using ANOVA (α = 0.05) and a Tukey multiple mean comparison test (Dowdy and Wearden, 1983). Statistical analyses were performed using the SPSS Program, version 10, 2003.
Results
Enzymatic activity
Protease activity, in culture supernatants of synthetic liquid medium supplemented with colloidal shrimp cuticle, was detected at 72 h in almost all the isolates tested, except for EH515 (data not shown). During the course time of the experiment (0312 h), the highest protease activities were observed at 120 (F17, 53=190.2; ρ<0.05), 168 (F17,53=120.6; ρ<0.05), 264 (F17,53=67.7; ρ<0.05) or 312 h (F17,53=129.3; ρ<0.05) for all isolates (Table 2). In six of 18 isolates (EH506, EH511, EH512, EH514, EH517, EH518) a peak of maximum activity was reached at 120 h. In the rest of the isolates, levels of maximum activity varied at 168, 264 and 312 h of the experiment. The highest significant (ρ<0.05) activity was registered by EH506, which exhibited 39.8, 23.6, 23.5 and 25.0 PU/mL at 120, 168, 264 and 312 h, respectively (Table 2).
Chitinase activity in culture supernatants of synthethic liquid medium supplemented with remazol bright blue stained colloidal chitin was detected since 72 h but in lower levels than protease activity (data not shown). Chitinase activities were detected at 120 (F17,53= 26.8 ρ<0.05), 168 (F17,53=52.6; ρ<0.05), 264 (F1753 = 89.1; ρ< 0.05) and 312 h (F17,53= 39.9; ρ<0.05). In general, the highest activities were reached at the end of the experiment, specifically at 264 or 312 h. Only one isolate, EH511, had the maximum activity at 168 h (Table 3). The highest activity was exhibited by EH503 with 28.1, 47.2 and 90.4 CU/mL at 120, 168 and 264 h respectively, and by EH518 (62.3 CU/mL) at 312 h.
Nine isolates (EH503, EH504, EH506, EH509, EH511, EH516, EH518, EH519, EH520) were selected according to a high, medium and low protease and chitinase activities to obtain monospore cultures (see Table 4) and perform the virulence bioassay with second instar whitefly nymphs.
Virulence bioassay
The results of LC50 of monospore isolates EH506/3 (high virulence), EH503/3 (medium virulence) and EH520/3 (low virulence) were reported previously in Castellanos et al. (2007), but for a better understanding of enzyme activities in our present results we have included them with the other six monospore isolates of this work.
All MCs infected second instar nymphs exhibited different virulences (Table 4). The mortality criterion of whiteflies was instar change, because nymphs are immobile, and at day 10, nymphs that had not shown change from second to third instar, were considered dead. At the end of the experiment (day 10), selected nymphs were transferred to wateragar and all of them showed mycelial growth, suggesting an insect mycosis and considered killed by the fungus. Control nymphs reached the adult stage in the same time period of the bioassay. Considering high virulence isolates those with the lower conidia concentration required to kill 50% of insects, LC50 calculated showed high virulent isolates as EH506/3 and EH511/3; medium virulence isolates as EH518/6, EH503/3, EH509/3; and low virulence isolates as EH519/2, EH520/3 and EH516/2 (Table 4). Isolate EH504/1 exhibited a very low virulence because the LC50 could not be calculated with the five conidial concentrations used in the bioassay (Table 4).
Insect instar percentage change was recorded for all isolates, the percentage of nymphs that remained in second instar corresponded mainly to nymphs infected with the higher concentrations, 4.7 x 105 and 4.7 x 106 conidia/mL. For each MC the higher conidial concentrations stopped instar change, while lower conidial concentrations permitted the changes. The most virulent isolates (EH506/3 and EH511/3) showed that more than 50% of second instar nymphs remained in this instar, even with low conidial concentrations (4.7 x 103), while medium and low virulence isolates, less than 50% of nymphs remained in second instar. In comparison, the percentage of nymphs that reached fourth instar, before changing to adults, are shown in Figure 1, and the high virulence isolates (EH506/3 and EH511/3) exhibited very low percentages (<20%) that reached fourth instar. The conidial concentrations that allowed nymphs to reach fourth instar were 4.7 x 102, 4.7 x 103 and 4.7 x 104 conidia/mL (Figure 1).
The maximum activity time and PU or CU/mL of protease and chitinase activity, respectively, with virulence LC50 is shown in Table 4. Protease production (39.8 and 14.9 PU/mL) of high virulence isolates (EH506/3 and EH511/3, respectively) was reached early in the experiment (120 h). In medium and low virulence isolates, with the exception of EH518/6 and EH504/1, protease was produced later in the experiment (168, 264 and 312 h). High chitinase production of all isolates, with exception of the high virulence isolate EH511/3, was observed at the end of the experiment at 264 and 312 h.
Discussion
The whitefly life cycle is approximately between 5 and 11 days depending on the insect species and environmental conditions (Byrne and Bellows, 1991). At this time (120 h) in vitro protease activity was the highest for the two most virulent isolates of our study and agrees with St. Leger et al. (1996) that report an early protease cuticular degradation allowing a faster colonization of the host. The tendency of the protease and chitinase activities in submerged cultures of the isolates, reflect a starting protease followed by the chitinase activity. The most virulent isolates (EH506/3 and EH511/3) had an early high production of protease (Table 4).
Concerning the other enzymatic activity, high chitinase appeared, in almost all isolates, at the end of the experiment (264 and 312 h). In other entomopathogens, i.e., Metarhizium anisopliae, high chitinase activities have been observed at the end of the experimenting cultures when the fungus was grown with chitin as the sole source of carbon and nitrogen (ChulKang et al., 1999).
The LC50 of isolate EH504/1 could not be calculated with the five P. fumosoroseus conidial concentrations assayed and used for the other isolates. This fact points out to the very low virulence of this isolate, that would probably be in need of higher conidial concentrations to achieve the LC50.
The different concentrations and appearance of both relevant enzymes (protease and chitinase) for fungal pathogenesis of insects (St. Leger et al., 1986a; 1986b; 1987) in liquid cultures with the specific substrates shows the variability among different isolates of the same fungus. The same was observed concerning the virulence of the studied isolates.
Fungal pathogenesis of insects involves many different factors among host, parasite and environment. In the initial contact between fungus and insect, after the adhesion process, the penetration of the fungal pathogen into the cuticle depends relevantly on the action of the enzymatic activities (St. Leger et al, 1986a; 1986b; 1987). Under the experimental conditions of this study, the results suggest a relationship between a high protease activity and the high virulence isolates, EH506/3 and EH511/3. Protease and chitinase activities in entomopathogenic fungi are important due to the great amount of protein and chitin in insect cuticle (St. Leger et al., 1986a).
The development of mycoinsectides depends largely on the selection of a virulent strain for the insect pest, among other characteristics. This virulence trait must be well conserved in the selected strain, thus different authors have shown that monospore cultures are capable of decreasing the danger of a potential variation because of its homogenous genetic fungal population (Veen, 1967; Samsinakova y Kalalova, 1983). In a recent study, CortezMadrigal et al. (2003) also showed differences with the median germination time and conidial production between monospore and multispore cultures of Lecanicillium lecanii, monospore cultures exceeding their multispore isolates. The abovemetioned facts support the use of monospore cultures in order to attain a homogenous genetic fungal population in candidate strains for biological control.
In addition, the total protease and chitinase activities determined in the original multispore isolates are representative of the enzymatic potential of the monospore cultures due to their homogenous genetic population.
Broadspectrum proteases are the main enzymes produced by entomopathogens during infection and degradation of the insect cuticle (St. Leger, 1995), and this protease activity has been related to virulence (Bidochka et al., 1999; Jackson et al., 1985; St Leger et al., 1986a; 1986b; 1987). However, this is the first time that an ample intraspecific variability in protease and chitinase activities as well as in virulence has been reported in a representative sample of P. fumosoroseus strains from Mexico, which suggest that protease activities could be used in the selection of candidates for whiteflies biological control.
Acknowledgments
The authors thank Víctor Hernández Velázquez and Angélica Berlanga from the National Reference Center on Biological Control, Mexico (Centro Nacional de Referencia en Control Biológico, México), for the original P. fumosoroseus isolates, and Laura Lina for the maintenance of whitefly colonies. This research was supported by Grant No. G31451B from CONACyT, Mexico. JCM acknowledges Conacyt the granted M. in Sc. scholarship. This research is part of JCM's M. in Sc. Thesis, student of the Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional.
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