Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 20 - 23 (201 2 ) Res.J. Recent .Sci. International Science Congress Associa tion 20 Factors Influencing the Interaction of three Fungi and Mycotoxin Production Surekha M., Kiran S., Naresh A. and Reddy S.M. Toxicology Laboratory, Department of Botany, Kakatiya University, Warangal, Andhra Pradesh, INDIA Available online at: www.isca.in Received 1 9 th October 2012, revised 31 st December 2012, accepted 28 th January 201 3 Abstract The interaction of three fungi viz, Penicillium griseofulvum P. crustosum and Aspergillus terreus in relation to mycotoxins pr oduction was studied. Production of penitrem A, CPA, patulin and terreic acid by above fungi varied with the environmental conditions. In general glucose, sucrose and potassium nitrate were favoured carbon and nitrogen sources. The biomass production also vari ed with environmental factors. Keywords: Interaction of fungi, Penicillium griseofulvum , P. crustosum , Aspergillus terreus , cyclopiazonic acid, penitrem A, patulin and terreic acid. Introduction In nature organisms compete either for space or for n utrients which may result in the elimination of weak organism or may settle down in coexistence due to a balance of power. In the process of interaction one organism affects the other in variety of ways. In recent times, some attempts have been made to und erstand such phenomena with a hope that such information may be helpful in formulating biocontrol agent for prevention and control of mycotoxins. A part from interacting fungi, a third factor i.e. environment play a significant role exerting its influence on individual or both the fungi. Though, the interaction of different fungi in relation to mycotoxins production have studied by some earlier workers 1 - 8 but practically no information is available on factors influencing interaction of mycotoxigenic fungi a nd mycotoxin production. Therefore, in the present investigations influence of different factors on the interaction of fungi and mycotoxin production was studied. Material and Methods Monosporic cultures of Penicillium griseofulvum P. crustosum and Asperg illus terreus isolated from paddy straw were grown in 50ml buffered Czapek’s dox medium (Sucrose 30g; NaNo 3 3.0g; KH 2 Po 4 1g; KCl 0.5g; FeSo 4 7 H 2 O 0.01g; MgSo 4 7H 2 O 0.5g; distilled water 1000ml; pH 5.5) contained in 250 ml Erlenmeyer conical flasks for 15 days at 27 2C and amount of mycotoxins production by respective fungi was assessed. The pH changes and biomass produced was also assessed. The influence of carbon and nitrogen source on interaction of above mycotoxic fungi was studied by substituting su crose and sodium nitrate of basal medium by different carbon and nitrogen sources so as to supply equivalent amount of carbon and nitrogen sources respectively. The pH of the basal medium was adjusted to desired level (2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5 an d 9.5) with the help of 6N HCl/NaOH for studying the influence of pH, and temperature (15, 20, 25, 30, 35 and 40C) on interaction of mycotoxigenic fungi and its impact on mycotoxin elaboration was also studied. At the end of 15days incubation period, the cultures were harvested on previously dried and weighed Whatman No. 42 filter paper to determine the biomass of the fungus. The filter paper along with mycelial mat was dried at 70C for 2 days and weighed to a constant weight after cooling to room temper ature in a desiccator. The culture filtrate was employed for extraction and estimation of different mycotoxins. Patulin 9 , terreic acid 10 , cyclopiazonic acid 11 and penitrem A 12 were estimated by standard methods. Mycelial mat was used for extraction and est imation of penitrem A. The experiments were conducted in triplicate. The results obtained are statistically analysed using SPSS software (Version 12.0). The details of extraction and estimation of other mycotoxins were similar to those described earlier 13 . The results are presented in table 1 and 2. Results and Discussion A critical study of results presented in Table 1 reveals that P. griseofulvum, P. crustosum and A. terreus interacted intimately during their growth resulting in disturbances in their met abolism. Sucrose favoured the production of CPA by P. griseofulvum , while xylose was ideal carbon source for production of penitrem A by P. crustosum. On the other hand, patulin production by A. terreus was maximum in medium containing starch, where as suc rose and starch were responsible for increased production of terreic acid. Lactose inhibited the production of all mycotoxins by the fungi under investigations. Glycerol inhibited the production of penitrem A and patulin by P. crustosum and A. terreus resp ectively. Sorbitol exerted varied influence on three fungi under study for production of respective mycotoxins. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 20 - 23 (201 2 ) Res.J.Recent.Sci . International Science Co ngress Association 21 Table - 1 Statistical analysis of effect of different carbon and nitrogen sources on production of biomass and mycotoxins by three mycotoxigenic fungi Source Statistical Parameters Final P H P. griseofulvum P. crustosum A. terreus Dry weight (mg/ml) CPA (g/ml) Penitrem A (mg/g) Terreic acid (ppb) Patulin (ppb) Carbon source Glucose MeanSt.d S.E 6.000.70 0.50 106.516.2 11.5 1.050 .71 0.50 152.524.7 17.5 36.59.19 6.50 20.80.98 0.70 Xylose MeanSt.d S.E 6.351.20 0.85 103.021.2 15.0 1.340.31 0.22 132.553.0 37.5 30.517.6 12.5 18.34.52 3.20 Sucrose (C) MeanSt.d S.E 5.650.21 0.15 112.08.48 6.00 1.150.57 0.40 155.021.2 15. 0 33.014.1 10.0 20.80.98 0.70 Lactose MeanSt.d S.E 6.251.06 0.75 73.063.6 45.0 0.890.94 0.66 112.581.3 57.5 29.019.7 14.0 17.65.51 3.90 Starch MeanSt.d S.E 6.251.06 0.75 76.558.6 41.5 1.020.76 0.59 164.081.3 57.5 31.516.2 11.5 19.13.39 2. 40 Sorbitol MeanSt.d S.E 6.000.70 0.50 91.537.4 26.5 0.960.84 0.60 125.063.6 45.0 34.012.7 9.00 19.33.04 2.15 Glycerol MeanSt.d S.E 5.450.07 0.50 84.048.0 34.0 0.920.90 0.64 115.077.7 55.0 29.019.7 14.0 20.31.69 1.20 Nitrogen source Sodium nitrate (C) MeanSt.d S.E 5.650.21 0.15 111.09.89 7.00 1.120.62 0.44 153.024.0 17.0 31.516.2 11.5 20.80.98 0.70 Potassium nitrate MeanSt.d S.E 5.550.70 0.50 102.022.6 16.0 1.280.39 0.28 142.538.8 27.5 32.514.8 10.5 20.52.05 1.45 Amm onium nitrate MeanSt.d S.E 5.000.70 0.50 72.564.3 45.5 0.850.99 0.70 102.595.4 67.5 27.521.9 15.5 16.86.57 4.65 L - aspartic acid MeanSt.d S.E 5.900.56 0.40 107.015.5 11.0 0.960.84 0.60 121.069.2 49.0 29.019.7 14.0 19.33.04 2.15 L - asparagine MeanSt.d S.E 6.251.06 0.75 114.05.65 4.00 0.940.87 0.62 117.574.2 52.5 30.517.0 12.5 20.31.69 1.20 L - tyrosine MeanSt.d S.E 5.250.35 0.25 102.521.9 15.5 0.791.08 0.76 155.021.2 15.0 26.523.3 16.5 18.44.38 3.10 Urea MeanSt.d S.E 5.750.35 0 .25 69.568.5 48.5 0.900.92 0.65 110.084.8 60.0 29.019.7 14.0 17.35.93 4.20 S.E= Standard Error, St.d= Standard Deviation. Table - 2 Effect of temperature and pH on biomass and mycotoxin production by three mycotoxigenic fungi Temperature (C) Final p H P. griseofulvum P. crustosum A. terreus Dry weight (mg/ml) CPA (g/ml) Penitrem A (mg/g) Terreic acid (ppb) Patulin (ppb) 15 5.8 58.0 0.21 20.0 0.00 15.7 20 6.2 80.0 0.32 120.0 0.00 18.1 25 5.8 105.0 0.70 142.0 25.0 20.0 30 6.8 90.0 0.52 135 .0 20.0 19.1 35 6.5 85.0 0.22 50.0 15.0 15.1 40 5.6 20.0 0.00 25.0 0.00 13.1 pH 2.5 5.0 0.00 0.00 28.0 0.00 3.15 3.5 5.5 75.0 0.15 38.0 5.00 10.1 4.5 5.0 90.0 0.20 100.0 8.00 13.1 5.5 (C) 5.8 105.0 0.72 140.0 23.0 21.1 6.5 7.0 82.0 0.32 143.0 20.0 20.1 7.5 7.8 50.0 0.18 138.0 6.00 18.1 8.5 8.5 70.0 0.00 120.0 0.00 16.2 9.5 8.5 0.00 0.00 40.0 0.00 5.12 Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 20 - 23 (201 2 ) Res.J.Recent.Sci . International Science Co ngress Association 22 Out of seven nitrogen sources tested, L - asparagine supported maximum amount of CPA production by P. griseofulvum, wh ile P. crustosum opted potassium nitrate for penitrem A production. L - tyrosine induced the maximum production of terreic acid. However, it supported minimum amount of penitrem A and patulin by P. crustosum and A. terreus respectively . The present fungi al so exhibited preferential response towards L - aspartic acid and L - asparagine. Amino form of nitrogen was preferred for production of CPA and patulin by P. griseofulvum and A. terreus respectively. On the other hand, for production of penitrem A and terreic acid, the acid form was preferred. Such preferential utilization of acid and amino forms of nitrogen has also reported for other fungi also 14,15,16 . Urea inhibited the production of CPA by P. griseofulvum to a significant level. Out of three nitrates test ed ammonium nitrate was poor as it supported least amount of growth and respective mycotoxin production. Sodium nitrate followed by L - asparagine favoured the growth of the present three fungi. On the other hand, urea and ammonium nitrate inhibited the biom ass production by the fungi under investigations. It is evident from the results presented in table 2, that production of CPA and penitrem A production by P. griseofulvum and P. crustosum respectively was maximum at 20 - 25 C, while A. terreus preferred 2 5 - 30C for patulin and terreic acid production. P. crustosum failed to produce penitrem A at 40C, while production of CPA by P. griseofulvum was adversely affected to a significant level at this temperature. A. terreus failed to produce patulin at incubat ion temperature of 15, 20 and 40C. The amount of CPA produced by P. griseofulvum was almost same at 20 and 35C. The biomass production was maximum at incubation temperature of 25C to 30C, while 40C temperature inhibited the growth of interacting fungi to some extent. Temperature below and above 25 0 C affected the biomass production by the present fungi. It is also evident from the table that all the three fungi understudy opted pH 5.5 for production of all the mycotoxins. The amount of toxin produced by respective fungi decreased both with the increase of alkalinity or acidity. At pH 2.5 and 9.5 production of CPA, penitrem A and patulin production by P. griseofulvum, P. crustosum and A. terreus respectively was totally inhibited, while A. terreus could produce a little amount of terreic acid, at pH 2.5. pH 5.5 to 7.5 was found to be favourable for biomass production, which decreased with further increase in acidity or alkalinity. The final pH of the medium varied with the environmental conditions. No po sitive correlation could be observed between mycotoxin production and pH of the medium. Conclusion From the present investigations it is clear that fungi living in an environment encounter interact and influence the living of other fungi. Further, the fun gal interaction is also influenced by nutrients (carbon and nitrogen sources) present, pH of the substratum and temperature prevailing. When D - xylose favoured the production of penitrem A by P. crustosum, starch stimulated the production of terreic acid. O n the other hand, patulin produced by A. terreus remained unaffected. L - tyrosine and sodium nitrate were responsible for maximum production of Terreic acid, while L - asparagine favoured production of CPA by P. griseofulvum . pH of 5.5 and incubation temperat ure of 252 ˚C were favourable for production respective of mycotoxins by the fungi understudy. Thus situation in an open environment is complex and needs to be studied more critically. 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