Isolation and characterization of Gibberellic acid 3 producing Fusarium sp. from Belgaum agriculture land and its impact on green pea and rice growth promotion
Worldwide ultimate aim of any agriculture sector or farmer is to take maximum yield. Sufficient supply of nutrients and fertilizer are not able to give maximum yield. There are numerous factors which are responsible for low yield, among that one is the environment stress or the unstable climate conditions. To increase the yield there are numerous approaches like use of genetically modified crops, but in India it is controversial approach and another approach is the use of multifunctional plant hormone like Gibberellic acid 3 (GA3). This research mainly involves the production of GA3 from fungal species and to apply it on crop plants. Fusarium species were isolated from Belgaum agriculture soil and screened for GA3 production under submerged fermentation. Strain showing maximum GA3 yield (strain M104) was taken to study the effect of various parameters on GA3 production, like incubation time (1 – 12 days), initial pH (5.0 -8.0), incubation temperature (20 – 40 °C), pH (5.0 -8.0), and carbon and nitrogen sources. The maximum production of GA3 was observed on day 8 at 30 °C, and pH 5.5 with glucose and ammonium chloride as good carbon and nitrogen sources, respectively. After optimization, a 6.56-increase in GA3 production was observed. The GA3 production was confirmed by thin layer chromatography. The GA3 crude extract obtained using submerged fermentation was then used to study its effect on germination and growth of green pea plant and paddy crops. It was observed that GA3 treated crops showed uniform growth and they were taller than non-treated plants, suggesting its application in increasing the crop plant harvests.
Key words: Fusarium sp, isolation, gibberellic acid, optimization, submerged fermentation, crop plants.
Gibberellic acids, also known as gibberellins, are the complex organic molecules acting as plant growth hormones. They are chemically known as diterpenoid acids having molecular formula C19H22O6. They regulate the functions like cell division, cell elongation, sex expression, seed germination, breakdown of seed dormancy and flowering etc. In microorganisms such as bacteria and fungi, gibberellic acid 3 is the principal product of gibberellins, act as secondary metabolite (Bruckner and Blecschmidt, 1991; Karakoc and Aksoz, 2006). Till now, 136 gibberellins were isolated from various plants, and among that gibberellic acid 3 shows maximum biological activity (Rodrigues et al., 2011). The use of GA3 has been approved by food and drug administration (FDA) because of its tremendous application and nontoxic properties, and its safety for environment and human was confirmed by Material Safety Data Sheet (MSDS) (Rodrigues et al., 2011).
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In counties under development where mainly the economy relies on agriculture, the farmers have to use fertilizers and plant hormones to increase production. As most of fertilizers are associated with environmental pollution, plant growth hormones like gibberellic acid 3 have to be produced cost-effectively in huge amounts in order to enhance the quantity of agricultural products (Bilkay et al., 2010). Three routes to obtain GA3 have been reported, viz. extraction from plants, chemical synthesis and microbial fermentation. Among this the third method is the most common method to produce GA3 (Rios-Iribe et al., 2011). GA3 is industrially produced by Gibberella fujikuroi / Fusarium moniliforme under submerged (Santos et al., 2003; Karakoc and Aksoz, 2006). It is also produced by several other fungal species such as Aspergillus niger and Fusarium species and some bacteria such as Pseudomonas, Rhizobium, Azobactor, and Azospirillu species (Rademacher, 1994). All above species produced very low yield of GA3 except Fusarium species in which most of the strains show the highest yield of GA3 than any other microbes (Rangaswamy, 2012). The search for new fungal species like Fusarium species capable of producing an important amount of GA3 is a continuous exercise. The aim of the present study was therefore to isolate and characterize a GA3 producing Fusarium sp. from soil, optimize the culture conditions for maximum GA3 production, and to evaluate its effect on green pea and rice growth promotion.
Materials and Methods
Soil sample selection
To isolate strains of Fusarium, the soil sample was taken from Belgaum agriculture area (Karnataka state, India). This soil was black coloured having high water holding capacity, good fertility and also best soil for crops like paddy, all types of beans, sugarcane and all types of vegetables.
Isolation of Fusarium species
The soil sample collected from Belgaum agriculture land was taken, serially diluted in distilled water and inoculated in a Malachite green agar (MGA). Petri plates containing 15 g of peptone, 0.01 g of Malachite Green (triaryl methane dye), 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulphate, and 20 g of agar per 1000 ml of distilled water were prepared. The incubation was carried out at 30 °C for 5 days (Castellá et al., 1977). The resulted various colonies were picked up and further inoculated in a potato dextrose agar (PDA) plate and incubated for a week for secondary pigmentation. The colony with different morphology and colour pigmentation were sub cultured on PDA slants and labelled (Avinash et al., 2003). The lactophenol cotton blue technique was used to study the characteristics of the fungal isolate (William and Cross, 1971).
Screening of the isolates for GA3 production under submerged fermentation
The Czapack Dox media (CD broth) containing sucrose (30 g), sodium nitrate (3 g), dipotassium hydrogen phosphate (1 g), potassium dihydrogen phosphate (0.5 g), magnesium sulphate (0.5 g), potassium chloride (0.5 g) and ferrous sulphate (0.1 g) per 1000 ml of distilled water was used. The CD broth was prepared in conical flask and adjusted the pH to 7.0, and sterilised in an autoclave for 20 min at 15 psi. After cooling the medium, it was aseptically inoculated (1 × 108 spores / ml) with individual isolated strains. The fermentation flasks were kept on a rotary shaker (100 rpm) at 30 °C for 12 days (Kahlon et al., 1986; Karakoc et al., 2006; Rangaswamy, 2012).
GA3 pre-treatment, extraction and estimation
The fermented broth was taken and centrifuged at 13200 rpm for 10 min and the supernatant was taken and acidified to pH 2-2.5 using 1N HCl. GA3 was extracted trice using equal amount of ethyl acetate/NaHCO3 (Cho et al., 1979). The ethyl extract was kept on hot air oven at 50 °C overnight to remove ethyl acetate and obtain crystals of GA3 (Kahlon et al., 1986; Karakoc and Aksoz, 2006; Karakoc et al., 2006; Bilkay et al., 2010; Rangaswamy, 2012). It was estimated by Berrios et al. (2004) spectrophotometric method and absorption was read at 254 nm in UV-VIS spectrophotometer (Elico, SL-159 model, India).
Confirmation of GA3 by thin layer chromatography (TLC)
The slurry of silica gel was poured on a TLC plates, air dried, and the matrix was activated by keeping the plates on hot air oven at 80 °C for 1 h. GA3 dissolved in ethanol was added as a spot and plates were run using mobile phase containing isopropanol : ammonia : water (10:1:1) for 2 h. The plates were removed, sprayed with 3% sulphuric acid containing 50 mg FeCl3 and heated in oven at 80 °C for 10 min. The GA3 appeared as greenish black/spot fluorescence under UV light (Cavell et al., 1967; Srivastava et al., 2003).
Optimization of culture conditions for maximum GA3 production by Fusarium sp. (isolate M-104).
The incubation time for GA3 production by the fungal isolate under submerged fermentation at 30 °C and at initial pH 7.0 was analysed by inoculating CD broth with 1 ml of fungal spores and incubating on a rotary shaker (100 rpm) for 12 days. The sample was taken every day as the fermentation proceeds in order to find the most suitable incubation time for GA3 production. The effect of pH on GA3 production was studied by adjusting CD broth at different pH, viz. 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The cultivation flasks were inoculated with 1.5% (v/v) inoculum, and incubated for 8 days on rotary shaker (100 rpm) at 30 °C. The effect of temperature on GA3 production was investigated by inoculating the fungal spores in CD broth of pH 7.0 and by incubating at three different temperatures 20, 30, 40, and 50 °C with other conditions remained same. The effect of carbon sources on GA3 secretion was analysed by replacing the sucrose in the CD broth of pH 5.5 by dextrose, glucose, mannitol, and starch, and by incubating at 30 °C for 8 days. The effect of nitrogen sources on GA3 secretion was analysed by replacing the sodium nitrate in the CD broth of pH 5.5 by glycine, ammonium chloride and ammonium sulfate at 30 °C for 8 days.
Effect of GA3 on pea plant and paddy crops
Seeds of pea plants were soaked in 200 ppm of GA3 fermented filtrate for 12 h and then sown in autoclaved soil. After a period of 8 days, 100 ppm of GA3 was sprayed on the plant for each alternative day for another 8 days. The control was soaked in water and sown in autoclaved soil and sprayed with distilled water only. The growth of both the control and test pea plants was monitored over a period of 15 days. 10 paddy seeds were soaked in 300 ppm of GA3 solution for about 2 days and sown in soil. They were sprayed with 200 ppm of GA3 after growth. The control seeds were soaked in water for the same period and sprayed with only water. The observation was carried out for 25 days (Tiwari et al., 2011; Susilawati et al., 2014).
The experiments were carried out in triplicate. ANOVA and DMRT at 5% significance level were used to give the differences between mean values, using SPSS statistical software.
Results and Discussion
Isolation of Fusarium species
Four strains of Fusarium species were isolated from agriculture soil sample and labelled as M101, M102, M104 and M110. The present labelling was based on following pigmentation black, grey, blue and red, respectively. All strains had cottony growth appearance which is one of the important morphological characteristic of the Fusarium species. By staining the fungi with lactophenol cotton blue dye, it was observed that they had non septate hyaline mycelium/ hyphae as shown in figure 1a. The macrospores of banana shape were reseptated which is a unique microscopic feature of Fusarium species as shown in the figure1b. The isolation medium containing malachite green was chosen since malachite green inhibits the radial colony growth of the saprophytes and allows only growth of Fusarium species (Castellá et al., 1997).
Screening for isolates for GA3 production
GA3 can be commercially produced by submerged fermentation using different media but the most common synthetic medium is the Czapack Dox medium (CD broth) (Rangaswamy, 2012). The isolated strains M101, M102, M104 and M110 were subjected to submerged fermentation to check their ability for GA3 production. The different amounts of GA3 produced are given in the table 1 and Figure 2, and the strain M104 was the highest producer of GA3 among the four isolates. Similarly, Aspergillus niger strains produced different amounts of GA3 with the highest of 150.35 mg/l for A. niger Fursan (Cihangir and Aksoz, 1993). Likewise, various amounts of GA3 were produced by Lentinus tigrinus and Laetiporus (Ozcan, 2001).
Optimization of culture conditions for maximum GA3 production by Fusarium isolate M104
The optimization of cultural parameters like incubation time, temperature, and pH, and nutritional conditions like nitrogen and carbon sources, is necessary to produce GA3 in a significant amount. Time course for GA3 production by the isolate M104 was studied. GA3 production started on day 3 and maximum production was observed at day 8, although statistically at par with day 9 and 10 (Table 2). Similar incubation time was noted for GA3 production by Fusarium monilifome (Rangaswamy, 2012). 9 days was optimal time for GA3 secretion by Fusarium fujikuroi SG2 (Uthandi et al., 2010) and Fusarium monilifome (Kobomoje et al., 2013). In contrast, a higher incubation time of 12 days was observed by for Fusarium moniliforme(Kahlon and Malhotra, 1986) and Aspergillus niger (Bilkay et al., 2010). The optimum incubation time for GA3 production by various fungal species depend therefore on the strain used. The short incubation period observed for GA3 production by fungal isolate M104 makes the fermentation cost-effective.
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Among all pH investigated, the pH 5.5 showed the maximum production of GA3 which was 1478.2 mg/L (Table 2). pH 5.5 was also optimum for GA3 production by Fusarium monilifome (Kahlon and Malhotra, 1986; Kobomoje et al., 2013) and Fusarium fujikuroi SG2 (Uthandi et al., 2010). Bilkay et al. (2010) reported pH 5.0 as optimal time for GA3 production by Aspergillus niger, whereas pH 7.0 was optimum for GA3 production by Fusarium monilifome (Rangaswamy, 2012).
The effect of temperature on GA3 production was analysed, and maximum production was observed at 30 °C (Table 3). The production of GA3 by various fungal species was also seen at an optimum temperature of 30 °C (Bilkay et al., 2010, Uthandi et al., 2010; Rangaswamy, 2012; Kobomoje et al., 2013). 25 °C was also optimum for GA3 production by Gibberella fujikuroi (Gelmi et al., 2002). A low GA3 yield at higher temperature was also recorded for GA3 production by Aspergillus niger (Bilkay et al., 2010). A low GA3 production was observed at higher temperatures because metabolic activities get stopped due to enzyme denaturation. The decrease in GA3 secretion by microbial species was ascribed to the variation in enzyme activity or thermal denaturation (Karakoc and Aksoz, 2006).
The effect of carbon sources on GA3 production was investigated. Maximum GA3 production was seen when glucose was used as carbon source (Table 2). Similarly, glucose was best carbon source for GA3 production by Fusarium moniliforme (Rangaswamy, 2012; Kobomoje et al., 2013). However, a mixture of glucose and rice flour was necessary to get GA3 production by Fusarium fujikuroi SG2 (Uthandi et al., 2010). When the concentration of glucose was increased, a decrease in enzyme production is observed due to catabolite repression (Tudzynski, 1999).
After analysing the effect of nitrogen sources on GA3 production, a significant yield was observed with ammonium chloride (Table 2). Similarly, an important yield was seen when ammonium chloride was used as nitrogen source for GA3 production by Fusarium fujikuroi SG2 (Uthandi et al., 2010). A low amount was seen when glycine was used as nitrogen source (Table 2). This can be attributed to the fact that glycine is a slowly consumed organic nitrogen source (Rodrigues et al., 2011). After exhaustion of nitrogen source, GA3 secretion starts and an important amount of carbon source is consumed (Tudzynski, 1999; Rodrigues et al., 2011).
The submerged fermentation for GA3 production by the isolate M104 was carried out under shaking conditions (100 rpm) to allow proper mixing of nutrients, favouring oxygen circulation and GA3 production. A 3-fold increase was recorded for GA3 production by Aspergillus niger when the culture flasks were agitated (Bilkay et al., 2010). Rodrigues et al. (2011) reported that GA3 production has to be carried with aeration since GA3 biosynthesis requires various oxidative steps catalysed by different oxygenases. After optimization, a 6.56-enhancement in GA3 secretion was observed
Thin layer chromatography (TLC)
After GA3 extraction, crystals of GA3 were obtained as shown on the figure 3. After carrying TLC, the value of resolution factor (Rf) of GA3 was calculated as follow: Rf = distance from origin to solvent peak / distance from origin to sample spot detected = 7.9 cm / 10.8 cm = 0.7315 (Figure 4). The value was closing approximate to the GA3 standard value. Similarly, an approximate Rf value was recorded for the GA3 extracted from Fusarium monilifome (Rangaswamy, 2012). The TLC was also used to confirm the GA3 produced by Fusarium solani (Bhalla et al., 2010).
Effect of GA3 on pea plants
It is was observed that the pea plants sprayed with GA3 was 7 cm taller than the pea plants without the GA3 within a period of two weeks (Fig. 5). Similarly, size of the lily plants was increased following exogenous GA3 treatment and this was attributable to the protein synthesis stimulation (Mahmoody and Noori, 2014). Likewise, the hybridized rice plant height was increased after GA3 extract application (Srivastava et al., 2003).
Effect of GA3 on paddy crops
All the 10 paddy seeds treated with GA3 were able to germinate and have uniform growth, colour and height and average height was 9.5 cm within a total period of 25 days. The untreated seeds were able to germinate and had unequal growth and average height was 8.5 cm (Fig. 7). Similarly, the shoot and root heights, and the yield of chana and wheat crops were increased after GA3 extract application (Pandya and Desai, 2014). After GA3 application, an important productivity was seen for hybrid rice plant, following a better plant growth and physiological properties (Susilawati et al., 2014). The GA3 application also led to a significant yield for faba bean, compared to Ca2+ ion, and this was attributed to the improvement of growth and photosynthetic activity by the plant hormone (Al-Whaibi et al., 2010).
Figure 7: Effect of GA3 on paddy crops: Uniform growth (left) and non-uniform growth (right). Paddy seeds were soaked in 300 ppm of GA3 solution for about 2 days and sown in soil. They were sprayed with 200 ppm of GA3 after growth. The control seeds were soaked in water for the same period and sprayed with only water. The observation was carried out for 25 days
Four strains of Fusarium were screened from Belgaum agriculture land by using a selective medium malachite green agar. They were confirmed as belonging to Fusarium species by lactophenol cotton blue spore staining method. The GA3 production depends on nutritional and physicochemical conditions. Strain M104 showed the highest GA3 production in CD broth. After optimization, a 5.56-increase in GA3 production was achieved. The pea plant sprayed with GA3 fungal extract was taller than unsprayed one. The effect of GA3 on paddy seeds showed uniform and more growth than control (without GA3). The isolate M104 can thus be used as a potent fungal species for GA3 production.
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