- Email: [email protected]
Oxygen Consumption by Metarhizium anisopliae during Germination and Growth on Different Carbon Sources
Journal of Invertebrate Pathology 74, 112–119 (1999) Article ID jipa.1999.4872, available online at http://www.idealibrary.com on
Oxygen Consumption by Metarhizium anisopliae during Germination and Growth on Different Carbon Sources Gilberto U. L. Braga,1 Ricardo H. R. Deste´fano, and Claudio L. Messias Departamento de Gene´tica e Evoluc¸a˜o, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, CEP 13083-970, SP, Brazil Received July 6, 1998; accepted April 14, 1999
Respirometry was used to monitor the germination and growth of the entomopathogenic deuteromycete Metarhizium anisopliae on media containing carbon sources of different kinds (monosaccharides, polysaccharides, amino acids, and proteins). As also observed in several other species of fungi, M. anisopliae germination was found to be marked by a significant increase in O2 consumption, which started a few hours before germ tube emergence. The exponential consumption of the carbon source and O2 coincided with the exponential growth phase of the cultures. QO2 reached a maximum value during the exponential growth phase and was drastically reduced after the depletion of the exogenous carbon source. Taking glucose as reference, we observed that casein, hydrolyzed casein, and Nacetylglucosamine accelerated germination, reduced the lag phase, and increased the growth rate. This fact demonstrates that the fungus can readily use amino acids and N-acetylglucosamine, which are the monomers of the major constituents of the insect cuticle (proteins and chitin), a property that represents an important physiological adaptation to entomopathogenicity. r 1999 Academic Press Key Words: Metarhizium anisopliae; fungal germination; fungal growth; energy requirement; respirometric analysis.
INTRODUCTION
The understanding of the basic aspects of germination and growth of entomopathogenic hyphomycete fungi has greatly helped programs seeking to use these organisms as biological control agents. Some entomopathogenic fungi, like Metarhizium anisopliae, must be adapted to two very different situations: they must be able to utilize different types of organic substrates as a source of energy and nutrients
1 To whom correspondence should be addressed at current address: Department of Biology, Utah State University, Logan, UT, 843225305, U.S.A.
0022-2011/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
during their saprophytic life, and they must also be able to establish a link with and germinate on a susceptible host insect using the nutrients present on its cuticle or the substances mobilized from the latter by the action of hydrolytic exoenzymes in the early stages of infection (St. Leger et al., 1987, 1991; Goettel et al., 1989; Braga et al., 1994). Several studies have shown that the mechanisms of host recognition are linked, at least in part, to the chemical constitution of the cuticle or to the types of nutrients available on it. Boucias and Latge´ (1988) reported that cuticle extracts were able to induce germination in the fungi Conidiobolus obscurus and Nomuraea rileyi. Woods and Grula (1984) observed that amino acids and glucosamine are present on the surface of Heliothis zea in sufficient amounts to support the germination and limited growth of Beauveria bassiana. St. Leger et al. (1992) observed marked differences in nutritional requirements for germination between strains of M. anisopliae var. anisopliae and M. anisopliae var. majus isolated from different hosts. Samuels et al. (1989) attributed the high specificity of various strains of M. anisopliae against Oryctes rhinoceros to the presence of indeterminate nutrients on or inside the host’s cuticle. Butt et al. (1995) observed that the high availability of nutrients on the cuticle of aphids stimulates the rapid germination of M. anisopliae conidia. The virulence of entomopathogenic deuteromycetes has been correlated with their rapid germination and high growth rate (Al-Aidroos and Seifert, 1980; Samuels et al., 1989; Hassan et al., 1989; Dillon and Charnley, 1985, 1990). The importance of the speed of germination can be attributed to several factors. During the period of conidium exposure on the insect’s cuticle before penetration, the fungus may suffer desiccation, antibiosis of saprophytic microorganisms, inhibition by cuticular lipids, or removal together with the cuticle during the molts. According to Hassan et al. (1989), any method that would reduce the time of germination on the host’s cuticle may increase the efficiency of a mycoinsecticide. Since the growth of an entomopathogen, especially during infection of the host, occurs by the utilization of complex substrates (such as cuticle, internal tissues,
112
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
and hemolymph), an understanding of this type of situation may facilitate the understanding of its pathogenicity. Campbell et al. (1978) emphasized that studies on fungal nutrition and growth could help in the production of large quantities of virulent inoculum and in the appropriate selection of virulent strains. The differential utilization of carbon sources has also helped phylogenetic and strain characterization studies on the genus Metarhizium (Li and Holdom, 1995; Rath et al., 1995). The aim of this study was to determine the basic parameters of germination and growth of M. anisopliae on different organic substrates. MATERIALS AND METHODS
Maintenance of Fungi and Inoculum Preparation The strain used in the respirometric evaluations was CLII, obtained from the germ plasm bank of the laboratory of entomopathogenic fungi of the State University of Campinas (UNICAMP). This strain was isolated from the sugar cane borer Mahanarva posticata (Homoptera, Cercopidae) in the state of Alagoas, Brazil. For conidial production, the strain was grown for 12 days at 28°C on plates containing complete solid medium (CSM) (Pontecorvo et al., 1953). The conidia were suspended in a solution containing 0.85% (w/v) NaCl and 0.002% (v/v) Tween 80. The conidial suspension was then filtered through multiple gauze layers, and the conidia were washed twice and resuspended in a 0.85% NaCl (w/v) solution. Conidial concentration was adjusted to 3 ⫻ 107 ml⫺1. Substrates Evaluated Oxygen consumption during germination and the subsequent phases of culture development was evaluated in media containing different organic substrates which served as the only carbon and energy source. The following substrates were used: (a) sugars: glucose, N-acetylglucosamine (Sigma), and chitin (purified powder from crab shells; Sigma) prepared as described by Braga et al. (1998); (b) proteins: casein (Hammarsten; vitamin free), hydrolyzed casein (Bacto casamino acids; Difco), gelatin (type A from porcine skin; Sigma), and elastin (a powder from bovine neck ligament; Sigma); and (c) amino acids: L-cystine, DL-serine, L-proline, Dproline, L-alanine, L-glutamic acid, and L-tyrosine (Sigma). The substrates were individually added to minimum liquid medium (MLM) (Pontecorvo et al., 1953) with no glucose, at the concentration of 0.25% (w/v). The pH was adjusted to 6.5 in all cases using 2 N NaOH. The media were autoclaved in 50-ml volumetric round flasks at 121°C for 15 min. After autoclaving, the volume was readjusted with sterile distilled water.
113
Determination of Oxygen Consumption Oxygen consumption was determined with a Gilson differential respirometer. The respirometer has 14 reaction flasks connected to individual reference chambers through a differential manometer. The reference chamber eliminates the effect of variations in atmospheric pressure. The variation in the internal pressure of each reaction flask due to oxygen consumption can be monitored with digital micrometers. The entire system remains immersed in water at controlled temperature and can be submitted to shaking. The basic procedures adopted in the respirometry experiments were described by Umbreit et al. (1972). In order to retain the CO2 released during respiration, 400 µl of a 30% KOH solution (w/v) was used and distributed as follows: 200 µl were placed in the central vessel of the reaction flask and 200 µl were placed in the lateral arm. In order to increase the retention surface, rectangular pieces of filter paper were placed in the central vessel (3 ⫻ 2 cm) and in the lateral arm (2 ⫻ 1 cm). An aliquot of 4.5 ml of culture medium and 0.5 ml inoculum were added to each reaction flask so that the final concentration of the carbon source was 0.225% (w/v) and the conidial concentration was 3 ⫻ 106 ml⫺1. The respirometer was prepared and turned on 1 h before the beginning of the experiment so that the entire system could enter thermal equilibrium. During the experiments, the system was maintained at 28°C under shaking at 50 oscillations/min. Readings were taken directly as microliters of consumed oxygen at mean intervals of 2 h. As a control, the conidial suspension was inoculated into MLM without the exogenous carbon source in order to estimate endogenous conidial respiration. The experimental design was completely randomized with three replications. Data were submitted to analysis of variance. One analysis was performed for each postinoculation period. The Tukey test was used for comparing treatment means (Steel and Torrie, 1960). Culture Filtrates and Dry Mass Determination When necessary, the dry mass produced in the respirometry experiments was determined as described below. The reaction flasks were disconnected from the apparatus and the culture medium was removed and filtered through previously washed and weighed paper (Inlab type 10). The KOH solution and the filter papers were then carefully removed. The central vessel and the lateral arm were washed with distilled water and dried on absorbent paper. The remainder of the mycelium was then removed, washed with distilled water, and dried for 72 h at 70°C. The presence of possible contaminants was determined by observations under the microscope and by plating aliquots of the medium onto plates containing CSM. The concentration of residual glucose present in medium containing glucose as carbon source was deter-
´ FANO, AND MESSIAS BRAGA, DESTE
114
mined throughout growth and autolysis as described by Nelson (1944). Determination of Germination Specific experiments were conducted with a respirometer to monitor conidial germination in media containing the various carbon sources and in the control. After periods of incubation ranging from 6 to 14 h, aliquots were removed from the media and three random samples of at least 100 conidia were observed to determine the germination percentage. The conidia were considered to have germinated when they presented germ tubes greater than the diameter of the conidia. RESULTS AND DISCUSSION
Oxygen Consumption, Glucose Consumption, and Biomass Production The growth of a filamentous fungus can be evaluated in a direct manner on the basis of linear growth, fresh biomass, dry mass, or the amount of some mycelial constituents, or in an indirect manner on the basis of metabolic activities, such as respiratory activity (O2 consumption or CO2 release), formation of products such as acids or pigments, substrate consumption, or specific enzyme activities. Respirometry has been widely used both to monitor colony development and to evaluate the utilization of specific substrates by fungi (Lyda, 1976; Belsky et al., 1984; Taber and Taber, 1982, 1987; Hill et al., 1992; Janda et al., 1993; Tan and Moore,
1995). Since it is not a destructive technique and since it permits continuous evaluation, respirometry permits the construction of much more detailed growth curves than those obtained by analysis of biomass production. The dry mass production and glucose consumption by strain CLII growing in medium whose initial glucose concentration was 2.25 mg ml⫺1 are shown in Fig. 1. Maximum biomass production was reached 96 h postinoculation and coincided with the depletion of the carbon source. Autolysis started soon after the depletion of the exogenous carbon source and, as observed in other experiments, provoked a reduction in mycelial mass of more than 60% (G. U. L. Braga, unpublished results). Oxygen consumption under the same growth conditions and oxygen consumption by the conidia in the absence of the exogenous carbon source are shown in Fig. 2. It can be seen that, after a phase of acceleration lasting about 40 h, oxygen consumption increased exponentially up to the 90th hour and was abruptly reduced thereafter. Biomass and oxygen consumption increased concomitantly in an exponential manner (Figs. 1 and 2). The depletion of the exogenous carbon source coincided with the abrupt fall in oxygen consumption. The oxygen utilized for the oxidation of endogenous conidial reserves represented only 4.6% of total consumption. Analysis of variance showed that a significant increase compared to control in conidial respiration occured after the 7th hour in medium containing glucose (F ⫽ 28.09, significant at the 0.01 level of probability). The increase in respiratory activity occurred several
FIG. 1. Dry mass production and glucose consumption in a respirometer by strain CLII growing in medium containing 0.225% glucose (w/v) as the only carbon source. Each point represents the mean of two replicates. The standard errors of the means were 0.58 for dry mass production and 0.11 for glucose consumption.
115
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
FIG. 2. Oxygen consumption by strain CLII growing in medium containing 0.225% glucose (w/v) as the only carbon source. The graph shows the results of three replicates of the same experiment and respiration in the absence of an exogenous source. The standard errors of the means were 46.64 for glucose and 6.42 for the control.
hours before the germ tube emergence. The increase in respiration is one of the more marked physiological changes occurring during the germination of fungal spores (Mandels et al., 1956; Cochrane et al., 1963; Martı´n and Nicola´s, 1970; Hill et al., 1992). After 14 h, only 3.9% of the conidia incubated in media containing glucose produced a detectable germ tube (Table 1). The emergence of the germ tubes could not be monitored for a longer period of time due to the strong aggregation of conidia and germinants during development in liquid medium under shaking (Dute et al., 1989; Milner et al., 1991). The respirometry data show that it is possible to detect the physiological effect of the exogenous carbon source on the germlings a few hours before the germ tube emergence. There was a linear increase in oxygen consumption up to the 36th hour, followed by an exponential increase up to the 72nd hour. A gradual decrease occurred thereafter until the end of the experiment (Table 2). As shown in Fig. 1, after 96 h of growth, exogenous glucose was already depleted. On this basis, we may assume that, starting from that point, mycelial respiration occurred at the expense of its endogenous reserves accumulated during the growth phase. Table 2 also
shows the estimates of QO2 during the various stages of culture development. The evaluations performed during the 31st h of growth (9.1 µl O2 mg⫺1 h⫺1 ) and the 51st h (9.7 µl O2 mg⫺1 h⫺1 ) showed that QO2 apparently remained constant during this period. The highest QO2 value (30.9 µl O2 mg⫺1 h⫺1 ) was observed during the 73rd h, in the full exponential growth phase. The QO2 values estimated after depletion of the exogenous carbon source were perceptibly lower and decreased until the end of the experiment, clearly indicating a progressive depletion of the endogenous reserves of the mycelium. Oxidative Metabolism on the Different Carbon Sources Figure 3 shows oxygen consumption by strain CLII during germination and development on the different carbon sources. The most interesting facts were the great reduction in lag phase, the increase in growth rate, and the slight increase in total oxygen consumption provoked by casein compared to the growth curve on medium containing glucose. This type of response
TABLE 1 Percentage Germinating Conidia of Strain CLII in Medium Containing 0.225% (w/v) of the Substrates Indicated as the Only Carbon Source
8h 14 h
Basal
Glucose
Casein
Gelatin
Hydr.Cas
N-acetyl.
Chitin
0.0 (0.0) 0.3 (0.5)
0.0 (0.0) 3.9 (0.9)
8.8 (2.0) 47.5 (0.6)
0.3 (0.5) 3.1 (2.0)
4.0 (1.1) 28.6 (0.8)
2.1 (1.9) 8.8 (1.1)
0.3 (0.5) 4.6 (1.3)
Note. Each point represents the mean of three replicates. The standard deviations are given in parenthesis.
´ FANO, AND MESSIAS BRAGA, DESTE
116
TABLE 2 Total Oxygen and QO2 Consumption during Different Periods of Growth and Autolysis of Strain CLII in Medium Initially Containing 0.225% Glucose (w/v) as the Only Carbon Source Period (h)
O2 consumption (µl/h)
0–12 12–24 24–36 36–48 48–60 60–72 72–84 84–96 96–108 108–120 120–132 132–144 144–156 156–168
3.8 (1.1) c 5.4 (1.8) 9.5 (1.7) 17.35 (3.2) 55.2 (8.5) 89.3 (4.6) 61.6 (11.4) 24.17 (3.7) 12.3 (1.6) 9.8 (1.4) 6.2 (0.3) 4.4 (0.8) 3.5 (0.7) 2.3 (0.9)
QO2 (µl/mg h) a,b
(30–31) 9.1 (50–51) 9.7 (72–73) 30.9 (102–103) 2.0
(139–140) 0.9 (171–172) 0.2
Note. Each value represents the mean of three replicates. a We used the oxygen consumption of the last hour immediately preceding the determination of dry matter. The indication of the hour appears in parenthesis. b Each value represents the mean of two replicates. c Standard deviation.
characterizes the presence of one or more nonessential growth factors, which, however, had promoting actions (Fries, 1965). The nature of this promoter has not been established; it could be a peptide, an amino acid, or an appropriate set of amino acids initially present in the medium or released during casein hydrolysis. Studies
of amino acid metabolism in fungi have shown that some amino acids can act as precursors for the synthesis of a family of amino acids. On this basis, we may assume that in media containing these amino acids or an appropriate amino acid combination, the work for protein biosynthesis will be considerably reduced (Lilly, 1965; Tripp and Paznokas, 1982). This may explain the reduction in the duration of the lag and acceleration phases and the increase in growth rate on medium containing casein. These ideas are supported by the observation that amino acid mixtures are frequently better nitrogen sources than a single amino acid alone (Smith and Grula, 1981; Li and Holdom, 1995). Some studies have associated the high virulence of certain M. anisopliae strains with their rapid germination and high growth rate (Al-Aidroos and Seifert, 1980; Samuels et al., 1989; Hassan et al., 1989). The fact that casein stimulated the germination and early development of the fungus opens perspectives for the use of this substance to obtain more efficient commercial formulations of M. anisopliae spores. The other protein substrates evaluated (gelatin and elastin) did not produce the same effects. Both required a longer acceleration phase than observed with glucose and, in the case of gelatin, there was also a significant reduction in growth rate. Total oxygen consumption with the two substrates, as observed with casein, was slightly higher than observed with glucose. These results show that the nature of the protein utilized as carbon source directly affects the duration of the lag and acceleration phases and the growth rate. The difference in amino
FIG. 3. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v) and in control media (endogenous). Each point represents the mean of three replicates. The standard errors of the means were 66.13 for casein, 53.09 for gelatin, 68.09 for elastin, 19.56 for chitin, 46.64 for glucose, and 6.48 for the control.
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
117
FIG. 4. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v). Each point represents the mean of three replicates. The standard errors of the means were 46.64 for glucose, 19.56 for chitin, and 29 for N-acetylglucosamine.
acid composition, as well as the differences in solubility, may explain part of the variations in the growth rates on the three protein substrates. Figure 3 also shows that growth on chitin was characterized by the long duration of the acceleration phase. Cultures were still in acceleration phase until 7 days postinoculation.
However, it was observed that conidia increased their respiratory rate in medium containing chitin, this being the major characteristic of the break of dormancy, which was more rapid than in the presence of glucose. Total oxygen consumption in medium containing chitin was higher than observed in glucose for several hours
FIG. 5. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v). Each point represents the mean of three replicates. The standard errors of the means were 66.13 for casein, 65.72 for hydrolyzed casein, and 46.64 for glucose.
118
´ FANO, AND MESSIAS BRAGA, DESTE
(Fig. 4). Chitin, when autoclaved in liquid medium, undergoes some hydrolysis with release of the Nacetylglucosamine monomer (Smith and Grula, 1983), which may have stimulated germination of M. anisopliae conidia. By being present at low concentration in the medium, it was rapidly depleted and the germination and/or development of the conidia was interrupted. Germination and development resumed only after the mobilization of the substrates by the germlings and adaptation to this situation probably was the factor responsible for the long duration of the acceleration phase. The presence of N-acetylglucosamine in the medium accelerated germination and reduced the duration of the lag and acceleration phases (Fig. 4). Although casein and hydrolyzed casein accelerated germination and development, these effects were more pronounced when casein was used as a carbon source (Fig. 5). The germ tube emergence also occurred earlier in medium containing casein than in medium containing hydrolyzed casein (Table 1). Oxidative metabolism was also studied when the major amino acids present in casein (Nolan, 1971) were utilized as the only carbon source. Some of these amino acids, such as alanine, are also present in substantial amounts in the proteins existing in the cuticle of insects (St. Leger et al., 1986; Andersen et al., 1995). None of the amino acids used individually as carbon source reproduced the effects of casein or hydrolyzed casein on germination and growth. The amino acids used as carbon sources proved to be inadequate for germination and growth of the CLII strain, causing the cultures to remain in the lag phase for several days. Analysis of variance showed a significant difference in oxygen consumption among the different amino acids (F ⫽ 13.16, significant at the 0.01 level of probability). The highest consumption after 50 h was observed for L-glutamic acid (325 ⫾ 92 µl), L-alanine (187 ⫾ 9 µl), and L-cystine (179 ⫾ 20 µl) and the lowest for L-tyrosine (108 ⫾ 20 µl), L-proline (108 ⫾ 9 µl), D-proline (109 ⫾ 5 µl), and DL-serine (124 ⫾ 22 µl). The least significant difference (LSD; Tukey’s test at 0.01 level of probability) for comparing treatment means was 133. The inability to germinate and develop on individual amino acids cannot be generalized. Some studies have reported the ability of other M. anisopliae strains to germinate and develop utilizing different amino acids at different concentrations as the only carbon source (Campbell et al., 1978; St. Leger et al., 1986, 1989). Growth curves for the fungus M. anisopliae presenting this level of resolution are reported here for the first time. Culture development was monitored for periods up to 170 h, with observations made at 2-h intervals. The results permitted not only the precise determination of the duration of each developmental phase on the various substrates but also of the behavior of the
cultures during the phase transitions. The results obtained provide information for the understanding of M. anisopliae development under conditions increasingly closer to those found by the fungus inside its hosts. We also expect these results to be useful for studies of gene regulation and differentiation and for the elaboration of more efficient culture media and commercial formulations. ACKNOWLEDGMENTS This work was supported in part by the following Brazilian organizations: CNPq, FAEP, CAPES, and PADCT II-FINEP. REFERENCES Al-Aidroos, K., and Seifert, A. M. 1980. Polysaccharide and protein degradation, germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 36, 29–34. Andersen, S. O., Højrup, P., and Roepstorff, P. 1995. Insect cuticular proteins. Insect. Biochem. Mol. Biol. 26, 153–176. Belsky, M. M., Goldstein, S., and Sesnowitz-Horn, S. 1984. Factors affecting endogenous respiration in a freshwater fungus. Mycologia 76, 804–809. Boucias, D. G., and Latge´, J. P. 1988. Nonspecific induction of germination of Conidiobolus obscurus and Nomuraea rileyi with host and non-host cuticle extracts. J. Invertebr. Pathol. 51, 168– 171. Braga, G. U. L., Messias, C. L., and Vencovsky, R. 1994. Estimates of genetic parameters related to protease production by Metarhizium anisopliae. J. Invertebr. Pathol. 64, 6–12. Braga, G. U. L., Vencovsky, R., and Messias, C. L. 1998. Estimates of genetic parameters related to chitinase production by Metarhizium anisopliae. Genet. Mol. Biol. 21, 171–177. Butt, T. M., Ibrahim, L., Clark, S. J., and Beckett, A. 1995. The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles. Mycol. Res. 99, 945–950. Campbell, R. K., Perring, T. M., Barnes, G. L., Eikenbary, R. D., and Gentry, C. R. 1978. Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae on media containing various amino acids. J. Invertebr. Pathol. 31, 289–295. Cochrane, V. W., Cochrane, J. C., Collins, C. B., and Serafin, F. G. 1963. Spore germination and carbon metabolism in Fusarium solani. II. endogenous respiration in relation to germination. Am. J. Bot. 50, 806–814. Dillon, R. J., and Charnley, A. K. 1985. A technique for accelerating and synchronizing germination of conidia of the entomopathogenic fungus Metarhizium anisopliae. Arch. Microbiol. 142, 204–206. Dillon, R. J., and Charnley, A. K. 1990. Initiation of germination in conidia of the entomopathogenic fungus, Metarhizium anisopliae. Mycol. Res. 94, 299–304. Dute, R. R., Weete, J. D., and Rushing, A. E. 1989. Ultrastructure of dormant and germinating conidia of Aspergillus ochraceus. Mycologia 81, 772–782. Fries, N. 1965. The chemical environment for fungal growth. 3. Vitamins and other organic growth factors. In ‘‘The Fungi, Vol. 1, The Fungal Cell’’ (G. C. Ainsworth and A. S. Sussman, Eds.), pp. 491–523. Academic Press, New York. Goettel, M., St. Leger, R. J., Rizzo, N. W., Staples, R. C., and Roberts, D. W. 1989. Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca sexta) cuticle. J. Gen. Microbiol. 135, 2233–2239.
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH Hassan, A. E. M., Dillon, R. J., and Charnley, A. K. 1989. Influence of accelerated germination of conidia on the pathogenicity of Metarhizium anisopliae for Manduca sexta. J. Invertebr. Pathol. 54, 277–279. Hill, E. P., Plesofsky-Vig, N., Paulson, A., and Brambl, R. 1992. Respiration and gene expression in germinating ascospores of Neurospora tetrasperma. FEMS Microbiol. Lett. 90, 111–116. Janda, S., Sigler, K., and Ho¨fer, M. 1993. Endogenous respiration reflects the energy load imposed by transport of nonmetabolizable substrates and by induced de novo protein synthesis in Rhodotorula glutinis. Arch. Microbiol. 159, 541–544. Li, D. P., and Holdom, D. G. 1995. Effects of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J. Invertebr. Pathol. 65, 253– 260. Lilly, V. G. 1965. The chemical environment for fungal growth. 1. Media, macro- and micronutrients. In ‘‘The Fungi, Vol. 1, The Fungal Cell’’ (G. C. Ainsworth and A. S. Sussman, Eds.), pp. 465–478. Academic Press, New York. Lyda, S. D. 1976. Optimizing mycelial respiration of Phymatotrichum omnivorum. Mycologia 68, 1011–1019. Mandels, G. R., Levinson, H. S., and Hyatt, M. T. 1956. Analysis of respiration during germination and enlargement of spores of Bacillus megaterium and of the fungus Myrothecium verrucaria. J. Gen. Physiol. 39, 301–309. Martı´n, J. F., and Nicola´s, G. 1970. Physiology of spore germination in Penicillium notatum and Trichoderma lignorum. Trans. Br. Mycol. Soc. 55, 141–148. Milner, R. J., Huppatz, R. J., and Swaris, S. C. 1991. A new method for assessment of germination of Metarhizium conidia. J. Invertebr. Pathol. 57, 121–123. Nelson, N. 1944. A photometric adaptation of the Somogy method for the determination of glucose. J. Biol. Chem. 153, 375–380. Nolan, R. A. 1971. Amino acids and growth factors in vitamin-free casamino acids. Mycologia 63, 1231–1237. Pontecorvo, G., Roper, J. A., Hemmons, L. M., McDonald, K. F., and Bufton, A. W. J. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 5, 141–238. Rath, A. C., Carr, C. J., and Graham, B. R. 1995. Characterization of Metarhizium anisopliae strains by carbohydrate utilization (API50CH). J. Invertebr. Pathol. 65, 152–161. Samuels, K. D. Z., Heale, J. B., and Llewellyn, M. 1989. Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens. J. Invertebr. Pathol. 53, 25–31.
119
Smith, R. J., and Grula, E. A. 1981. Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana. J. Invertebr. Pathol. 37, 222–230. Smith, R. J., and Grula, E. A. 1983. Chitinase is an inducible enzime in Beauveria bassiana. J. Invertebr. Pathol. 42, 319–326. Steel, R. G. D., and Torrie, J. H. 1960. ‘‘Principles and Procedures of Statistics.’’ McGraw-Hill, New York. St. Leger, R. J., Butt, T. M., Goettel, M. S., Staples, R. C., and Roberts, D. W. 1989. Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13, 274–288. St. Leger, R. J., Cooper, R. M., and Charnley, A. K. 1986. Cuticledegrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens. J. Invertebr. Pathol. 47, 167–177. St. Leger, R. J., Cooper, R. M., and Charnley, A. K. 1987. Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vormitoria and Manduca sexta. J. Gen. Microbiol. 133, 1371–1382. St. Leger, R. J., Goettel, M., Roberts, D. W., and Staples, R. C. 1991. Prepenetration events during infection of host cuticle by Metarhizium anisopliae. J. Invertebr. Pathol. 58, 168–179. St. Leger, R. J., May, B., Allee, L. L., Frank, D. C., Staples, R. C., and Roberts, D. W. 1992. Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 60, 89–101. Taber, W. A., and Taber, R. A. 1982. Nutrition and respiration of basidiospores and mycelium of Pisolithus tinctorius. Phytopathology 72, 316–322. Taber, W. A., and Taber, R. A. 1987. Carbon nutrition and respiration of Pisolithus tinctorius. Trans. Br. Mycol. Soc. 89, 13–26. Tan, Y. H., and Moore, D. 1995. Glucose catabolic pathways in Lentinula edodes determined with radiorespirometry and enzymic analysis. Mycol. Res. 99, 859–866. Tripp, M. L., and Paznokas, J. L. 1982. Role of amino acids and endogenous protein in the germination of Mucor racemosus Sporangiospores. J. Gen. Microbiol. 128, 485–495. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. 1972. ‘‘Manometric Methods.’’ Burgess, Minneapolis, MN. Woods, S. P., and Grula, E. A. 1984. Utilizable surface nutrients on Heliothis zea available for growth of Beauveria bassiana. J. Invertebr. Pathol. 43, 259–269.
Oxygen Consumption by Metarhizium anisopliae during Germination and Growth on Different Carbon Sources Gilberto U. L. Braga,1 Ricardo H. R. Deste´fano, and Claudio L. Messias Departamento de Gene´tica e Evoluc¸a˜o, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, CEP 13083-970, SP, Brazil Received July 6, 1998; accepted April 14, 1999
Respirometry was used to monitor the germination and growth of the entomopathogenic deuteromycete Metarhizium anisopliae on media containing carbon sources of different kinds (monosaccharides, polysaccharides, amino acids, and proteins). As also observed in several other species of fungi, M. anisopliae germination was found to be marked by a significant increase in O2 consumption, which started a few hours before germ tube emergence. The exponential consumption of the carbon source and O2 coincided with the exponential growth phase of the cultures. QO2 reached a maximum value during the exponential growth phase and was drastically reduced after the depletion of the exogenous carbon source. Taking glucose as reference, we observed that casein, hydrolyzed casein, and Nacetylglucosamine accelerated germination, reduced the lag phase, and increased the growth rate. This fact demonstrates that the fungus can readily use amino acids and N-acetylglucosamine, which are the monomers of the major constituents of the insect cuticle (proteins and chitin), a property that represents an important physiological adaptation to entomopathogenicity. r 1999 Academic Press Key Words: Metarhizium anisopliae; fungal germination; fungal growth; energy requirement; respirometric analysis.
INTRODUCTION
The understanding of the basic aspects of germination and growth of entomopathogenic hyphomycete fungi has greatly helped programs seeking to use these organisms as biological control agents. Some entomopathogenic fungi, like Metarhizium anisopliae, must be adapted to two very different situations: they must be able to utilize different types of organic substrates as a source of energy and nutrients
1 To whom correspondence should be addressed at current address: Department of Biology, Utah State University, Logan, UT, 843225305, U.S.A.
0022-2011/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
during their saprophytic life, and they must also be able to establish a link with and germinate on a susceptible host insect using the nutrients present on its cuticle or the substances mobilized from the latter by the action of hydrolytic exoenzymes in the early stages of infection (St. Leger et al., 1987, 1991; Goettel et al., 1989; Braga et al., 1994). Several studies have shown that the mechanisms of host recognition are linked, at least in part, to the chemical constitution of the cuticle or to the types of nutrients available on it. Boucias and Latge´ (1988) reported that cuticle extracts were able to induce germination in the fungi Conidiobolus obscurus and Nomuraea rileyi. Woods and Grula (1984) observed that amino acids and glucosamine are present on the surface of Heliothis zea in sufficient amounts to support the germination and limited growth of Beauveria bassiana. St. Leger et al. (1992) observed marked differences in nutritional requirements for germination between strains of M. anisopliae var. anisopliae and M. anisopliae var. majus isolated from different hosts. Samuels et al. (1989) attributed the high specificity of various strains of M. anisopliae against Oryctes rhinoceros to the presence of indeterminate nutrients on or inside the host’s cuticle. Butt et al. (1995) observed that the high availability of nutrients on the cuticle of aphids stimulates the rapid germination of M. anisopliae conidia. The virulence of entomopathogenic deuteromycetes has been correlated with their rapid germination and high growth rate (Al-Aidroos and Seifert, 1980; Samuels et al., 1989; Hassan et al., 1989; Dillon and Charnley, 1985, 1990). The importance of the speed of germination can be attributed to several factors. During the period of conidium exposure on the insect’s cuticle before penetration, the fungus may suffer desiccation, antibiosis of saprophytic microorganisms, inhibition by cuticular lipids, or removal together with the cuticle during the molts. According to Hassan et al. (1989), any method that would reduce the time of germination on the host’s cuticle may increase the efficiency of a mycoinsecticide. Since the growth of an entomopathogen, especially during infection of the host, occurs by the utilization of complex substrates (such as cuticle, internal tissues,
112
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
and hemolymph), an understanding of this type of situation may facilitate the understanding of its pathogenicity. Campbell et al. (1978) emphasized that studies on fungal nutrition and growth could help in the production of large quantities of virulent inoculum and in the appropriate selection of virulent strains. The differential utilization of carbon sources has also helped phylogenetic and strain characterization studies on the genus Metarhizium (Li and Holdom, 1995; Rath et al., 1995). The aim of this study was to determine the basic parameters of germination and growth of M. anisopliae on different organic substrates. MATERIALS AND METHODS
Maintenance of Fungi and Inoculum Preparation The strain used in the respirometric evaluations was CLII, obtained from the germ plasm bank of the laboratory of entomopathogenic fungi of the State University of Campinas (UNICAMP). This strain was isolated from the sugar cane borer Mahanarva posticata (Homoptera, Cercopidae) in the state of Alagoas, Brazil. For conidial production, the strain was grown for 12 days at 28°C on plates containing complete solid medium (CSM) (Pontecorvo et al., 1953). The conidia were suspended in a solution containing 0.85% (w/v) NaCl and 0.002% (v/v) Tween 80. The conidial suspension was then filtered through multiple gauze layers, and the conidia were washed twice and resuspended in a 0.85% NaCl (w/v) solution. Conidial concentration was adjusted to 3 ⫻ 107 ml⫺1. Substrates Evaluated Oxygen consumption during germination and the subsequent phases of culture development was evaluated in media containing different organic substrates which served as the only carbon and energy source. The following substrates were used: (a) sugars: glucose, N-acetylglucosamine (Sigma), and chitin (purified powder from crab shells; Sigma) prepared as described by Braga et al. (1998); (b) proteins: casein (Hammarsten; vitamin free), hydrolyzed casein (Bacto casamino acids; Difco), gelatin (type A from porcine skin; Sigma), and elastin (a powder from bovine neck ligament; Sigma); and (c) amino acids: L-cystine, DL-serine, L-proline, Dproline, L-alanine, L-glutamic acid, and L-tyrosine (Sigma). The substrates were individually added to minimum liquid medium (MLM) (Pontecorvo et al., 1953) with no glucose, at the concentration of 0.25% (w/v). The pH was adjusted to 6.5 in all cases using 2 N NaOH. The media were autoclaved in 50-ml volumetric round flasks at 121°C for 15 min. After autoclaving, the volume was readjusted with sterile distilled water.
113
Determination of Oxygen Consumption Oxygen consumption was determined with a Gilson differential respirometer. The respirometer has 14 reaction flasks connected to individual reference chambers through a differential manometer. The reference chamber eliminates the effect of variations in atmospheric pressure. The variation in the internal pressure of each reaction flask due to oxygen consumption can be monitored with digital micrometers. The entire system remains immersed in water at controlled temperature and can be submitted to shaking. The basic procedures adopted in the respirometry experiments were described by Umbreit et al. (1972). In order to retain the CO2 released during respiration, 400 µl of a 30% KOH solution (w/v) was used and distributed as follows: 200 µl were placed in the central vessel of the reaction flask and 200 µl were placed in the lateral arm. In order to increase the retention surface, rectangular pieces of filter paper were placed in the central vessel (3 ⫻ 2 cm) and in the lateral arm (2 ⫻ 1 cm). An aliquot of 4.5 ml of culture medium and 0.5 ml inoculum were added to each reaction flask so that the final concentration of the carbon source was 0.225% (w/v) and the conidial concentration was 3 ⫻ 106 ml⫺1. The respirometer was prepared and turned on 1 h before the beginning of the experiment so that the entire system could enter thermal equilibrium. During the experiments, the system was maintained at 28°C under shaking at 50 oscillations/min. Readings were taken directly as microliters of consumed oxygen at mean intervals of 2 h. As a control, the conidial suspension was inoculated into MLM without the exogenous carbon source in order to estimate endogenous conidial respiration. The experimental design was completely randomized with three replications. Data were submitted to analysis of variance. One analysis was performed for each postinoculation period. The Tukey test was used for comparing treatment means (Steel and Torrie, 1960). Culture Filtrates and Dry Mass Determination When necessary, the dry mass produced in the respirometry experiments was determined as described below. The reaction flasks were disconnected from the apparatus and the culture medium was removed and filtered through previously washed and weighed paper (Inlab type 10). The KOH solution and the filter papers were then carefully removed. The central vessel and the lateral arm were washed with distilled water and dried on absorbent paper. The remainder of the mycelium was then removed, washed with distilled water, and dried for 72 h at 70°C. The presence of possible contaminants was determined by observations under the microscope and by plating aliquots of the medium onto plates containing CSM. The concentration of residual glucose present in medium containing glucose as carbon source was deter-
´ FANO, AND MESSIAS BRAGA, DESTE
114
mined throughout growth and autolysis as described by Nelson (1944). Determination of Germination Specific experiments were conducted with a respirometer to monitor conidial germination in media containing the various carbon sources and in the control. After periods of incubation ranging from 6 to 14 h, aliquots were removed from the media and three random samples of at least 100 conidia were observed to determine the germination percentage. The conidia were considered to have germinated when they presented germ tubes greater than the diameter of the conidia. RESULTS AND DISCUSSION
Oxygen Consumption, Glucose Consumption, and Biomass Production The growth of a filamentous fungus can be evaluated in a direct manner on the basis of linear growth, fresh biomass, dry mass, or the amount of some mycelial constituents, or in an indirect manner on the basis of metabolic activities, such as respiratory activity (O2 consumption or CO2 release), formation of products such as acids or pigments, substrate consumption, or specific enzyme activities. Respirometry has been widely used both to monitor colony development and to evaluate the utilization of specific substrates by fungi (Lyda, 1976; Belsky et al., 1984; Taber and Taber, 1982, 1987; Hill et al., 1992; Janda et al., 1993; Tan and Moore,
1995). Since it is not a destructive technique and since it permits continuous evaluation, respirometry permits the construction of much more detailed growth curves than those obtained by analysis of biomass production. The dry mass production and glucose consumption by strain CLII growing in medium whose initial glucose concentration was 2.25 mg ml⫺1 are shown in Fig. 1. Maximum biomass production was reached 96 h postinoculation and coincided with the depletion of the carbon source. Autolysis started soon after the depletion of the exogenous carbon source and, as observed in other experiments, provoked a reduction in mycelial mass of more than 60% (G. U. L. Braga, unpublished results). Oxygen consumption under the same growth conditions and oxygen consumption by the conidia in the absence of the exogenous carbon source are shown in Fig. 2. It can be seen that, after a phase of acceleration lasting about 40 h, oxygen consumption increased exponentially up to the 90th hour and was abruptly reduced thereafter. Biomass and oxygen consumption increased concomitantly in an exponential manner (Figs. 1 and 2). The depletion of the exogenous carbon source coincided with the abrupt fall in oxygen consumption. The oxygen utilized for the oxidation of endogenous conidial reserves represented only 4.6% of total consumption. Analysis of variance showed that a significant increase compared to control in conidial respiration occured after the 7th hour in medium containing glucose (F ⫽ 28.09, significant at the 0.01 level of probability). The increase in respiratory activity occurred several
FIG. 1. Dry mass production and glucose consumption in a respirometer by strain CLII growing in medium containing 0.225% glucose (w/v) as the only carbon source. Each point represents the mean of two replicates. The standard errors of the means were 0.58 for dry mass production and 0.11 for glucose consumption.
115
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
FIG. 2. Oxygen consumption by strain CLII growing in medium containing 0.225% glucose (w/v) as the only carbon source. The graph shows the results of three replicates of the same experiment and respiration in the absence of an exogenous source. The standard errors of the means were 46.64 for glucose and 6.42 for the control.
hours before the germ tube emergence. The increase in respiration is one of the more marked physiological changes occurring during the germination of fungal spores (Mandels et al., 1956; Cochrane et al., 1963; Martı´n and Nicola´s, 1970; Hill et al., 1992). After 14 h, only 3.9% of the conidia incubated in media containing glucose produced a detectable germ tube (Table 1). The emergence of the germ tubes could not be monitored for a longer period of time due to the strong aggregation of conidia and germinants during development in liquid medium under shaking (Dute et al., 1989; Milner et al., 1991). The respirometry data show that it is possible to detect the physiological effect of the exogenous carbon source on the germlings a few hours before the germ tube emergence. There was a linear increase in oxygen consumption up to the 36th hour, followed by an exponential increase up to the 72nd hour. A gradual decrease occurred thereafter until the end of the experiment (Table 2). As shown in Fig. 1, after 96 h of growth, exogenous glucose was already depleted. On this basis, we may assume that, starting from that point, mycelial respiration occurred at the expense of its endogenous reserves accumulated during the growth phase. Table 2 also
shows the estimates of QO2 during the various stages of culture development. The evaluations performed during the 31st h of growth (9.1 µl O2 mg⫺1 h⫺1 ) and the 51st h (9.7 µl O2 mg⫺1 h⫺1 ) showed that QO2 apparently remained constant during this period. The highest QO2 value (30.9 µl O2 mg⫺1 h⫺1 ) was observed during the 73rd h, in the full exponential growth phase. The QO2 values estimated after depletion of the exogenous carbon source were perceptibly lower and decreased until the end of the experiment, clearly indicating a progressive depletion of the endogenous reserves of the mycelium. Oxidative Metabolism on the Different Carbon Sources Figure 3 shows oxygen consumption by strain CLII during germination and development on the different carbon sources. The most interesting facts were the great reduction in lag phase, the increase in growth rate, and the slight increase in total oxygen consumption provoked by casein compared to the growth curve on medium containing glucose. This type of response
TABLE 1 Percentage Germinating Conidia of Strain CLII in Medium Containing 0.225% (w/v) of the Substrates Indicated as the Only Carbon Source
8h 14 h
Basal
Glucose
Casein
Gelatin
Hydr.Cas
N-acetyl.
Chitin
0.0 (0.0) 0.3 (0.5)
0.0 (0.0) 3.9 (0.9)
8.8 (2.0) 47.5 (0.6)
0.3 (0.5) 3.1 (2.0)
4.0 (1.1) 28.6 (0.8)
2.1 (1.9) 8.8 (1.1)
0.3 (0.5) 4.6 (1.3)
Note. Each point represents the mean of three replicates. The standard deviations are given in parenthesis.
´ FANO, AND MESSIAS BRAGA, DESTE
116
TABLE 2 Total Oxygen and QO2 Consumption during Different Periods of Growth and Autolysis of Strain CLII in Medium Initially Containing 0.225% Glucose (w/v) as the Only Carbon Source Period (h)
O2 consumption (µl/h)
0–12 12–24 24–36 36–48 48–60 60–72 72–84 84–96 96–108 108–120 120–132 132–144 144–156 156–168
3.8 (1.1) c 5.4 (1.8) 9.5 (1.7) 17.35 (3.2) 55.2 (8.5) 89.3 (4.6) 61.6 (11.4) 24.17 (3.7) 12.3 (1.6) 9.8 (1.4) 6.2 (0.3) 4.4 (0.8) 3.5 (0.7) 2.3 (0.9)
QO2 (µl/mg h) a,b
(30–31) 9.1 (50–51) 9.7 (72–73) 30.9 (102–103) 2.0
(139–140) 0.9 (171–172) 0.2
Note. Each value represents the mean of three replicates. a We used the oxygen consumption of the last hour immediately preceding the determination of dry matter. The indication of the hour appears in parenthesis. b Each value represents the mean of two replicates. c Standard deviation.
characterizes the presence of one or more nonessential growth factors, which, however, had promoting actions (Fries, 1965). The nature of this promoter has not been established; it could be a peptide, an amino acid, or an appropriate set of amino acids initially present in the medium or released during casein hydrolysis. Studies
of amino acid metabolism in fungi have shown that some amino acids can act as precursors for the synthesis of a family of amino acids. On this basis, we may assume that in media containing these amino acids or an appropriate amino acid combination, the work for protein biosynthesis will be considerably reduced (Lilly, 1965; Tripp and Paznokas, 1982). This may explain the reduction in the duration of the lag and acceleration phases and the increase in growth rate on medium containing casein. These ideas are supported by the observation that amino acid mixtures are frequently better nitrogen sources than a single amino acid alone (Smith and Grula, 1981; Li and Holdom, 1995). Some studies have associated the high virulence of certain M. anisopliae strains with their rapid germination and high growth rate (Al-Aidroos and Seifert, 1980; Samuels et al., 1989; Hassan et al., 1989). The fact that casein stimulated the germination and early development of the fungus opens perspectives for the use of this substance to obtain more efficient commercial formulations of M. anisopliae spores. The other protein substrates evaluated (gelatin and elastin) did not produce the same effects. Both required a longer acceleration phase than observed with glucose and, in the case of gelatin, there was also a significant reduction in growth rate. Total oxygen consumption with the two substrates, as observed with casein, was slightly higher than observed with glucose. These results show that the nature of the protein utilized as carbon source directly affects the duration of the lag and acceleration phases and the growth rate. The difference in amino
FIG. 3. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v) and in control media (endogenous). Each point represents the mean of three replicates. The standard errors of the means were 66.13 for casein, 53.09 for gelatin, 68.09 for elastin, 19.56 for chitin, 46.64 for glucose, and 6.48 for the control.
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH
117
FIG. 4. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v). Each point represents the mean of three replicates. The standard errors of the means were 46.64 for glucose, 19.56 for chitin, and 29 for N-acetylglucosamine.
acid composition, as well as the differences in solubility, may explain part of the variations in the growth rates on the three protein substrates. Figure 3 also shows that growth on chitin was characterized by the long duration of the acceleration phase. Cultures were still in acceleration phase until 7 days postinoculation.
However, it was observed that conidia increased their respiratory rate in medium containing chitin, this being the major characteristic of the break of dormancy, which was more rapid than in the presence of glucose. Total oxygen consumption in medium containing chitin was higher than observed in glucose for several hours
FIG. 5. Oxygen consumption by strain CLII growing in media containing the substrates indicated at the concentration of 0.225% (w/v). Each point represents the mean of three replicates. The standard errors of the means were 66.13 for casein, 65.72 for hydrolyzed casein, and 46.64 for glucose.
118
´ FANO, AND MESSIAS BRAGA, DESTE
(Fig. 4). Chitin, when autoclaved in liquid medium, undergoes some hydrolysis with release of the Nacetylglucosamine monomer (Smith and Grula, 1983), which may have stimulated germination of M. anisopliae conidia. By being present at low concentration in the medium, it was rapidly depleted and the germination and/or development of the conidia was interrupted. Germination and development resumed only after the mobilization of the substrates by the germlings and adaptation to this situation probably was the factor responsible for the long duration of the acceleration phase. The presence of N-acetylglucosamine in the medium accelerated germination and reduced the duration of the lag and acceleration phases (Fig. 4). Although casein and hydrolyzed casein accelerated germination and development, these effects were more pronounced when casein was used as a carbon source (Fig. 5). The germ tube emergence also occurred earlier in medium containing casein than in medium containing hydrolyzed casein (Table 1). Oxidative metabolism was also studied when the major amino acids present in casein (Nolan, 1971) were utilized as the only carbon source. Some of these amino acids, such as alanine, are also present in substantial amounts in the proteins existing in the cuticle of insects (St. Leger et al., 1986; Andersen et al., 1995). None of the amino acids used individually as carbon source reproduced the effects of casein or hydrolyzed casein on germination and growth. The amino acids used as carbon sources proved to be inadequate for germination and growth of the CLII strain, causing the cultures to remain in the lag phase for several days. Analysis of variance showed a significant difference in oxygen consumption among the different amino acids (F ⫽ 13.16, significant at the 0.01 level of probability). The highest consumption after 50 h was observed for L-glutamic acid (325 ⫾ 92 µl), L-alanine (187 ⫾ 9 µl), and L-cystine (179 ⫾ 20 µl) and the lowest for L-tyrosine (108 ⫾ 20 µl), L-proline (108 ⫾ 9 µl), D-proline (109 ⫾ 5 µl), and DL-serine (124 ⫾ 22 µl). The least significant difference (LSD; Tukey’s test at 0.01 level of probability) for comparing treatment means was 133. The inability to germinate and develop on individual amino acids cannot be generalized. Some studies have reported the ability of other M. anisopliae strains to germinate and develop utilizing different amino acids at different concentrations as the only carbon source (Campbell et al., 1978; St. Leger et al., 1986, 1989). Growth curves for the fungus M. anisopliae presenting this level of resolution are reported here for the first time. Culture development was monitored for periods up to 170 h, with observations made at 2-h intervals. The results permitted not only the precise determination of the duration of each developmental phase on the various substrates but also of the behavior of the
cultures during the phase transitions. The results obtained provide information for the understanding of M. anisopliae development under conditions increasingly closer to those found by the fungus inside its hosts. We also expect these results to be useful for studies of gene regulation and differentiation and for the elaboration of more efficient culture media and commercial formulations. ACKNOWLEDGMENTS This work was supported in part by the following Brazilian organizations: CNPq, FAEP, CAPES, and PADCT II-FINEP. REFERENCES Al-Aidroos, K., and Seifert, A. M. 1980. Polysaccharide and protein degradation, germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 36, 29–34. Andersen, S. O., Højrup, P., and Roepstorff, P. 1995. Insect cuticular proteins. Insect. Biochem. Mol. Biol. 26, 153–176. Belsky, M. M., Goldstein, S., and Sesnowitz-Horn, S. 1984. Factors affecting endogenous respiration in a freshwater fungus. Mycologia 76, 804–809. Boucias, D. G., and Latge´, J. P. 1988. Nonspecific induction of germination of Conidiobolus obscurus and Nomuraea rileyi with host and non-host cuticle extracts. J. Invertebr. Pathol. 51, 168– 171. Braga, G. U. L., Messias, C. L., and Vencovsky, R. 1994. Estimates of genetic parameters related to protease production by Metarhizium anisopliae. J. Invertebr. Pathol. 64, 6–12. Braga, G. U. L., Vencovsky, R., and Messias, C. L. 1998. Estimates of genetic parameters related to chitinase production by Metarhizium anisopliae. Genet. Mol. Biol. 21, 171–177. Butt, T. M., Ibrahim, L., Clark, S. J., and Beckett, A. 1995. The germination behaviour of Metarhizium anisopliae on the surface of aphid and flea beetle cuticles. Mycol. Res. 99, 945–950. Campbell, R. K., Perring, T. M., Barnes, G. L., Eikenbary, R. D., and Gentry, C. R. 1978. Growth and sporulation of Beauveria bassiana and Metarhizium anisopliae on media containing various amino acids. J. Invertebr. Pathol. 31, 289–295. Cochrane, V. W., Cochrane, J. C., Collins, C. B., and Serafin, F. G. 1963. Spore germination and carbon metabolism in Fusarium solani. II. endogenous respiration in relation to germination. Am. J. Bot. 50, 806–814. Dillon, R. J., and Charnley, A. K. 1985. A technique for accelerating and synchronizing germination of conidia of the entomopathogenic fungus Metarhizium anisopliae. Arch. Microbiol. 142, 204–206. Dillon, R. J., and Charnley, A. K. 1990. Initiation of germination in conidia of the entomopathogenic fungus, Metarhizium anisopliae. Mycol. Res. 94, 299–304. Dute, R. R., Weete, J. D., and Rushing, A. E. 1989. Ultrastructure of dormant and germinating conidia of Aspergillus ochraceus. Mycologia 81, 772–782. Fries, N. 1965. The chemical environment for fungal growth. 3. Vitamins and other organic growth factors. In ‘‘The Fungi, Vol. 1, The Fungal Cell’’ (G. C. Ainsworth and A. S. Sussman, Eds.), pp. 491–523. Academic Press, New York. Goettel, M., St. Leger, R. J., Rizzo, N. W., Staples, R. C., and Roberts, D. W. 1989. Ultrastructural localization of a cuticle-degrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca sexta) cuticle. J. Gen. Microbiol. 135, 2233–2239.
RESPIROMETRIC ANALYSIS OF M. anisopliae GROWTH Hassan, A. E. M., Dillon, R. J., and Charnley, A. K. 1989. Influence of accelerated germination of conidia on the pathogenicity of Metarhizium anisopliae for Manduca sexta. J. Invertebr. Pathol. 54, 277–279. Hill, E. P., Plesofsky-Vig, N., Paulson, A., and Brambl, R. 1992. Respiration and gene expression in germinating ascospores of Neurospora tetrasperma. FEMS Microbiol. Lett. 90, 111–116. Janda, S., Sigler, K., and Ho¨fer, M. 1993. Endogenous respiration reflects the energy load imposed by transport of nonmetabolizable substrates and by induced de novo protein synthesis in Rhodotorula glutinis. Arch. Microbiol. 159, 541–544. Li, D. P., and Holdom, D. G. 1995. Effects of nutrients on colony formation, growth, and sporulation of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes). J. Invertebr. Pathol. 65, 253– 260. Lilly, V. G. 1965. The chemical environment for fungal growth. 1. Media, macro- and micronutrients. In ‘‘The Fungi, Vol. 1, The Fungal Cell’’ (G. C. Ainsworth and A. S. Sussman, Eds.), pp. 465–478. Academic Press, New York. Lyda, S. D. 1976. Optimizing mycelial respiration of Phymatotrichum omnivorum. Mycologia 68, 1011–1019. Mandels, G. R., Levinson, H. S., and Hyatt, M. T. 1956. Analysis of respiration during germination and enlargement of spores of Bacillus megaterium and of the fungus Myrothecium verrucaria. J. Gen. Physiol. 39, 301–309. Martı´n, J. F., and Nicola´s, G. 1970. Physiology of spore germination in Penicillium notatum and Trichoderma lignorum. Trans. Br. Mycol. Soc. 55, 141–148. Milner, R. J., Huppatz, R. J., and Swaris, S. C. 1991. A new method for assessment of germination of Metarhizium conidia. J. Invertebr. Pathol. 57, 121–123. Nelson, N. 1944. A photometric adaptation of the Somogy method for the determination of glucose. J. Biol. Chem. 153, 375–380. Nolan, R. A. 1971. Amino acids and growth factors in vitamin-free casamino acids. Mycologia 63, 1231–1237. Pontecorvo, G., Roper, J. A., Hemmons, L. M., McDonald, K. F., and Bufton, A. W. J. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 5, 141–238. Rath, A. C., Carr, C. J., and Graham, B. R. 1995. Characterization of Metarhizium anisopliae strains by carbohydrate utilization (API50CH). J. Invertebr. Pathol. 65, 152–161. Samuels, K. D. Z., Heale, J. B., and Llewellyn, M. 1989. Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens. J. Invertebr. Pathol. 53, 25–31.
119
Smith, R. J., and Grula, E. A. 1981. Nutritional requirements for conidial germination and hyphal growth of Beauveria bassiana. J. Invertebr. Pathol. 37, 222–230. Smith, R. J., and Grula, E. A. 1983. Chitinase is an inducible enzime in Beauveria bassiana. J. Invertebr. Pathol. 42, 319–326. Steel, R. G. D., and Torrie, J. H. 1960. ‘‘Principles and Procedures of Statistics.’’ McGraw-Hill, New York. St. Leger, R. J., Butt, T. M., Goettel, M. S., Staples, R. C., and Roberts, D. W. 1989. Production in vitro of appressoria by the entomopathogenic fungus Metarhizium anisopliae. Exp. Mycol. 13, 274–288. St. Leger, R. J., Cooper, R. M., and Charnley, A. K. 1986. Cuticledegrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens. J. Invertebr. Pathol. 47, 167–177. St. Leger, R. J., Cooper, R. M., and Charnley, A. K. 1987. Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vormitoria and Manduca sexta. J. Gen. Microbiol. 133, 1371–1382. St. Leger, R. J., Goettel, M., Roberts, D. W., and Staples, R. C. 1991. Prepenetration events during infection of host cuticle by Metarhizium anisopliae. J. Invertebr. Pathol. 58, 168–179. St. Leger, R. J., May, B., Allee, L. L., Frank, D. C., Staples, R. C., and Roberts, D. W. 1992. Genetic differences in allozymes and in formation of infection structures among isolates of the entomopathogenic fungus Metarhizium anisopliae. J. Invertebr. Pathol. 60, 89–101. Taber, W. A., and Taber, R. A. 1982. Nutrition and respiration of basidiospores and mycelium of Pisolithus tinctorius. Phytopathology 72, 316–322. Taber, W. A., and Taber, R. A. 1987. Carbon nutrition and respiration of Pisolithus tinctorius. Trans. Br. Mycol. Soc. 89, 13–26. Tan, Y. H., and Moore, D. 1995. Glucose catabolic pathways in Lentinula edodes determined with radiorespirometry and enzymic analysis. Mycol. Res. 99, 859–866. Tripp, M. L., and Paznokas, J. L. 1982. Role of amino acids and endogenous protein in the germination of Mucor racemosus Sporangiospores. J. Gen. Microbiol. 128, 485–495. Umbreit, W. W., Burris, R. H., and Stauffer, J. F. 1972. ‘‘Manometric Methods.’’ Burgess, Minneapolis, MN. Woods, S. P., and Grula, E. A. 1984. Utilizable surface nutrients on Heliothis zea available for growth of Beauveria bassiana. J. Invertebr. Pathol. 43, 259–269.