Physiological and Cultural Variabilities among Fusarium oxysporum f.sp. cepae Isolates in Onion

The effect of culture media, carbon sources, nitrogen sources, temperature and pH on the mycelial growth of Fusarium oxysporum f.sp. cepae was investigated. The fungus grew the best on Czapek’s dox medium and glucose was the best source of carbon whereas peptone was the best source of nitrogen. Growth was maximum at 30°C which was reduced drastically below 20°C and above 35°C. The most suitable pH for optimum growth of fungus was 6.5.

Control of this disease was possible to a limited extent, with the help of fungicides, biocontrol agents and cultural practices. Biocontrol agents like Trichoderma harzianum (TH3) and Pseudomonas sp. (Pf 12) effectively reduced the growth of the Fusarium oxysporum sp. cepae (Malathi and Mohan, 2011). For the development of cultural management practices, there is necessity to identify the nutritional requirements and environmental conditions for the infection and survival of the pathogen (Imran Khan et al., 2011). Hence, the present investigation was conducted to study the effect of physiological factors on the mycelial growth of the fungus.

Isolation of pathogen
Onion bulbs infected with basal rot disease were collected from fifteen different places of Tamil Nadu (Table 1). The pathogen was isolated from the diseased tissues of onion by tissue segment method (Rangaswami, 1958). The infected portions of onion bulbs were cut into small pieces using sterilized scalpel and these were surface sterilized with 0.1 per cent mercuric chloride for one minute and washed thrice with sterile distilled water and then placed on previously poured and solidified Petri dish containing Potato Dextrose Agar (PDA) medium. These plates were incubated at room temperature (28 ± 2ºC) for five days and observed for the growth of the fungus. The hyphal tips of fungi grown from the pieces were transferred aseptically to PDA slants for maintenance of the culture.

Growth characters of Fusarium oxysporum f.sp.cepae isolates on different solid media
In order to compare the growth of Fusarium oxysporum f.sp. cepae on nine solid media viz., Potato dextrose, oatmeal, carrot dextrose, radish dextrose, beet root dextrose, czapek's dox, modified czapek's dox, richard's medium and rose bengal. The sterilized warm medium @ 15 ml was poured in sterilized Petri dishes (10 cm) and allowed to solidify. The pathogen was inoculated at the centre of the plate by placing a five days old nine mm culture disc. Plates were incubated at room temperature (28±2°C) and three replications were maintained in each medium and the radial growth of the mycelia was measured seven days after inoculation.

Growth characters of Fusarium oxysporum f.sp.cepae isolates on different liquid media
Potato dextrose, oatmeal, carrot dextrose, radish dextrose, Czapek's dox, modified Czapek's dox, richard's, rose bengal and beetroot dextrose broth were prepared (without adding agar). From the prepared medium 100 ml was distributed in 250 ml Erlenmeyer flasks and autoclaved at 1200C at 15 psi for 20 minutes. The flasks were separately inoculated with a five days old nine mm culture disc of the pathogen and three replications were maintained in each medium. The mycelial mat was filtered through a preweighed Whatman No.1 filter paper, dried in hot air oven at 100oC until constant weight was obtained and the mycelia dry weight was measured. *Corresponding author email: malathi_agri@redifmail.com.

Effect of carbon and nitrogen sources
The czapek's dox medium was substituted with carbon sources viz., carboxy methyl cellulose, glucose, fructose, mannitol, sucrose, starch and nitrogen sources such as ammonium nitrate, ammonium oxalate, ammonium sulphate, urea, sodium nitrate, peptone and potassium nitrate and sterilized. The medium without nitrogen and carbon source served as control. The sterilized warm medium was poured in the sterilized Petri plates and allowed to solidify. Petri plates were inoculated with five days old nine mm culture disc of the pathogen and incubated at the room temperature (28±2°C) for seven days. Three replications were maintained for each carbon and nitrogen source and the diameter of mycelial growth was recorded (Lily and Barnet, 1951).

Effect of pH
Czapek's dox medium was prepared and distributed in 250 ml conical flask @ 100 ml/flask and the pH of the medium was adjusted to different pH levels viz., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 with 0.1 N HCl or 0.1 N NaOH using digital pH meter and sterilized in autoclave at 1200C at 15 psi for 20 minutes. Fifteen ml of the medium from each pH level were poured onto sterilized Petri dishes and allowed to solidify. A nine mm PDA culture disc of actively growing Fusarium oxysporum f.sp.cepae was placed at the center of each Petri dish containing medium under aseptic conditions. The plates were incubated at room temperature (28±2°C) for seven days. Three replications were maintained for each pH level and the mycelial growth of the pathogen was measured at seven days after incubation (Gangadhara et al., 2010).

Effect of temperature
Isolates were inoculated in Czapek's dox medium and different temperature was maintained viz., 5, 10, 15, 20, 25, 30, 35, and 40°C (Gangadhara et al., 2010). Three replications were maintained for each temperature and the mycelial growth of the pathogen was measured at seven days after incubation.

Effect of culture media
Fungi secure food and energy from the substrate upon which they live in nature. In order to culture the In the present investigation the isolates exhibited variations of growth in different solid media tested. All fifteen isolates grew well in Czapek's dox medium (8.66 cm) followed by Potato Dextrose Agar (8.15 cm) while the lowest growth was recorded in beet root agar medium (6.01 cm). Among the isolates, DNSFOC1 collected from Sempatti recorded maximum mycelial growth 8.90 cm in czapek's dox medium (Table 2) followed by EROFOC3 (8.87 cm) collected from perumalkovil pudhur.
Among nine liquid media tested, czapek's dox liquid medium recorded the highest mean mycelial 603 dry weight (507.89 mg) followed by potato dextrose liquid medium (467.44 mg) while beet root broth showed the least dry weight (280.43 mg) (Table 3). Among the isolates, DNSFOC1 collected from Sempatti recorded maximum dry mycelial weight 546.24 mg in czapek's dox broth (   Haware et al., (1986) modified the czapek's dox agar medium by adding PCNB, streptomycin and malachite green which was highly effective for the growth of Fusarium oxysporum. Similarly, czapeks dox agar was the best for the radial growth of Fusarium oxysporum as this fungus gave maximum growth of 85 mm (Farooq et al., 2005). Imran Khan et al. (2011) reported that five isolates of Fusarium oxysporum f.sp.ciceri produced maximum growth on potato dextrose agar medium (85.76 mm) followed by Richard's medium (84.62 mm) and Czapek's dox medium (72.56 mm).

Effect of carbon and nitrogen sources
Carbon source is important for the growth and development of fungi. The results of this experiment indicated that all the carbon sources were suitable for the growth of Fusarium oxysporum f.sp.cepae. Glucose recorded maximum mean mycelial growth (8.43 cm) followed by sucrose (8.16 cm). Mannitol recorded the minimum mean mycelial growth (5.45 cm) (Table 4). Among the isolates of Fusarium oxysporum f.sp.cepae, ERKFOC1 isolate from Kodumudi showed the highest mycelial growth (8.85 cm) in glucose as a carbon sources.
Nitrogen is required for protein synthesis and other essential functions in fungal cell. Peptone amended medium allowed maximum mean mycelial growth (8.53 cm) followed by potassium nitrate (8.05 cm) (Table 5). Among the isolates of Fusarium oxysporum f.sp.cepae, MDPFOC2 collected from Palamedu showed the highest mycelial growth (8.79 cm) in peptone as a nitrogen source. Such variability occurs among growth pattern of the isolates in respect to carbon and nitrogen sources, perhaps due to difference in their environment conditions where they were collected. Similar results were obtained by Farooq et al. (2005) who showed that glucose and peptone were the best carbon and nitrogen sources for Fusarium oxysporum f.sp.ciceri. Glucose, maltose and sucrose were excellent for maximum mycelial growth of Fusarium avenaceum. Glucose (483.26 mg) was found to be good carbon source for the growth of Fusarium oxysporum f.sp. ciceri followed by maltose (476.19 mg) (Imran Khan et al., 2011). Results of the study indicated that the role of C:N ratio was very important to colonize organic substances in the soil. Increased inoculum potential and disease severity were positively correlated with the food base of organic substances. Crop debris that served as a food base can also serve as an infection bridge. Increasing the C/N ratio of the medium resulted in reduced macroconidial formation and increased chlamydospore production.

Effect of pH
Change in pH plays an important role in hydrogen ion concentration which is essential for the growth of fungi. In the present study, maximum mean mycelial growth was observed at pH 6.5 (8.56 cm) followed by pH 7.0 (8.42 cm) and less growth was observed below pH 5.0 and above pH 7.5 (Table 6). Farooq et al. (2005) reported that the growth of F.oxysporum was maximum (80 mm) at pH 7. Fusarium species grew well at a pH range of 6.5 to 7.0 (El-Sayed et al., 2008). Imran Khan et al. (2011) reported that between pH 6.5 and pH 7.0 was found 605 to be optimum for the growth of Fusarium oxysporum f.sp. ciceri. Fusarium oxysporum had maximum radial growth at pH 6.5 followed by pH 7.5 (Bhale, 2012).
Among the isolates, DNSFOC1 from Sempatti recorded maximum mycelial growth 8.98 cm followed by DNOFOC2 (8.85 cm) collected from Ottanchathram at pH 6.5. Variation in growth and sporulation of isolates at a particular pH may be due to fact that a particular isolate grew better at a specific pH. The other possible reason may be that isolates collected from a particular location may have become adopted to particular pH for growth and sporulation (Mehta et al., 2005).

Effect of temperature
Each fungus has its own temperature range for growth and sporulation. Growth of all organisms is affected by temperature because it mediates metabolic reactions. The temperature range between 0°C and 40°C was the most suitable for growth of microorganisms, the minimum temperature being associated with the transition phase at freezing and the maximum temperature influenced the catabolism of microorganisms (Gilloly et al., 2001). In the present study, growth of the fungus was drastically reduced below 200C and above 350C. The fungus attained the mean maximum growth of 8.51 cm at 300C (Table 7).
Among the isolates, MDUFOC3 from Usilampatti recorded maximum mycelial growth 8.85 cm followed by DNSFOC1 (8.81 cm) from Sempatti at 300C. Farooq et al. (2005) observed that at 25oC and 30oC the Fusarium oxysporum attained the maximum growth 76.8 and 85.4 mm. Temperature above 37oC affected the hyphal tip elongation and appressorial formation (Agrios, 2006). A temperature of 25-30°C was found to be favourable for germination, germ-tube elongation, sporulation and growth, although the fungus could tolerate higher temperatures. Activity of the fungus was enhanced at high relative humidities. El-Sayed et al.(2008) reported that growth of Fusarium spp. was best between 25oC and 30oC. 30oC was optimum for the growth of Fusarium oxysporum f.sp.ciceri which was an important factor for growth, reproduction and survival of the fungus (Imran Khan et al., 2011).
The results of this study determined the culture conditions required to optimize the growth better understanding of Fusarium oxysporum f.sp.cepae isolates. These results will be helpful for the cultural management of the disease.