INFLUENCE OF SOME GROWTH FACTORS ON IN-VITRO GROWTH OF FUSARIUM OXYSPORUM F. SP. PHASEOLI CAUSING SEEDLING MORTALITY OF BUSH BEAN
Sharmin Siddique, M. K. A.Bhuiyan, M. R. Uddin and M. B. Anwar. 2012. Influence of some growth factors on in-vitro growth of Fusarium oxysporum f. sp. phaseoli causing seedling mortality of bush bean. Bangladesh J. Plant Pathol. Vol. 28 (1&2): 13-18.
The present experiment was conducted to determine the influence of different temperature regimes (15, 20, 25, 30 and 35C), pH levels (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5), nitrogen sources (peptone, L-asparagines, NaNO3, NH4NO3, (NH4)2SO4,), carbon sources (dextrose, D-xylose, sucrose, glycerol and D-mannitol) and C/N ratio (5, 10, 20, 40, 80, and 100) on in-vitro growth of Fusarium oxysporum f.sp. phaseoli isolated from diseased bush bean (Phaseolus vulgaris L.) seedlings. The growth was measured in terms of radial colony diameter on semi solid medium and dry mycelial weight grown in liquid medium. The pathogen grew well at the temperature range of 15-35 C, pH range of 4.0-8.5 and C/N ratio range of 5-100. The maximum colony diameter of 85.47 mm and 272.89 mg mycelial dry weight per plate were found at 30C. The highest colony diameter of 78.67 mm/plate was recorded from pH 6.0 and maximum dry mycelial weight of 246.06 mg/plate was found at pH 6.5. The highest colony diameter and dry mycelial weight were recorded when C/N ratio was maintained at 20. The best source of nitrogen was peptone and that of carbon was sucrose. Based on results of the present study it may be concluded that the optimum temperature, pH and C/N ratio for mycelial growth of the fungus are 30 C, pH 6.0-6.5 and C/N ratio 20, respectively. Peptone and sucrose are the best sources of nitrogen and carbon, respectively.
Fusarium oxysporum f. sp. phaseoli is a major cause of seedling mortality of vegetables including bush bean (Phaseolus vulgaris L.). It causes seed deterioration and root rot or wilt (Cavalacanti et al. 2002). Ellanskaia (1969) demonstrated that different sources of carbon had remarkable influence on the growth and conidia formation of fungi under the genus Fusarium. Warner (1990) studied the effect of temperature and medium composition on growth and sporulation of F. oxysporum. He found that the maximum growth and sporulation of the fungus occurred on potato dextrose agar and malt agar at 25-30C. Chlamydospore germination is influenced by the presence of exogenous sources of carbon and nitrogen (Ciotola et al. 2000). The effects of physiological and pathological factors on growth, development and pathogenicity of F. oxysporum have been well documented by Ciotola et al. (2000). Systematic researches on physiological aspects of F. oxysporum f. sp. phaseoli causing seedling mortality of bush bean are not available in Bangladesh. But such information is essential to develop an appropriate control strategy against seedling mortality of vegetable crops caused by F. oxysporum f.sp. phaseoli.
The present study was undertaken to determine the influence of different growth factors on in-vitro growth of F. oxysporum f. sp. phaseoli infecting bush bean.
MATERIALS AND METHODS
Influence of five important growth factors namely temperature during incubation, and pH, nitrogen source, carbon sources and C/N ratio of the culture medium were tested following in-vitro method (Dhingra and Sinclair 1985). First two experiments were conducted to test five temperature regimes (15, 20, 25, 30 and 35 C) and ten levels of pH (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5). In both the experiments, potato dextrose agar (PDA) and potato dextrose broth (Tuite 1969) were used. Another three experiments were conducted to test the effect of five nitrogen sources (NaNO3, NH4NO3, (NH4)2SO4, peptone and L-Asparagine), five carbon sources (Dextrose, D-Xylose, Sucrose, Glycerol and D- mannitol) and six levels of C/N ratio (5, 10, 20, 40, 80, and 100) on mycelium growth of F. oxysporum f.sp. phaseoli. Czapek’s solution with agar (semisolid) and without agar (liquid) was used as basal media. The test fungus, F. oxysporum f.sp. phaseoli was isolated from foot rot infected bush bean seedling following tissue planting method (Tuite 1969). To prepare its inocula, the fungus was grown on PDA medium in Petri dishes. Mycelial discs were cut from growing edge of 4 days old culture of the fungus with a flame sterilized 5 mm diameter cork borer. Except the experiment with pH levels, the pH of the culture medium was adjusted to 6 using 0.1N HCl and 0.1N NaOH and sterilized in an autoclave at 120C under 1kg/cm2 pressure for 20 minutes. The liquid medium (broth) was prepared using all ingredients except agar.
To test carbon sources, Czapek’s medium was prepared by mixing each carbon source at 30 g per 1 liter. Czapek’s medium without any carbon source was used as control. To study the influence of different nitrogen sources, the Czapek’s medium was prepared by mixing requisite quantity of each nitrogen source which is equivalent to the amount of nitrogen obtainable from 3g of NaNO3. The Czapek’s medium without any nitrogen source was used as a control. To test the effect of C/N ratio on growth of the pathogen, Czapek’s medium was prepared by mixing requisite quantity of NaNO3 as a source of nitrogen at two fixed levels of sucrose (2g and 5g) as a source of carbon. The levels of the C/N ratio were 5, 10, 20, 40, 80, and 100.
The growth of the fungus was measured in terms of colony diameter and mycelial dry weight. To measure colony diameter, agar medium (semisolid) was used and the fungus was grown in 90 mm glass Petri dishes. For the measurement of mycelium dry weight, the fungus was grown in 100 ml conical flasks containing liquid medium (without agar). The agar media were dispensed into the Petri dishes and the liquid media were also poured into the conical flasks at 20 ml per dish or flask.
To measure colony diameter, Petri plates containing agar medium were inoculated after solidification with the mycelium discs (0.5 cm) of the test fungus. The inoculum was placed at the center of each plate. Except the experiment to test different temperature regimes, the inoculated Petri plates under all experiment were incubated at 25C in incubators. The plates were arranged in the incubators following completely randomized design with three replications. The colony growth was measured by averaging the two diameters taken at right angle for each colony after 4 days of incubation.
To measure mycelium dry weight, conical flaks containing potato dextrose broth or liquid Czapeck’s medium (40 C) were inoculated with the inoculum at one disc per flask. The inoculated flasks were incubated at room temperature (25-28 C) for 14 days. The flasks were arranged on the laboratory desks following completely randomized design with three replications (flasks). At the end of incubation period, the cultures in all flasks were filtered through dry (at 70 C for 12 hr) and pre-weighed filter paper. Dry weight of mycelium was determined after drying the mycelium along with the filter paper in an oven at 70 C for constant weight. Dry weight of mycelium was obtained by subtracting weight of only filter paper from weight of filter paper plus mycelium.
Data collected from different experiments were analyzed for ANOVA using MSTAT-C program. Duncan’s Multiple Range Test (DMRT) was performed to compare treatment means. Whenever necessary, data were transformed following appropriate method before statistical analysis.
RESULTS AND DISCUSSION
Effect of temperature on mycelial growth
Results on the effect of temperature regimes on radial colony diameter and mycelium dry weight of F. oxysporum f. sp. phaseoli are presented in Figure 1. Significantly the highest radial growth was observed at 30C followed by room temperature, 25 and 20C. The lowest radial growth was observed at 15C. The optimum temperature for the radial colony growth was 30C. An increase or decrease from the optimum temperature, the radial growth of the pathogen was significantly reduced. The relationship between temperature regimes and radial colony diameter was polynomial. Similar trend was observed in case of mycelium dry weight. The highest mycelium dry weight was found at 30C followed by room temperature (25-28 C), 25 and 20C. However, the mycelium dry weight at those levels was statistically similar but significantly higher compared to 35 and 15C. Relationship between mycelium dry weight and temperature level was also polynomial. Findings of the present study suggest that temperature regime play an important role on the growth of the F. oxysporum f.sp. phaseoli.
Effect of pH on mycelium growth
The pH of the culture medium showed appreciable influence on radial colony diameter and mycelium dry weight of F. oxysporum f. sp. phaseoli. The highest radial growth was observed at pH 6.5, which was statistically similar to the growth at pH 5.0, 5.5, 6.0 and 7.0. The lowest radial hyphal growth was recorded at pH 4.0 followed by pH 8.5. The highest mycelium dry weight was found at pH 6.5, which was statistically similar to pH 5.5, 6.0, 6.5 and 7.0. The lowest mycelium dry weight was observed at pH 4.0, which was statistically similar to pH 8.5. The relationship between radial colony diameter and pH level, and mycelium dry weight and pH level was polynomial. The results of the present experiment indicate that F. oxysporum f. sp. phaseoli can grow at a wide range (4.0-8.5) of pH levels of culture medium (Fig. 2).
Effect of nitrogen sources on mycelial growth
The effect of five nitrogen sources on radial colony diameter and mycelium dry weight of F. oxysporum f. sp. phaseoli is shown in Figure 3. All the sources of nitrogen increased radial growth significantly over control (Czapek’s medium). The highest radial colony diameter of the pathogen was obtained from Czapek’s basal medium supplemented with peptone as a nitrogen source. The radial growth obtained from the medium containing NaNO3 and NH4NO3 was statistically similar but significantly higher compared to (NH4)2SO4. The highest mycelial dry weight was observed in peptone medium followed by L-Asparagine. But their differences were not significantly different. Other nitrogen sources also gave higher mycelial dry weight compared to control.
Effect of carbon sources
Use of different carbon sources in the growth medium gave significant increase in colony growth of F. oxysporum f. sp. phaseoli compared to control. The highest radial growth was obtained with Czapek’s medium containing sucrose as a carbon source followed by glucose and dextrose. Among the carbon sources, glycerol appeared to be a poor source of carbon for in vitro the growth of the pathogen. Similar growth trend was also observed incase of mycelium dry weight. Sucrose was appeared to be the best source of carbon for mycelium dry weight of F. oxysporum f. sp. phaseoli followed by glucose, dextrose, mannitol, glycerol and PDA (Fig. 4).
Effect of C/N ratio on colony growth
At 2 g sucrose, the highest radial colony diameter was found at C/N ratio 20, which was statistically similar to C/N ratio 10 but significantly higher compared to other levels of C/N ratio. At C/N ratios 5, 10 and 20, the radial colony diameter was statistically similar but significantly higher compared to only two higher C/N ratios. At 5 g sucrose, the highest radial colony diameter was also observed at C/N ratio 20, which was statistically similar to C/N ratio of 10 but significantly higher compared to other levels of C/N ratio. The second highest radial colony growth was recorded from C/N ratio 10 followed by C/N ratio 5, 40 and 80. The differences in colony diameter at those three levels of C/N ratio were not significant. At both 2 and 5 g sucrose, the lowest colony diameter was recorded from C/N ratio 100, which was statistically similar to C/N 80 (Fig. 5).
In presence of 2 g as well as 5 g sucrose, the mycelium dry weight of the fungus increased gradually with increased levels of C/N ratio up to 20 and decrease thereafter. However, the mycelium dry weight at C/N ratios 5, 10 and 20 was statistically similar and significantly higher compared to all other of C/N ratios. The lowest dry weight was found at the highest C/N ratio followed by C/N ratio 80 (Fig. 6).
The colony diameter and mycelium dry weight increased gradually with the increase of C/N ratio up to 20 and decreased thereafter. Relationship of two parameters with level of C/N ratio was polynomial (Fig. 5 and 6).
Results of the present study reveal that all growth factors tested have shown considerable influence on the radial colony diameter and mycelial dry weight of F. oxysporum f. sp. phaseoli. The pathogen can grow at a temperature range of 15-35 C, pH range 4.0-8.5 and C/N ratio 5-100. Influence of nitrogen and carbon sources on mycelial growth of the pathogen is also considerable. The fungus grows well in culture medium containing peptone, L-Asparagine, NaNO3 and NH4NO3 as nitrogen sources, and manitol, dextrose and glucose as carbon sources. The best source of nitrogen is peptone and that of carbon is sucrose. More or less similar findings have been reported by many other researchers. Gaikwad and Pachpande (1992) and Osman et al. (1992) observed that the optimum temperature range for better growth of F. oxysporum is 20 to 35C with the highest growth at 30C. Other investigators also found optimum colony growth of Fusarium at a temperature rage of 24 -27C (Chen et al. 2003, Dwievedi and De 2003). Raghuwanshi (1995), Kumar et al. (2000) and Ragazzi (1992) found that various formae speciales of F. oxysporum grow well at a range of pH of 5.0-7.5.
The results on the effect of nitrogen sources on the growth of the pathogen recorded in the present study are in agreement with findings of other investigators (Rahman et al. 1993, Chen et al. 2003, Kumar et al. 2000, Ciotola et al 2003). The results on the effect of nitrogen sources on the growth of the pathogen recorded in the present study are in agreement with findings of other investigators (Rahman et al. 1993, Chen et al. 2003, Kumar et al. 2000, Ciotola et al 2003). Griffin (1970) demonstrated that carbon and nitrogen are the most vital nutrients for the growth of F. oxysporum. Kumar et al. (2000) and Desai et al. (1994) obtained maximum growth of F. oxysporum f. sp. lentis and F. oxysporum f. sp. ciceri with mannitol and maltose used as carbon sources in culture medium.
Based on findings of the present investigation it may be concluded that the optimum temperature and pH for mycelial growth of F. oxysporum f. sp. phaseoli is 30 C and pH 6.0-6.5, respectively. The optimum C/N ratio for its mycelial growth is 20. Peptone and sucrose are the best sources of nitrogen and carbon, respectively for mycelial growth of the fungus.
Cavalcanti, L. S., Cohelo, R. S. B. and Perez, J. O. 2002. Use of two inoculation methods to cultivate the resistance of common bean cultivars and lines to Fusarium oxysporum f. sp. phaseoli. Ciencia- Rural. 32(1): 1-5.
Chen, F. R., Yang, X. J., Li, T., Xie, S. Y. and Ruan, H. C. 2003. Studies on the biological characteristics and control of banana vascular wilt (Fusarium oxysporum f. sp. cubense). Acta. Agric. Univ. Jiangxiensis. 25(6): 900-903.
Ciotola, M., DiTommaso, A. and Watson, A. K. 2000. Chlamydospore production, inoculation methods and Pathogenicity of Fusarium oxysporum M12-4A, a biocontrol for Striga hermonthica. Bio. Sci. Tech. 10(2): 129-145.
Desai, S., Nene, Y. L. and Reddy, A. G. R. 1994. Races of Fusarium oxysporum causes wilt in chickpea: growth variability. Indian J. Mycol. Plant Pathol. 24(2) :120-127.
Dhingra, O. D. and Sinclair, J. B. 1985. Basic plant pathology methods. CRC Press. Inc. Boca Raton Florida. pp.13-44.
Dwivedi, R. P. and De, R. K. 2003. Effect of different culture media and temperature on growth and sporulation of Fusarium oxysporum f. sp. lentis. Indian J. Pul. Res. 16(1): 50-53.
Ellanskaia, I. A. 1969. Effect of different sources of carbon nutrition on growth and conidium formation of fungi of the genus Fusarium. Mikrobiol Zh. 31(1):22-27.
Gaikwad, S. J., and Pachpande, S. M. 1992. Effects of temperature on wilt of sesame caused by of Fusarium oxysporum f. sp. sesame. J. Mharastra Agric. Univ. 17(1): 76-78.
Griffin, G. J. 1970. Exogenous carbon and nitrogen requirements for chlamydospore germination by Fusarium solani: dependence on spore density. Can J Microbiol. 16(12):1366-1368.
Kumar, R., Jha, D. K. and Dubey, S. C. 2000. Influence of nutrition and pH on growth and sporulation of Fusarium oxysporum. J. Research, Birsa Agric. Univ. 12(1): 61-65.
Osman, M., El-Sayed, M. A., Mohamed, Y. A. H. and Metwally, M. 1992. Effect of Various culture conditions on Alternaria alternate and Fusarium oxysporum. 1. Culture media, temperature, age and carbon source. Microbios 71(286): 15-26.
Ragazzi, A. 1992. Different strains of Fusarium oxysporum f. sp. vasinfectum from cotton in Angola: biological aspects and pathogenicity. Zeitschrift fur Pflanzenkrankheiten und Pflan-zenscutz 99(5): 499-504.
Raghuwanshi, K. S. 1995. Cultural; and physiological studies of Fusarium oxysporum f sp. sesami causing wilt disease of sesamum. Madras Agric. J. 82 (11): 605-607.
Rahman, M. Z., Ayub, A., Dey, T. K. and Alam, K. B. 1993. Effect of nitrogen and carbon sources on growth of Fusarium oxysporum and Sclerotium rolfsii. Bangladesh J. Plant Pathol. 9(1&2): 23-25.
Tuite, J. 1969. Plant Pathological Method. Fungi and Bacteria. Burgress Pub. Minnesota, Minn. USA. 293 pp.
Warner, M. 1990. Effect of temperature and medium composition on growth and sporulation of Formae speciales of Fusarium oxysporum Schlecht. Rockzniki Akademii Rolniczez W. Poznaniu, Ogrodnictwo. 217(18): 107-12