Introduction

Cyanobacteria belong to the prokaryotic photosynthetic group of organisms, with many species possessing nitrogen fixation ability. Cyanobacteria growing under extreme environments offer the opportunity of growing and harvesting them in open outdoor ecosystems. Out of these, the halotolerant cyanobacteria being primary producers, proved to be useful for amelioration of salt affected soils (Kaushik 2007). Cyanobacteria have also been searched for other biotechnological potentials including pharmaceutically and agriculturally important bioactive compounds (Skulberg 2000; Soltani et al. 2005). In general, isolation of bioactive compounds from cyanobacteria is done with two objectives: (i) to discover new compounds and (ii) to understand the interactions of individual organisms within their natural communities (Schlegel et al. 1999).

Several species of cyanobacteria produce substances with antibiotic activity. They include Tychonema bourrellyi, Aphanizomenon flos-aquae, Cylindrospermopsis (Østensvik et al. 1998), Anabaena, Calothrix, Leptolyngbya, Lyngbya, Microcystis, Nostoc, Phormidium, Pseudoanabaena, Synechococcus, Synechocystis, Tolypothrix (Ördög et al. 2004). In spite of the studies carried out so far, many cyanobacterial compounds are still largely unexplored, thus giving a rich opportunity for discovery of new bioactive compounds. The expected rate of rediscovery is far lower than that for other better-studied group of organisms (Olaizola 2003). Cyanobacteria produce a wide variety of toxins and other bioactive compounds including 40% lipopeptides, 5.6% amino acids, 4.2% fatty acids, 4.2% macrolides, and 9% amides (Singh et al. 2005).

Numerous screening programs have revealed the potential of cyanobacteria in the production of novel antimicrobial compounds (Schlegel et al. 1999; Mian et al. 2003; Ghazala and Shameel 2005; Soltani et al. 2005). Antifungal effects from aqueous and organic solvent extracts of cyanobacteria have been reported in bioassays comprising selected fungal test organisms (Mian et al. 2003; Ördög et al. 2004; Ghazala and Shameel 2005; Soltani et al. 2005). Numerous bioassays have been reported for screening of antimicrobial compounds, which include minimum inhibitory concentration (MIC) broth assay (Østensvik et al. 1998; Ördög et al. 2004), agar disc diffusion method (Soltani et al. 2005), agar well method (Ördög et al. 2004), double layer agar method (Schlegel et al. 1999), and silica gel plates (Mian et al. 2003; Quiroga et al. 2004; Rahmani et al. 2004).

The aim of the current venture was to investigate the antifungal activities of halotolerant cyanobacterial strains isolated from terrestrial and freshwater habitats of Maharashtra state, India. This paper reports the comparative evaluation of four methods of bioassay for visualizing antifungal activity.

Materials and methods

Cultivation of halotolerant cyanobacteria

Halotolerant cyanobacterial cultures were isolated from diverse habitats such as saline soils (sandy loam and black cotton soils), fresh waters (water springs and stagnant water bodies), paddy fields, and an alkaline lake (Lonar lake) of North Maharashtra region of India. Cyanobacterial cultures were isolated by enriching water or soil samples in BG11/Gerloff medium (Kaushik 1987) amended with 200 mmol l−1 sodium chloride. Axenic cultures of filamentous cyanobacteria were obtained by repeated liquid transfer of small amounts of material followed by antibiotic treatment (streptomycin, chloramphenicol, and penicillin (10 mg ml−1) (Kaushik 1987). Unicellular cultures were purified by successive transfer from liquid to solid media. Isolates were grown axenically in 100 ml of BG11 or Gerloff’s medium containing 200 mmol l−1 sodium chloride at pH 7.8 and 24°C temperature in 500 ml Erlenmeyer flasks under continuous illumination at 2000 lux.

Preparation of culture extracts

Cyanobacterial biomass was harvested after 10 and 25 days of incubation for unicellular and filamentous cultures, respectively. Biomass was separated by centrifugation of 100 ml culture broth at 5000g for 15 min (Sorvall, USA, Model RC2). Water extracts were made by resuspending cyanobacterial biomass in distilled water (10 mg ml−1) and ultrasonicating at 20 KHz frequency (Sonics and Materials Inc., USA) for 2 min. The biomass was also extracted simultaneously with organic solvents viz. methanol, n-propanol, and petroleum ether. The cell mass was separated by centrifugation at 5000g for 20 min and the extraction procedure was repeated thrice. The pooled supernatants were dried at 40°C under reduced pressure in a rotary evaporator (Büchi Rotavapor R.214, Switzerland). The dried extracts were resuspended in 3 ml of each solvent and preserved at 4°C till further use in antifungal assays.

Isolation of test cultures

Five phytopathogenic fungal cultures viz. Aspergillus flavus, Aspergillus niger, Colletotrichum musae, Fusarium oxysporum, and Paecilomyces lilacinus isolated from diseased plants and seeds were used in the study. Identification of these cultures was carried out from Agharkar Research Institute, Pune, India. Test cultures were grown on 50 ml Sabouraud’s dextrose agar of composition (in g l−1) peptone, 10, dextrose, 20, and agar, 15 at pH 5.6 for sporulation in 500 ml Erlenmeyer flasks. Spore suspension was prepared by pouring 5 ml sterile 0.01% triton X-100 solution, vortexing for 1 min and sieving through cheesecloth. Final inoculum density of 106 spores ml−1 was calibrated using a hemocytometer.

Screening of isolates for antifungal activity

Double layer agar method

Four to six cyanobacterial cultures were spot inoculated on Petri plates containing 15 ml growth medium (either BG11 or Gerloff’s medium) solidified with 1.5% bacteriological agar and incubated for 3–5 days at 24 ± 3°C under illumination of light (2000 lux). The spot grown cultures were overlaid with a 10 ml of double strength Sabouraud’s dextrose medium containing 1% agar and 0.1 ml spore suspension containing 106 spores ml−1 of fungal test cultures. The plates were incubated under dark for 3 days at 30°C. Antifungal activity was assessed by measuring the diameter of clearance zone around the cyanobacterial colony (Schlegel et al. 1999).

Disc diffusion assay

Whatman No.1 paper discs (6 mm) were saturated with 50 μl of cyanobacterial extracts, dried, and placed on Sabouraud’s dextrose agar plates, prior inoculated with 0.1 ml fungal spore suspension (1 × 106 spores ml−1). Antifungal activity was assessed by measuring the diameter of growth inhibition zone after incubation at 30°C for 48 h.

Silica gel method

Antifungal activities of selected cyanobacterial extracts were checked using silica gel plate. Slurry of silica gel (S.D. Fine Chemicals, Mumbai, India) prepared in water was spread as a thin layer in the base of Petri dish (9″ × 9″). The plates were activated at 90°C for 1 h. Cyanobacterial extracts were spot inoculated on the silica gel plate. Fungal spore suspensions (1 × 106 spores ml−1) prepared in sterile Sabouraud’s broth were spread over the silica gel using glass spreader and plates were incubated under moist conditions at 30°C for 3 days. Antifungal activity was measured as diameter of inhibition zones on the silica gel plate.

Minimum inhibitory concentration

The MIC was determined by inoculating 9 ml of Sabouraud’s broth with 1 ml of various dilutions of cyanobacterial extracts and 0.05 ml of spore suspension (1 × 106 spores ml−1) of fungal cultures. Set of tubes was incubated at 30°C for 3 days. Fungal growth in tubes was assessed visually. The MIC was determined as the lowest concentration of cyanobacterial extract resulting in complete inhibition of fungal growth in tubes after 3 days of incubation.

Filter sterilized fluconazole (Zuventus Healthcare Ltd, Mumbai, India), considered to be the standard antifungal antibiotic was used as the positive control at varying concentrations (50 to 500 μg ml−1) in disc diffusion, silica gel, and MIC assays.

Results and discussion

Numerous studies have shown that cyanobacteria produce substances with antifungal activities, thereby indicating a high commercial potential of these primitive prokaryotes (Mian et al. 2003). Numerous studies report the screening of cyanobacteria for variety of antimicrobial activities (Skulberg 2000; Soltani et al. 2005; Volk 2005). The present study is an attempt to screen halotolerant cyanobacterial cultures isolated from terrestrial and freshwater habitats.

The cyanobacterial isolates were identified on the basis of morphological traits according to Desikachary (1959) and Castenholz and Waterbury (1989). Distribution of isolates in genera of cyanophyta is given in Table 1. Forty strains belonging to nine genera were isolated by culturing axenically either in BG.11 or Gerloff’s medium containing 200 mmol l−1 sodium chloride. Eighteen isolates belonging to genus Oscillatoria dominated the population of halotolerant cyanobacterial cultures. Genera Aphanocapsa, Trichodesmium, and Synechocystis were among the minority.

Table 1 Halotolerant cyanobacterial isolates investigated in preliminary screening for antifungal activity
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Screening by double layer agar method

Primary screening of isolates for antifungal activities was performed using double layer agar method (Fig. 1a). Table 2 shows the antifungal activities of cyanobacterial isolates against five fungal pathogens viz. A. niger, A. flavus, C. musae, P. lilacinus, and F. oxysporum. Among the cyanobacterial cultures tested, five isolates could inhibit growth of A. flavus while 12 isolates could inhibit growth of C.  musae. Twenty cyanobacterial cultures appeared to possess inhibitory activity against F. oxysporum. From a set of 40 cultures tested, 20 isolates showed no activity against all of the fungal test cultures. Hyper-antifungal activity (>21 mm zone of diameter) was seen in case of three isolates. Based on the performance of cultures in agar double layer diffusion method, 10 cultures exhibiting zone of inhibition >15 mm were selected for further screening.

Fig. 1
figure 1

Demonstration of the antifungal effect of Oscillatoria limosa NMU-31 against Aspergillus flavus in three different bioassay methods, (a) Double layer agar method; (b) Disc diffusion assay; and (c) Silica gel method

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Table 2 Distribution of cyanobacterial cultures on the basis of antifungal activity against test organisms in double layer agar method
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Screening by disc diffusion assay

Antifungal activities of selected ten cyanobacterial isolates extracted with methanol, n-propanol, and petroleum ether along with aqueous extracts were tested using disc diffusion method (Table 3). In all, 86 inhibitions were noted for 200 combinations of 10 cyanobacterial cultures, four extraction solvents, and five fungal test cultures. Among the solvents used, methanol extracts showed 34.9% inhibition followed by n-propanol, petroleum ether, and water extracts exhibiting 30.2, 18.6, and 16.2% inhibition, respectively. Out of five fungal test cultures, 12.8% inhibition was noted for A. flavus and a maximum of 24.4% inhibition was observed for C. musae. Highest number of inhibitions i.e. 14, was recorded for Synechocystis sp. NMU-17, whereas, lowest number of inhibitions i.e. 4 could be seen for Nostoc commune NMU-73 and Anaebaena orientalis NMU-79. A maximum zone of inhibition of 28 mm diameter was obtained for methanol extract of Oscillatoria limosa NMU-31 (Fig. 1b). Marginal antifungal activities (<10 mm diameter of inhibition zone) were obtained in case of 26 test combinations (Table 3).

Table 3 Activity of cyanobacterial extracts against fungal test cultures by disc diffusion method
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Antifungal activities of cyanobacterial extracts were found to differ with the type of solvent used for extraction. Methanol, n-propanol, petroleum ether, ethanol, chloroform, and water were employed for the extraction antimicrobial compounds from cyanobacteria (Soltani et al. 2005). Maximum antifungal activities in case of methanol extraction as observed in the present study are in accordance with earlier reports (Østensvik et al. 1998; Soltani et al. 2005). Decreased activities obtained for aqueous and petroleum ether extracts may be due to the different polarities of antifungal substances.

Screening by silica gel method

Performance of disc diffusion method and silica gel method were compared using varying concentrations of methanol extracts of O. limosa NMU-31 and Phormidium tenue NMU-55 against test cultures, A. niger (Fig. 2) and F. oxysporum (Fig. 3). In both these methods, the diameter of inhibition zones was found to be increased with increasing concentrations of cell extracts. The regression coefficient of >0.89 were observed for both disc diffusion and silica gel methods. The slopes of regression lines were 0.24 and 0.34 for disc diffusion method for extracts of O. limosa NMU-31 and P. tenue NMU-55, respectively, while with silica gel method they were 0.56 and 0.89. Increase in inhibition zone was more proportionate with concentration of cell extract in silica gel method in comparison with disc diffusion method (Fig. 1b and c).

Fig. 2
figure 2

Dose–response relationship of methanol extract (A), Oscillatoria limosa NMU-31 (■,□) and (B), Phormidium tenue NMU-55 (●,◯) against test culture, Aspergillus niger in the antifungal bioassay performed in disc diffusion method (■,●) and silica gel method (□,◯)

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Fig. 3
figure 3

Dose–response relationship of methanol extract (A), Oscillatoria limosa NMU-31 (■,□) and (B), Phormidium tenue NMU-55 (●,◯) against test culture, Fusarium oxysporum in the antifungal bioassay performed in disc diffusion method (■,●) and silica gel method (□,◯)

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The comparative assessment of disc diffusion and silica gel method of antifungal assays showed that silica gel method was a more appropriate method of the two. Elimination of solvent effect in bioassays was the main advantage with silica gel method. The method demands less quantity of bioactive compounds and results in proportionate increase in zone of inhibition. Sharp and clear zone of inhibitions could not be seen with agar diffusion methods due to increased diffusion of bioactive compounds with incubation time, whereas, in silica gel method, the extent of diffusion and dilution could be reduced and sharper zones were obtained.

Minimum inhibitory concentration

On the basis of antifungal activities obtained in disc diffusion assay, five cyanobacterial cultures were selected for determination of the MICs. Methanol extracts, recording maximum inhibitions (34%) in disc diffusion assay were used in further bioassays. Antifungal activity higher than that obtained for antibiotic fluconazole was observed in case of P. tenue NMU-55 against A. niger, Trichodesmium hildebrantii NMU-62 against C. musae, Oscillatoria ornata NMU-64 against P. lilacinus and O. limosa NMU-31 and P. tenue NMU-55 against F. oxysporum (Table 4).

Table 4 Minimum inhibitory concentrations of the cyanobacterial extracts prepared in methanol
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Skulberg (2000) has emphasized on exercising imagination and creativity in investigating organisms from untried biotopes and develop new methodologies to exploit them. Different methods reported so far for the screening of microbial cell extracts, cell free broth and plant extracts for antifungal activity mostly rely on disc diffusion, agar well diffusion and broth assays (Østensvik et al. 1998; Mian et al. 2003; Ördög et al. 2004). The double agar layer method reported here has more advantages in screening cyanobacterial cultures. In the present study 10 out of 40 cultures tested could be selected with a positive result. The success of the method lies in the fact that preliminary screening could test a large number of cultures in one step without extraction. Schlegel et al. (1999) reported screening of cyanobacterial isolates for antialgal-cyanobacterial activity using double layer agar technique. In the present study, the proportion of isolates with antifungal activity was between 12–50%. None of the cyanobacterial isolates exhibited inhibition of all test fungal cultures. The variation in antifungal activities could be due to different permeabilities of bioactive substances into the test organisms. The production of bioactive compounds and expression of antimicrobial activity depends on physiological factors such as stage of growth and culture conditions (Schlegel et al. 1999). Two fold increase in antibiotic production under optimum light conditions have been reported by Chetsumon et al. (1994). Different environmental conditions may result in expression of entirely different suites of biological activities (Robles Centeno and Ballantine 1999). Fungal cultures used in present study are among the phytopathogens causing major damage to agricultural products in North Maharashtra region of India.

Conclusions

The results presented in this paper show that halotolerant cyanobacterial cultures possess promising antifungal activities. The success of screening program was also found to be dependent on appropriate selection of bioassay method. The double agar layer method was a suitable method of preliminary screening for handling large number of cultures without extraction of compounds. However, further screening experiments proved that silica gel method was advantageous over MIC and agar disc diffusion methods.