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Some Cultural Conditions that Control Production of Verrucarin J, a Cytotoxic Metabolite of Stachybotrys chartarum
Zbl. Mikrobiol. 137 (1982), 241-246 [Department of Botany, Faculty of Science, Assiut University, Assiut, Egypt]
Some Cultural Conditions that Control Production of Verrucarin J, a Cytotoxic Metabolite of Stachybotrys chartarum 1. A. EL-KADY and M. H. MOUBASHER
Summary A suitable chemically defined culture medium was selected and some optimal conditions for the biosynthesis of the highly cytostatic and antifungal compound verrucarin J were reported. Medium of the following composition was favourable for the production of verrucarin J by Btachybotrys chartarum: sucrose, 50; NaNO a, 2.0; KH2P04 , 1.0; MgS0 4 , 0.5; KCI, 0.5; leucine, 1.0 and FeS0 4 , 0.01 (g/l of distilled water). Biosynthesis of verrucarin J was maximal (U.8 mg/l) at pH 6.5 -7.0 and after incubation for 14 days at 25°C.
Zusammenfassung Es wird tiber ein geeignetes, chemisch definiertes Nahrmedium und optimale Bedingungen fUr die Biosynthese der hoch zytostatischen und fungiziden Verbindung Verrucarin J durch Btachybotrys chartarum berichtet. Als vorteilhaft erwies sich ein Medium der Zusammensetzung (g/l dest. Wasser): Glukose 50, NaNO a 2, KH2P04 1, MgS0 4 0,5, KCI 0,5, Leuzin 1 und FeS0 4 0,01. Eine maximale Bildung von Verrucarin J in Hohe von U,8 mg/l wurde bei pH 6,5 -7 und einer 14tagigen Inkubation bei 25°C erzielt.
Antifungal and antibacterial properties of Stachybotrys metabolites have been studied by BUTT and GHAFFAR (1972, 1974). They reported that S. atra had an inhibitory effect on the growth of 97.5 % of 52 fungus species, on all six actinomycetes tested, and on 83.3 %of the species of bacteria investigated. Recently the toxic metabolites produced by S. atra (Syn. S. alternans, S. chartarum) had been identified by EpPLEY et al. (1973) as roridin E, verrucarin J and satratoxin H, members of macrocylic ester of trichothecene-type mycotoxins. Roridins and verrucarins have a wide spectrum of biological activities; they possess antibacterial, fungistatic as well as insecticidal activities (HARRI et al. 1962, KISHABA et al. 1962, LOEFLER et al. 1965). Most of the trichothecene toxins have some antitumor activities, and roridins and verrucarins have been reported as mong the most active cytostatic agents known (HARRI et al. 1962). In a previous study (MOUBASHER 1980), some culture conditions that control biosynthesis of two members of the roridins series (roridin E and satratoxin H) by Stachybotrys chartarum isolate No. 128 were reported. In this work, the role of some culture conditions on growth and verrucarin J biosynthesis of S. chartarum isolate No. 61 was aimed.
Materials and Methods Organism: B. chartarum isolate No. 61 was selected from 54 toxigenic isolates as a high producer of verrucarin J (MOUBASHER 1980).
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1. A. EL-KADY and M. H. MOUBASHER
Culti vation: The fungus was inoculated into autoclaved (121°C, 20 min, 15lb/in2) 250-mlErlenmeyer flask containing 50 ml of the desired medium. After inoculation with two ml inoculum suspension of two-week-old cultures of the pure organism, the flasks were incubated at 28°C or other mentioned temperatures for 10 days without shaking. Preparation of the crude toxin: The method described by U:mNO et al. (1970) for preparation of Fusarium toxin was applied. After incubation, cultures were filtered using vacuum and four layers of cheese-cloth on Buchner funnel. After washing with 10-15 ml distilled water, the filtrate was mixed with 1 % (w/v) of active charcoal and, after standing overnight at 4°C, the charcoal was filtered and immersed in about 10-15 ml of methanol for about 12 hours. After filtration of the charcoal, the methanol was evaporated to dryness, and remain materials were referred to as "crude toxin". Quantitative analysis of the toxins: The amounts of the toxins were determined by the preparative thin-layer chromatographic technique with authentic samples as controls. A known volume of the methanol eluate was separated, using chloroform-methanol (98: 2, v/v) as solvent system. After development the bands containing the toxins were outlined under long ultraviolet irradiation (365 nm), scraped off, and eluted with methanol. The methanol extract was completed to a certain volume with methanol. The exact concentration of each toxin was then determined after making the necessary dilution by ultraviolet spectrophotometric measurement at 262 nm,
Results Effect of different carbon sources: Table 1 shows that very weak growth was observed when malate, acetate, pyruvate, fumarate, mannose, arabinose, glucose and raffinose were used as carbon sources. Best mycelial growth was obtained using starch, maltose and sucrose. The majority of TeA acid intermediates proved to be unsuitable for verrucarin J biosynthesis, when used as sole carbon sources, but oxalate and citrate yielded good mycelial growth. Verruoarin J was detected when arabinose, glucose, fructose, mannose, lactose, sucrose, maltose, raffinose, mannitol and starch were used as carbon sources. However, the maximum yield (5 mgjl) was obtained in presence of sucrose. Using different concenTable 1. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. (1) as affected by the type of carbon source Carbon source
Mycelial dry weight (g/liter)
Verrucarin J (mg/liter)
Arabinose Glucose Fructose Mannose Lactose Sucrose Maltose Raffinose Starch Mannitol Acetate Fumarate Malate Oxalate Citrate Pyruvate
0.093 0.126 1.124 0.086 0.236 0.314 0.330 0.138 0.494 0.230 0.028 0.086 0.026 0.222 0.260
1.0 1.8 1.7 1.9 1.8 5.0 1.0 2.0 3.0 3.0
Some Cultural Conditions that Control Production of Verrucarin J
243
trations of sucrose, it was noticed that mycelial growth increased with the increase of sucrose concentration and the maximum yield was obtained at 50 gil of sucrose (0.914 gil), which was followed by sharp decrease at higher sucrose concentrations, ranging from 60 to 100 gil (0.72 to 0.13 gil). Verrucarin J concentration increased parallel with the increase of mycelial growth, reaching maximum (6.8 mg/l) at 50 gil of sucrose, and regularly declined at higher concentrations. Effect of different nitrogen sources: Modified Czapek's medium with sucrose (50 gil) as carbon source was used during this study. The different nitrogen sources were added as nitrogen, equivalent to 2 gil NaN0 3 . Nitrate nitrogen was more favourable than ammonium or nitrite nitrogen for both cultural growth and verrucarin J biosynthesis (Table 2). Verruearin J was undetected when NH4Cl, (NH4)2S04 or (NH4hP04 was used as nitrogen source, and a very low yield (1.8 mg/l) was estimated when NH4N0 3 was supplied. Peptone and yeast extract supported the best fungal growth but with comparatively low yield of verrucarin J. Increasing the nitrogen level of the culture medium (using NaN0 3 ) induced an increase of the mycelial growth, reaching maximum at 5 gil NaN0 3 . Almost equal values of mycelial growth were obtained at higher concentrations (6-10 gil). However, maximum yield of verrucarin J (7.2 mgjl) was obtained at 2 gil of NaN0 3 . Table 2. Biosynthesis of verrucarin J with Stachybotrys chartarum (isolate No. 61) as affected by the type of nitrogen source Nitrogen source
Nitrogen source omitted NaNO a KNO a Ca(NOa)2 NH4NO a NaN0 2 NH4 Cl (NH4hP 0 4 (NH4)2S0 4 Peptone Yeast extract
Mycelial Verrucarin J dry weight (mg/liter) (g/100 ml medium) 0.0317 0.925
6.6
0.874
4.5
0.208 0.188 0.307 0.145 0.132 0.096 1.382 1.113
1.8
3.9 2.4
Effect of hydrogen ion concentration: Modified medium of sucrose (50 gil) and NaN0 3 (2.0 gil) as carbon and nitrogen sources, respectively, was employed during this study. Aliquots of the culture medium were adjusted before sterilization to different pH values, ranging between pH 4 and pH 11 (using N/10 of both HCI and NaOH). The results in Table 3 reveal that verrucarin J biosynthesis increased gradually with the increase of pH values, reaching maximum at pH ranging from 6.5 to 7.0 which was followed by sharp decrease at higher pHs. It was observed that, when verruearin J production diminished at high pH values, verrucarol was detected and accumulated, reaching maximum at pH 8.5 and 9. Culture medium of pH 7 was selected for further investigations.
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1. A. EL-KADY and M. H. MouBAsHER
Table 3. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. 61) as affected by variation of pH pH value
Mycelial dry weight (g/100 ml medium)
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 11.0
0.105 0.195 0.245 0.453 0.783 0.980 1.285 1.338 1.375 1.415 1.285 0.925 0.395 0.195
Verrucarin J (mg/liter)
Verrucarol
0.5 2.5 4.6 6.8 8.3 8-:3 5.3 3.6
++ +++ ++++ ++++ ++ +
2.7 1.8
Effect of amino acids and derivatives: As shown in Table 4, the different amino acids (which were added individually as 1 gil) affected cultural growth and verrucarin J synthesis at different degrees. Most amino acids were suitable for the growth of the experimental organism to some extent, but glutamic acid, norvaline and phenylalanine were the most effective. On the other hand, cysteine retarded cultural growth and verrucarin J formation. Although glutamic acid supported the best mycelial growth, yet it was inhibitory to verrucarin J formation. The remaining amino acids were generally favourable for verrucarin J production, best with leucine (9.3 mg!l), followed by lysine (8.8 mg!l). Leucine was thus selected for further investigations. Table 4. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. 61) as affected by the addition of some amino acids Amino acids
Verrucarin J Mycelial dry weight (g/100 ml medium)
Control (without amino acids) Leucine Lysine Glutamine Asparagine Valine Norvaline Alanine Phenylalanine Arginine Glutamic acid Glycine Serine Cysteine
1.337
7.1
1.446 1.510 1.415 1.412 1.500 1.680 1.405 1.653 1.530 1.688 1.412 1.340 0.825
9.3 8.8 7.9 7.8 7.6 7.7 7.5 8.2 7.0 5.8 7.0 7.0 5.2
Some Cultural Conditions that Control Production of Verrucarin J
245
Effect of incubation temperature: Table 5 shows that both mycelial growth and verrucarin J formation regularly increased with the increase of incubation temperature. Mycelial growth reached maximum (1.87 mg/l) at 30 °C, while verrucarin J reached maximum (10.8 mg/l) at 25 °C. Mycelial growth gradually decreased, whereas verrucarin J concentration sharply declined at higher temperatures. No growth was detected at 5 °C or 45 °C. Table 5. Biosynthesis of verrucarin J by Stachybotrys chartarum at various temperatures1 ) Incubation temperature (OC)
Verrucarin J Mycelial dry weight (mg/liter) (g/lOO ml medium)
5 10 15 20 25
30 35 40 45
0.170 0.714 1.246 1.558 1.867 1.218 0.882
1.2 4.3 6.4 10.0 9.0 8.1 4.3
1) The composition of the medium for this and the following experiment was as follows: sucrose, 50.0; NaN0 3 , 2.0; KH2P04 , 1.0; MgS04 , 0.5; KCI, 0.5; leucine, 1.0; FeS0 4 , 0.01 (g/liter of distilled water); pH adjusted to 7.0.
Effect of incubation period: Maximum mycelial growth (1.72 gil) was gained after 12 days, followed by gradual decrease with the increase of incubation period. A maximum yield of verrucar n J (U.8 mg/l) was obtained after 14 days of incubation, which declined considerably with the lengthening of the experimental period so that only 20.3 % of the maximum was detected after 20 days of incubation. Discussion The results of the different experiments directed to find out suitable and chemically defined liquid medium for the production of verrucarin J, reported as one of the highly potent antitumor agents and having antifungal property, reveal that a medium of the following composition was suitable for this purpose: sucrose, 50; NaN0 3 , 2; KH 2P04 , 1; MgS0 4 , 0.5; KCI, 0.5; leucine, 1; FeS0 4 , 0.01 (gil of distilled water) and with pH adjusted to 7. Incubation at 25°C for 14 days was optimal for the production of verrucarin J. Reports on trichothecene-type mycotoxins by Stachybotrys fungi are few (EpPLEY et al. 1973, 1977). Further reports on verruearins or roridins by this fungus are completely absent. However, a number of liquid media have been used for production of these toxins from Myrothecium spp. Toxicity was first demonstrated in RaulinThom medium with 0.01 % yeast extract (BRIAN 1946). BRIAN et al, (1948) used a basal broth of glucose, KH 2P04 , MgS0 4 , and trace metals with either potassium nitrate, ammonium sulphate or ammonium tartrate as a nitrogen source. MORTIMER et al. (1971) used the broth by BOHNER et al, (1965) which contained 2 %glucose and 0.2 % of each of maltose, peptone, yeast extract, NH 4N0 3 , NaN0 3 , MgS0 4 and KH 2P04 • Incubation times and temperatures ranged from 14 days at 8 00 (BAMBURG
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1. A. EL-KADY and M. H. MOUBASHER, Some Cultural Conditions that Control Production
et al. 1946) or 14 days at 20°C (DI MENNA et al. 1973, MORTIMER et al. 1971) to 7 to 9 days at 27°C (BOWDEN and SCHANTZ 1955, HARRI et al. 1962). In a previous study (MOUBASHER 1980), some cultural conditions that control biosynthesis of roridin E and satratoxin H (closely related compounds to verrucarin J) by Stachybotrys chartarum isolate No. 128 were reported. Comparing the results of that study with the present ones, one can conclude th it the nutritional requirements for the biosynthesis of both toxins are almost similar. Sucrose, maltose, mannitol and starch supported the formation of both toxins. NaN0 3 and KN0 3 also promoted the biosynthesis of both roridin E and verrucarin J and Ca(N0 3 )2 retarded both. However, isolate No. 128 of S. chartarum utilized both ammonium and nitrate nitrogen, while isolate No. 61 preferred nitrate nitrogen. Leucine, lysine, valine, phenylalanine and glutamine were favourable for the biosynthesis of botn toxins. Both roridin E and verrucarin J were maximally produced at nearly the same pH (roridin at pH 7 and verrucarin J at pH 6.5-7.0) and at close temperatures (25°C and 30°C) and incubation periods (12 and 14 days). These similarities in the response of roridin E and verrucarin J formation to different factors are reasonable since all roridins and verrucarins are closely related to macrocyclic trichothecene ester, initially recorded in closely related soil fungi, Myrothecium roridum and Myrothecium verrucaria, and in the present and previous work (MOUBASHER 1980) are produced by two isolates of one fungus, Stachybotrys chartarum.
References BAMBURG, J. R., MARASAS, W. F., RIGGS, N. V., SMALLEY, E. B., and STRONG, F. M.: Biotechnol. Bioeng. 10 (1968),445. BOHNER, B., FETZ, E., HARBI, E., SIGG, H. P., STOLL, C., and TAMM, C.: Helv. Chim. Acta 48 (1965), 1079. BOWDEN, J. P., and SCHANTZ, E. J.: J. BioI. Chern. 214 (1955), 365. BRIAN, P. W., and MCGOWAN, J. C.: Nature (Lond.) 157 (1946), 334. BRtAN, P. W., HEMMING, H. G., and JEFFERYS, E. G.: Mycologia 40 (1948), 363. BUTT, Z. L., and GHAFFAR, A.: Mycopathol. Myeol, Appl, 47 (1972), 241. BUTT, Z. L., and GHAFFAR, A.: Zeitschr. Pflanzenphysiol. B 71 (1974), 463. DI MENNA, M. E., MORTIMER, P. H., SMITH, B. L., and TULLOCH, M.: J. Gen. Microbiol. 79 (1973), 81. EpPLEY, R. M., and BAILEY, W. J.: Science 181 (1973), 758. EpPLEY, R. M., MAZZOLA, E. P., HIGHET, R. J., and BAILEY, W. J.: J. Org. Chern. 42 (1977), 240. HARRI, E., LOEFFLER, W., SIGG, H. P., STARLIN, H., STOLlo, C., TAMM, C., and WIESINGER, D.: Helv, Chim. Acta 45 (1962), 839. KISHABA, A. N., SHANKLAND, D. L., CURTIS, R. w., and WILSON, M. C.: J. Econ. Entomol. 55 (1962), 211. LOEFLER, W., MAULl, E., RUSCHE, M. E., and STAHELIN, H.: French patent (1964) 1, 372, 122; Chern. Abstr. 62 (1965), 5856d. MORTIMER, P. H., CAMPBELL, J., DI MENNA, M. E., and WHITE, E. P.: Res. Vet. Sci. 12 (1971), 508. MOUBASHER, M. H.: Studies on toxin production by Stachybotrys species. M. Sc. Thesis, Botany Department, Faculty of Science, Assiut University, Assiut, Egypt (1980). UENO, Y., ISHIKAWA, Y., SAITO-AMAKAI, K., and TSUNODA, H.: Chern. Pharm. Bull. 18 (1970), 304. Eingegangen am 27. 4. 1981 Author's address: ISMAIL EL-KADY, Botany Department, Faculty of Science, Assiut University, Assiut, Egypt.
Some Cultural Conditions that Control Production of Verrucarin J, a Cytotoxic Metabolite of Stachybotrys chartarum 1. A. EL-KADY and M. H. MOUBASHER
Summary A suitable chemically defined culture medium was selected and some optimal conditions for the biosynthesis of the highly cytostatic and antifungal compound verrucarin J were reported. Medium of the following composition was favourable for the production of verrucarin J by Btachybotrys chartarum: sucrose, 50; NaNO a, 2.0; KH2P04 , 1.0; MgS0 4 , 0.5; KCI, 0.5; leucine, 1.0 and FeS0 4 , 0.01 (g/l of distilled water). Biosynthesis of verrucarin J was maximal (U.8 mg/l) at pH 6.5 -7.0 and after incubation for 14 days at 25°C.
Zusammenfassung Es wird tiber ein geeignetes, chemisch definiertes Nahrmedium und optimale Bedingungen fUr die Biosynthese der hoch zytostatischen und fungiziden Verbindung Verrucarin J durch Btachybotrys chartarum berichtet. Als vorteilhaft erwies sich ein Medium der Zusammensetzung (g/l dest. Wasser): Glukose 50, NaNO a 2, KH2P04 1, MgS0 4 0,5, KCI 0,5, Leuzin 1 und FeS0 4 0,01. Eine maximale Bildung von Verrucarin J in Hohe von U,8 mg/l wurde bei pH 6,5 -7 und einer 14tagigen Inkubation bei 25°C erzielt.
Antifungal and antibacterial properties of Stachybotrys metabolites have been studied by BUTT and GHAFFAR (1972, 1974). They reported that S. atra had an inhibitory effect on the growth of 97.5 % of 52 fungus species, on all six actinomycetes tested, and on 83.3 %of the species of bacteria investigated. Recently the toxic metabolites produced by S. atra (Syn. S. alternans, S. chartarum) had been identified by EpPLEY et al. (1973) as roridin E, verrucarin J and satratoxin H, members of macrocylic ester of trichothecene-type mycotoxins. Roridins and verrucarins have a wide spectrum of biological activities; they possess antibacterial, fungistatic as well as insecticidal activities (HARRI et al. 1962, KISHABA et al. 1962, LOEFLER et al. 1965). Most of the trichothecene toxins have some antitumor activities, and roridins and verrucarins have been reported as mong the most active cytostatic agents known (HARRI et al. 1962). In a previous study (MOUBASHER 1980), some culture conditions that control biosynthesis of two members of the roridins series (roridin E and satratoxin H) by Stachybotrys chartarum isolate No. 128 were reported. In this work, the role of some culture conditions on growth and verrucarin J biosynthesis of S. chartarum isolate No. 61 was aimed.
Materials and Methods Organism: B. chartarum isolate No. 61 was selected from 54 toxigenic isolates as a high producer of verrucarin J (MOUBASHER 1980).
242
1. A. EL-KADY and M. H. MOUBASHER
Culti vation: The fungus was inoculated into autoclaved (121°C, 20 min, 15lb/in2) 250-mlErlenmeyer flask containing 50 ml of the desired medium. After inoculation with two ml inoculum suspension of two-week-old cultures of the pure organism, the flasks were incubated at 28°C or other mentioned temperatures for 10 days without shaking. Preparation of the crude toxin: The method described by U:mNO et al. (1970) for preparation of Fusarium toxin was applied. After incubation, cultures were filtered using vacuum and four layers of cheese-cloth on Buchner funnel. After washing with 10-15 ml distilled water, the filtrate was mixed with 1 % (w/v) of active charcoal and, after standing overnight at 4°C, the charcoal was filtered and immersed in about 10-15 ml of methanol for about 12 hours. After filtration of the charcoal, the methanol was evaporated to dryness, and remain materials were referred to as "crude toxin". Quantitative analysis of the toxins: The amounts of the toxins were determined by the preparative thin-layer chromatographic technique with authentic samples as controls. A known volume of the methanol eluate was separated, using chloroform-methanol (98: 2, v/v) as solvent system. After development the bands containing the toxins were outlined under long ultraviolet irradiation (365 nm), scraped off, and eluted with methanol. The methanol extract was completed to a certain volume with methanol. The exact concentration of each toxin was then determined after making the necessary dilution by ultraviolet spectrophotometric measurement at 262 nm,
Results Effect of different carbon sources: Table 1 shows that very weak growth was observed when malate, acetate, pyruvate, fumarate, mannose, arabinose, glucose and raffinose were used as carbon sources. Best mycelial growth was obtained using starch, maltose and sucrose. The majority of TeA acid intermediates proved to be unsuitable for verrucarin J biosynthesis, when used as sole carbon sources, but oxalate and citrate yielded good mycelial growth. Verruoarin J was detected when arabinose, glucose, fructose, mannose, lactose, sucrose, maltose, raffinose, mannitol and starch were used as carbon sources. However, the maximum yield (5 mgjl) was obtained in presence of sucrose. Using different concenTable 1. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. (1) as affected by the type of carbon source Carbon source
Mycelial dry weight (g/liter)
Verrucarin J (mg/liter)
Arabinose Glucose Fructose Mannose Lactose Sucrose Maltose Raffinose Starch Mannitol Acetate Fumarate Malate Oxalate Citrate Pyruvate
0.093 0.126 1.124 0.086 0.236 0.314 0.330 0.138 0.494 0.230 0.028 0.086 0.026 0.222 0.260
1.0 1.8 1.7 1.9 1.8 5.0 1.0 2.0 3.0 3.0
Some Cultural Conditions that Control Production of Verrucarin J
243
trations of sucrose, it was noticed that mycelial growth increased with the increase of sucrose concentration and the maximum yield was obtained at 50 gil of sucrose (0.914 gil), which was followed by sharp decrease at higher sucrose concentrations, ranging from 60 to 100 gil (0.72 to 0.13 gil). Verrucarin J concentration increased parallel with the increase of mycelial growth, reaching maximum (6.8 mg/l) at 50 gil of sucrose, and regularly declined at higher concentrations. Effect of different nitrogen sources: Modified Czapek's medium with sucrose (50 gil) as carbon source was used during this study. The different nitrogen sources were added as nitrogen, equivalent to 2 gil NaN0 3 . Nitrate nitrogen was more favourable than ammonium or nitrite nitrogen for both cultural growth and verrucarin J biosynthesis (Table 2). Verruearin J was undetected when NH4Cl, (NH4)2S04 or (NH4hP04 was used as nitrogen source, and a very low yield (1.8 mg/l) was estimated when NH4N0 3 was supplied. Peptone and yeast extract supported the best fungal growth but with comparatively low yield of verrucarin J. Increasing the nitrogen level of the culture medium (using NaN0 3 ) induced an increase of the mycelial growth, reaching maximum at 5 gil NaN0 3 . Almost equal values of mycelial growth were obtained at higher concentrations (6-10 gil). However, maximum yield of verrucarin J (7.2 mgjl) was obtained at 2 gil of NaN0 3 . Table 2. Biosynthesis of verrucarin J with Stachybotrys chartarum (isolate No. 61) as affected by the type of nitrogen source Nitrogen source
Nitrogen source omitted NaNO a KNO a Ca(NOa)2 NH4NO a NaN0 2 NH4 Cl (NH4hP 0 4 (NH4)2S0 4 Peptone Yeast extract
Mycelial Verrucarin J dry weight (mg/liter) (g/100 ml medium) 0.0317 0.925
6.6
0.874
4.5
0.208 0.188 0.307 0.145 0.132 0.096 1.382 1.113
1.8
3.9 2.4
Effect of hydrogen ion concentration: Modified medium of sucrose (50 gil) and NaN0 3 (2.0 gil) as carbon and nitrogen sources, respectively, was employed during this study. Aliquots of the culture medium were adjusted before sterilization to different pH values, ranging between pH 4 and pH 11 (using N/10 of both HCI and NaOH). The results in Table 3 reveal that verrucarin J biosynthesis increased gradually with the increase of pH values, reaching maximum at pH ranging from 6.5 to 7.0 which was followed by sharp decrease at higher pHs. It was observed that, when verruearin J production diminished at high pH values, verrucarol was detected and accumulated, reaching maximum at pH 8.5 and 9. Culture medium of pH 7 was selected for further investigations.
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Table 3. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. 61) as affected by variation of pH pH value
Mycelial dry weight (g/100 ml medium)
4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 11.0
0.105 0.195 0.245 0.453 0.783 0.980 1.285 1.338 1.375 1.415 1.285 0.925 0.395 0.195
Verrucarin J (mg/liter)
Verrucarol
0.5 2.5 4.6 6.8 8.3 8-:3 5.3 3.6
++ +++ ++++ ++++ ++ +
2.7 1.8
Effect of amino acids and derivatives: As shown in Table 4, the different amino acids (which were added individually as 1 gil) affected cultural growth and verrucarin J synthesis at different degrees. Most amino acids were suitable for the growth of the experimental organism to some extent, but glutamic acid, norvaline and phenylalanine were the most effective. On the other hand, cysteine retarded cultural growth and verrucarin J formation. Although glutamic acid supported the best mycelial growth, yet it was inhibitory to verrucarin J formation. The remaining amino acids were generally favourable for verrucarin J production, best with leucine (9.3 mg!l), followed by lysine (8.8 mg!l). Leucine was thus selected for further investigations. Table 4. Biosynthesis of verrucarin J by Stachybotrys chartarum (isolate No. 61) as affected by the addition of some amino acids Amino acids
Verrucarin J Mycelial dry weight (g/100 ml medium)
Control (without amino acids) Leucine Lysine Glutamine Asparagine Valine Norvaline Alanine Phenylalanine Arginine Glutamic acid Glycine Serine Cysteine
1.337
7.1
1.446 1.510 1.415 1.412 1.500 1.680 1.405 1.653 1.530 1.688 1.412 1.340 0.825
9.3 8.8 7.9 7.8 7.6 7.7 7.5 8.2 7.0 5.8 7.0 7.0 5.2
Some Cultural Conditions that Control Production of Verrucarin J
245
Effect of incubation temperature: Table 5 shows that both mycelial growth and verrucarin J formation regularly increased with the increase of incubation temperature. Mycelial growth reached maximum (1.87 mg/l) at 30 °C, while verrucarin J reached maximum (10.8 mg/l) at 25 °C. Mycelial growth gradually decreased, whereas verrucarin J concentration sharply declined at higher temperatures. No growth was detected at 5 °C or 45 °C. Table 5. Biosynthesis of verrucarin J by Stachybotrys chartarum at various temperatures1 ) Incubation temperature (OC)
Verrucarin J Mycelial dry weight (mg/liter) (g/lOO ml medium)
5 10 15 20 25
30 35 40 45
0.170 0.714 1.246 1.558 1.867 1.218 0.882
1.2 4.3 6.4 10.0 9.0 8.1 4.3
1) The composition of the medium for this and the following experiment was as follows: sucrose, 50.0; NaN0 3 , 2.0; KH2P04 , 1.0; MgS04 , 0.5; KCI, 0.5; leucine, 1.0; FeS0 4 , 0.01 (g/liter of distilled water); pH adjusted to 7.0.
Effect of incubation period: Maximum mycelial growth (1.72 gil) was gained after 12 days, followed by gradual decrease with the increase of incubation period. A maximum yield of verrucar n J (U.8 mg/l) was obtained after 14 days of incubation, which declined considerably with the lengthening of the experimental period so that only 20.3 % of the maximum was detected after 20 days of incubation. Discussion The results of the different experiments directed to find out suitable and chemically defined liquid medium for the production of verrucarin J, reported as one of the highly potent antitumor agents and having antifungal property, reveal that a medium of the following composition was suitable for this purpose: sucrose, 50; NaN0 3 , 2; KH 2P04 , 1; MgS0 4 , 0.5; KCI, 0.5; leucine, 1; FeS0 4 , 0.01 (gil of distilled water) and with pH adjusted to 7. Incubation at 25°C for 14 days was optimal for the production of verrucarin J. Reports on trichothecene-type mycotoxins by Stachybotrys fungi are few (EpPLEY et al. 1973, 1977). Further reports on verruearins or roridins by this fungus are completely absent. However, a number of liquid media have been used for production of these toxins from Myrothecium spp. Toxicity was first demonstrated in RaulinThom medium with 0.01 % yeast extract (BRIAN 1946). BRIAN et al, (1948) used a basal broth of glucose, KH 2P04 , MgS0 4 , and trace metals with either potassium nitrate, ammonium sulphate or ammonium tartrate as a nitrogen source. MORTIMER et al. (1971) used the broth by BOHNER et al, (1965) which contained 2 %glucose and 0.2 % of each of maltose, peptone, yeast extract, NH 4N0 3 , NaN0 3 , MgS0 4 and KH 2P04 • Incubation times and temperatures ranged from 14 days at 8 00 (BAMBURG
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et al. 1946) or 14 days at 20°C (DI MENNA et al. 1973, MORTIMER et al. 1971) to 7 to 9 days at 27°C (BOWDEN and SCHANTZ 1955, HARRI et al. 1962). In a previous study (MOUBASHER 1980), some cultural conditions that control biosynthesis of roridin E and satratoxin H (closely related compounds to verrucarin J) by Stachybotrys chartarum isolate No. 128 were reported. Comparing the results of that study with the present ones, one can conclude th it the nutritional requirements for the biosynthesis of both toxins are almost similar. Sucrose, maltose, mannitol and starch supported the formation of both toxins. NaN0 3 and KN0 3 also promoted the biosynthesis of both roridin E and verrucarin J and Ca(N0 3 )2 retarded both. However, isolate No. 128 of S. chartarum utilized both ammonium and nitrate nitrogen, while isolate No. 61 preferred nitrate nitrogen. Leucine, lysine, valine, phenylalanine and glutamine were favourable for the biosynthesis of botn toxins. Both roridin E and verrucarin J were maximally produced at nearly the same pH (roridin at pH 7 and verrucarin J at pH 6.5-7.0) and at close temperatures (25°C and 30°C) and incubation periods (12 and 14 days). These similarities in the response of roridin E and verrucarin J formation to different factors are reasonable since all roridins and verrucarins are closely related to macrocyclic trichothecene ester, initially recorded in closely related soil fungi, Myrothecium roridum and Myrothecium verrucaria, and in the present and previous work (MOUBASHER 1980) are produced by two isolates of one fungus, Stachybotrys chartarum.
References BAMBURG, J. R., MARASAS, W. F., RIGGS, N. V., SMALLEY, E. B., and STRONG, F. M.: Biotechnol. Bioeng. 10 (1968),445. BOHNER, B., FETZ, E., HARBI, E., SIGG, H. P., STOLL, C., and TAMM, C.: Helv. Chim. Acta 48 (1965), 1079. BOWDEN, J. P., and SCHANTZ, E. J.: J. BioI. Chern. 214 (1955), 365. BRIAN, P. W., and MCGOWAN, J. C.: Nature (Lond.) 157 (1946), 334. BRtAN, P. W., HEMMING, H. G., and JEFFERYS, E. G.: Mycologia 40 (1948), 363. BUTT, Z. L., and GHAFFAR, A.: Mycopathol. Myeol, Appl, 47 (1972), 241. BUTT, Z. L., and GHAFFAR, A.: Zeitschr. Pflanzenphysiol. B 71 (1974), 463. DI MENNA, M. E., MORTIMER, P. H., SMITH, B. L., and TULLOCH, M.: J. Gen. Microbiol. 79 (1973), 81. EpPLEY, R. M., and BAILEY, W. J.: Science 181 (1973), 758. EpPLEY, R. M., MAZZOLA, E. P., HIGHET, R. J., and BAILEY, W. J.: J. Org. Chern. 42 (1977), 240. HARRI, E., LOEFFLER, W., SIGG, H. P., STARLIN, H., STOLlo, C., TAMM, C., and WIESINGER, D.: Helv, Chim. Acta 45 (1962), 839. KISHABA, A. N., SHANKLAND, D. L., CURTIS, R. w., and WILSON, M. C.: J. Econ. Entomol. 55 (1962), 211. LOEFLER, W., MAULl, E., RUSCHE, M. E., and STAHELIN, H.: French patent (1964) 1, 372, 122; Chern. Abstr. 62 (1965), 5856d. MORTIMER, P. H., CAMPBELL, J., DI MENNA, M. E., and WHITE, E. P.: Res. Vet. Sci. 12 (1971), 508. MOUBASHER, M. H.: Studies on toxin production by Stachybotrys species. M. Sc. Thesis, Botany Department, Faculty of Science, Assiut University, Assiut, Egypt (1980). UENO, Y., ISHIKAWA, Y., SAITO-AMAKAI, K., and TSUNODA, H.: Chern. Pharm. Bull. 18 (1970), 304. Eingegangen am 27. 4. 1981 Author's address: ISMAIL EL-KADY, Botany Department, Faculty of Science, Assiut University, Assiut, Egypt.