Impact of media composition and growth condition of antifungal production by Streptomyces ambofaciens S2

Anthracnose caused by fungus Colletotrichum gloeosporioides is an important disease of chilli in Asia countries such as Malaysia. This fungus makes the chilli fruits to be unmarketable. In this study, Streptomyces ambofaciens S2 which gave a high antifungal activity was selected and underwent cultural condition optimization. The optimization involved selecting the best carbon and nitrogen sources, seed age, incubation time, pH, percent NaCl (w/v), and fermentation temperature. It was observed that S. ambofaciens S2 gave the highest activity when grown in the present of chitin and peptone as carbon and nitrogen sources, respectively. S. ambofaciens S2 also gave optimal cultural growth and antifungal production at pH 8, incubation temperature of 28°C, 2% of NaCl, 6 days of seed age and 3 days of cultural incubation. By studying the impact of each of the cultural conditions, antifungal activity of S. ambofaciens S2 has been increased to about 30% in comparison with the use of original conditions.


INTRODUCTION
Anthracnose fruit rot of chillies is often caused by fungus from the genus of Colletotrichum sp. This disease often posed a major constraint for farmers to obtain good profit (Rajapaske, 1999). It is widely spread and often cause severe disease to chilli planted in lowland of Peninsular Malaysia (Mah, 1989). According to Poonpolgul and Kumphai (2007), Thailand suffer severe loses in their chilli production due to anthracnose infection. The use of chemical fungisides not only will pollute the environment but also may cause the microbes to develop resistance towards it (Lodish et al., 1995).
Actinomycetes had been known to be the producers of various antimicrobial activities. Actinomycetes, mainly from the genus of Streptomycetes are well known as biological control agents that inhibit or lyse plant pathogenic fungi (Nakai and Kanehisa, 1991;Nakashima and Nishikawa, 1994). Marten et al. (2000) reported that Rhizovit R from Streptomyces rimosus is used for the control of several plant fungal pathogens.
In a study done by Yu et al. (2008), the authors stated that antifungal activity of S. rimosus MY02 increased when the culture conditions were adjusted to the optimal

Isolation of soil streptomyces and screening for antifungal activity
Soil samples were collected from random areas at the agriculture land in Pontian, Johor. Soil samples were taken at the depth of about 15 cm from the top soil and kept in a double zip lock bag. The soil samples were transported back to the laboratory in a cooler box containing ice bags to maintain the natural condition of the soil. Soil samples were air dried for about 1-2 weeks.
Ten grams of the pre-treated soil samples were then added to 100 ml of sterile distilled water and was vigorously shaken with a orbital shaker at 200 rpm for about an hour. Hundred and fifty microliter of the supernatant was later pipetted and spread onto Strach Casien Agar (soluble starch; 10.0 g, casein hydrolysate; 0.3 g, KNO3; 2.0 g NaCl; 2.0 g; K2HPO4; 2.0 g; MgSO4.7H2O; 0.05 g, CaCO3; 0.02 g, FeSO4.7H2O; 0.01 g, Agar; 18.0 g, 1.0 ml of Cycloheximide (50mg/ml); at pH 7). The plates were later incubated at room temperature (28 ± 2°C) for 14 days. Emerging colonies of Streptomyces were later subcultured onto fresh SCA plates and further incubate for 14 days prior to used.
Streptomyces isolated were then tested for their antifungal activity against Colletotrichum gleosporidease in a dual culture test. In this test, the Colletotrichum was placed in the middle of the petri dish surrounded by the two plugs of same Streptomyces culture, a positive control disc (50 mg/ml) and a negative control disc. The Petri dish was then incubated at 28 ± 2°C for 5 days. Tests were conducted in triplicate manner.

Effect of incubation period on antifungal activity
To test the incubation period, one plug of the S. ambofaciens S2 was inoculated into a 250 ml Erlenmeyer flask contaning 100 ml Starch Casein Broth (SCB). S. ambofaciens S2 were grown in triplicate manner for 7 days at 28 ± 2°C to observe the growth pattern. The culture filtrates of each day were tested for their antifungal activity. All results were presented as mean inhibition zone ± standard deviation (SD).
Biomass of S. ambofaciens S2 for each day was collected and air dried before weighted. The weight observed for each day was then plot on graph. All results were presented as mean weight ± standard deviation (SD).

Effect of pH and temperature on antifungal activity
S. ambofaciens S2 was grown in a 250 ml Erlenmeyer flask with 100 ml SCB at pH 5.0, 6.0, 7.0, 8.0 and 9.0. S. ambofaciens S2 was also test for its ability to produce antifungal activity under controlled incubation temperature of 15, 30, 35 and 45°C. Tests were conducted in triplicate for 3 days at 28 ± 2°C and tested using disc diffusion method. All results were presented in mean inhibition zone ± standard deviation (SD) manner.

Effect of carbon and nitrogen sources on antifungal activity
To determine the effect of carbon sources on antimicrobial metabolites production, different carbon sources; D-glucose, molasses, mannitol, chitin and maltose were added to the SCB medium by replacing soluble starch in the SCB with these carbon sources (soluble starch; 10.0 g, casein hydrolysate; 0.3 g, KNO3; 2.0 g NaCl; 2.0 g; K2HPO4; 2.0 g; MgSO4.7H2O; 0.05 g, CaCO3; 0.02 g, FeSO4.7H2O; 0.01 g).
The effect of nitrogen sources such as soy bean, yeast extract, peptone, L-asparagine and beef extract was used to replace casein hydrolysate in the SCB medium. No change of nitrogen source concentration in the media was done. Tests were conducted under pH 7, for 3 days at 28 ± 2°C for both carbon and nitrogen sources. Antifungal activity was checked by disc diffusion method against C. gleosporioides after 5 days of incubation. All results were presented in mean inhibition zone ± standard deviation (SD) manner.

Effect of salt (NaCl) on antifungal activity
Salt concentration had been known to cause certain effect on the production of antibiotic from microorganism (Pelczar et al., 1993). The testing of salt concentration effect was done by altering the concentration of NaCl in the SCB. The tested concentrations were 0.1, 0.2, 0.3, 0.4, 0.5 and 1.0% (w/v). Tests were conducted under pH 7, for 3 days at 28 ± 2°C. Disc diffusion method against C. gleosporioides was done. Tests were conducted in triplicate and all results were presented as mean inhibition zone ± standard deviation (SD).

Effect of the culture seed age on antifungal activity
Seed age of S. ambofaciens S2 was evaluated for their effect on the antifungal production by using different seed age of S. ambofaciens S2. S. ambofaciens S2 was grown on SCA and one plug (diameter of 0.5 cm) of the culture was taken every 2, 4, 6, 8 and 10 days using a borer (size 5 mm in diameter). The plug of colony was then inoculated into a 250 ml of Erlenmeyer flask with 100 ml of SCB. Tests were conducted under pH 7, for 3 days at 28 ± 2°C. Each test was conducted in triplicate manner. All results were presented as mean inhibition zone ± standard deviation (SD).

Antifungal activity test
Fifteen microliter of S. ambofaciens S2 extract from original SCB media and the modified optimal media impregnated separately onto sterile paper disc and placed next to Coletotrichum gloesporidesea at a distance of 5 cm apart from each other. Cyloheximide (50 mg/ml) impregnated disc was used as positive control while disc impregnated with sterile distilled water act as negative control. Tests were conducted in triplicate manner and the plates were incubated at 28 ± 2°C for 5 days under sterile condition.

Isolation of actinomycetes
From the total of 110 isolates of Streptomyces spp. Isolated, observation on the aerial mycelium colour showed that 45.5 (50 isolates), 21.8 (24 isolates), 20 (22 isolates) and 12.7% (14 isolates) of the isolates were found to be white, grey, dark grey and red, respectively. A total of 38 isolates were observed to produce diffusible pigmentation on the agar plate. It was also observed that the colony forming unit per gram (cfu/g) of dry soil for actinomycetes was in the range of 4.2 x 10 3 -6.0 x 10 4 .

Antifungal activity screening
From the total of 110 Streptomyces spp. isolated,  preliminary screening showed that 44 isolates of Streptomyces spp. gave antifungal activity towards C. gleosporioides. From these, only one isolate of Streptomyces spp. gave the best antifungal activity with the inhibition zone of 15 mm. This Streptomyces sp. was later known as S. ambofaciens S2.

Impact of cultural condition on the antifungal activity
During the process, we observed that S. ambofaciens S2 produced better inhibition zone in these conditions: 3 days of incubation, pH 8, under 28°C, chitin as carbon source, peptone as nitrogen source, when 2% of NaCl was used and 6 days of seed age. Details of the tests are shown in Figures 1 to 7. S. ambofaciens S2 showed its lag phase on the 3 rd day and followed by the death phase on the 7 th day.

Effect of cultural media optimization on antifungal activity
When S. ambofaciens S2 was cultured in the optimal cultural media, we observed that S. ambofaciens S2 gave an inhibition zone of 20 mm as compared to initial  inhibition zone of 15 mm.

DISCUSSION
It has been widely known that soil rhizosphere present a unique biological niche with a diverse of microorganisms living in it (Merckx et al., 1987). The use of effective microorganisms or its secondary metabolite to control plant pathogens had open up a doorway to a more sustainable and environmentally friendly solution to pests and diseases issues. Gohel et al. (2006) stated that, fungal plant diseases accounted for approximately one  third of the world fungal-plant infection. By optimizing the cultural condition of the microbes, we can increase the antifungal activity for microbes of our choice. From the results we observed that S. ambofaciens S2 reached it stationary phase on the 3 rd day of incubation. This could be the reason why S. ambofaciens S2 gave the highest inhibition zones on the 3 rd day of incubation. Study done by Kavitha et al. (2010), showed that Streptomyces spp. (A2 and A4) gave the highest antifungal activity on the 5 th day and Streptomyces sp. (A1) gave the highest antifungal activity on the 4 th days although these Streptomyces spp. (A1, A2 and A4) reached their stationary phase on the 3 rd day of incubation. Study done by El-Mehalawy et al. (2005) showed that actinomycetes isolated, gave the biggest inhibition zone at pH 7. This however was different from our study whereby, largest inhibition zone were observed at pH 8. El-Abyad et al. (1996) showed that highly acidic or basic media, were not suitable for the antifungal production by many Streptomyces species and that neutral media (pH 7.0) were the most favorable for antifungal production. However, in this study this hypothesis was null as S. ambofaciens S2 produces the highest antifungal activity when grown on pH 8.
In this study, we observed that chitin and peptone were the two best carbon and nitrogen sources respectively. However study done by Yu et al. (2008) showed that Streptomyces rimosus MY02 gave the best antifungal activity when starch and defatted peanut powder were used. El-Mehalawaty et al. (2005), showed best antifungal activity were observed when glycerol was used as the carbon source for all 4 Streptomyces spp. used in the test. While ammonium sulphate was the best for Streptomyces lydicus and Streptomyces ederenses, glutamic acid for Streptomyces erumpens and soybean for Streptomyces antimycoticus. This shows that each of the Streptomyces spp. have their own choices of carbon and nitrogen sources that help them increased their antifungal activity.
S. ambofaciens S2 gave the highest inhibition zone when 0.2% of NaCl was used but the zone size become smaller as the NaCl concentration increased. This showed that S. ambofaciens strain S2 could grow on high percentage of NaCl but the secretion of antifungal compounds would be inhibited.
In the cultural age testing, S. ambofaciens S2 showed that the highest antifungal activity was obtained when 6 days old seed culture was used. This result however was parallel to the results obtained for S. rimosus MY02 which gave the highest antifungal activity when 4-6 days old seed were used (Yu et al., 2008) and S. hygroscopicus ATCC 29253 produced the highest amount of rapamycin when 5 and 7 days of seed culture were used (Hamdy et al., 2011).

Conclusion
S. ambofaciens S2 isolated from Malaysia soil showed its antifungal ability towards anthracnose on chilli. The optimized cultural condition showed an improvement in the activity of the antifungal compound to approximately 30%.