Inhibitory Effect of Algal Extracts on Mycelial Growth of the Tomato-Wilt Pathogen, Fusarium oxysporum f. sp. lycopersici

Abstract

The present study was undertaken to explore the inhibitory effect of cyanobacterial extracts of Nostoc commune FA-103 against the tomato-wilt pathogen, Fusarium oxysporum f. sp. lycopersici. In an optimal medium, cell growth, antifungal activity, and antifungal compound production could be increased 2.7-fold, 4.1-fold, and 13.4-fold, respectively. A crude algal extract had a similar effect as mancozeb at the recommended dose, both in laboratory and pot tests. In vitro and in vivo fungal growth, spore sporulation and fungal infection of wilt pathogen in tomato seeds were significantly inhibited by cyanobacterial extracts. Nostoc commune FA-103 extracts have potential for the suppression of Fusarium oxysporum f. sp. lycopersici.

Fusarium spp. are an abundant saprophyte in soil and organic matter and are found worldwide. Some strains cause a vascular wilt disease in crops, including vegetables, bananas, and date palms. However, some strains can protect plants from wilt. The pathogenic stains of this fungus have numerous specialized forms, known as formae specials (f. sp.), that cause diseases such as vascular wilt, corm rot, root rot, or damping-off (Olivain and Alabouvette, 1999). Fusarium oxysporum f. sp. lycopersici is known to affect tomato plants which is a crop plant of great economic importance (Suárez-Estrella et al., 2007). Tomato production is significantly reduced by Fusarium oxysporum f. sp. lycopersici because it can destroy roots of tomatoes at growth stages. Many strategies to control this fungal pathogen have been investigated in the field (Biondi et al., 2004; Khan et al., 2007). Currently, the most effective method in preventing tomato from Fusarium wilt is to mix the seed with chemical fungicides. The application of chemical fungicide induces other problems, such harm to other living organisms and the reduction of useful soil microorganisms (Khalifa et al., 1995; Lewis et al., 1996). Therefore, public concern is focused on alternative methods of pest control, which can play a role in integrated pest management systems to reduce our dependence on chemical pesticides (Sutton, 1996). As with other vascular plant-diseases and sanitation measures are difficult to apply (Brayford, 1992). A promising strategy for the replacement of chemical pesticides has been the implementation of biological control. The recent development in the commercialization of biological control products has accelerated this approach (Fravel et al., 2003).

Algae are one of the chief biological agents that have been studied for the control of plant pathogenic fungi, particularly soil borne disease (Abdel-Kadar, 1997; Hewedy et al., 2000). For example, cyanobacteria (blue-green algae) and eukaryotic algae produce biologically active compounds that have antifungal activity (Kulik, 1995; Schlegel et al., 1998), and antibiotic and toxic actvity (Bonjouklian et al., 1991; Kiviranta et al., 2006) against plant pathogens.

Anabaena spp. (Moore et al., 1986; Farnkmolle et al., 1992), Scytonema spp. (Chetsumon et al., 1993), and Nostoc spp. (Bloor and England, 1989) were shown to be efficient in the control of damping-off as well as the growth of soil fungus Cunninghamella blakesleana. Especially, culture filtrates or cell extracts from cyanobacteria and algae applied to seeds protect them from damping-off fungi such as Fusarium sp., Pythium sp. and Rhizoctonia solani (Kulik, 1995). In a previous study (Kim, 2006), antifungal activities were found in 29 strains of the 298 microalgal strains tested. Nostoc communm FA-103 was selected as the subject of this study because of its broad spectrum of antifungal activity on plant pathogenic fungi, especially F. oxysporum f. sp. lycopersici.

In this study, we investigated the optimal conditions for producing the antifungal compounds and the application of algal extracts for control of Fusarium wilt in tomato seeds.

Materials and Methods

Microorganisms

The N₂-fixing blue-green alga (cyanobacterium), _{Nostoc commune} Vaucher ex Bornet et Flahault strain FA-103, was isolated from rice paddy fields in Korea. The strain FA-103 was cultured at 25℃ in the N-free BG-11 (basal medium) liquid medium (Kim, 2006; Kim and Lee, 2006) under the illumination of cool-white fluorescent lamps at a photon flux density of 40 µE/m²/s. Algal growth was determined by measuring the cell concentration and chlorophyll a concentration according to the method of Parsons and Strickland (1963). The culture of the strain FA-103 was filtered by GF/C (Whatman, Uppsala, Sweden) and extracted with 90% methanol at 60℃ for 10min. After extraction, the solid suspension was removed by centrifugation. Then, absorbance of extracts was measured at 665, 645, and 635 nm using a spectrophotometer (model HP8453, Hewlett Packard, MI, USA). The concentration of chlorophyll a (Chl-a) was calculated using the following equation.

Chl-a (mg/l) = 11.6 A₆₆₅-1.31 A₆₄₅-0.14 A₅₁₅

The plant pathogenic fungus, Fusarium oxysporium f. sp. lycospersici KACC40032 was kindly supplied by the National Institute of Agricultural Biotechnology in Suwon, Korea. Tomato seeds (Lycopersicon esculentum cv. Pink charm) were purchased by Nongwoobio Co. Ltd., Suwon, Korea.

Optimization of antifungal compound production

To optimize culture conditions for production of antifungal compound, approximately 1.0 × 10⁴ cells/ml of algae in the exponential growth phase was inoculated into 500 ml-Erlenmeyer flasks containing 250 ml of medium. The flasks were incubated at 28℃ with agitation (150 rpm in a rotary shake incubator) under the continuous illumination of coolwhite fluorescent lamps with light intensity of 40 µE/m²/s. In this study, the culture media such as N-free BG-11 (Kim and Lee, 2006), BG-11 (Rippka et al., 1979), BG-11+N, and Allen's medium (Allen, 1952) were applied to algal cell culture for algal cell growth, antifungal activity and production of antifungal compounds and then we focused on the concentrations of NaNO₃, K₂HPO₄ and NaCl including initial pH. BG-11 included NaNO₃ 1.5 g, K₂HPO₄ 0.04 g, MgSO₄·7H₂O 0.075 g, CaCl₂·2H₂O 0.036 g, EDTA-disodium 0.001 g, Na₂CO₃ 0.02 g, citric acid 0.006 g, ferric ammonium citrate 0.006 g, and micronutrient 1 ml per liter (Rippka et al., 1979). The composition of micronutrient was H₃BO₃ 2.86 g, MnCl₂·4H₂O 1.81 g, ZnSO₄·7H₂O 0.222 g, Na₂MoO₄·2H₂O 0.39 g, CuSO₄·5H₂O 0.079 g, Co(NO₃)₂·6H₂O 0.0494 g per liter (Rippka et al., 1979).

The optimal culture condition found in each step was applied to the next step. After selection of the optimal medium for production antifungal compounds in Erlenmeyer flasks, The combined effects obtained from the optimization were consequently applied to antifungal compound production in 10 l bubble column photobioreactors (BCPs) (Lee et al., 2006). A pre-culture of 250 ml was prepared in a 500-ml Erlenmeyer flask. The cells were transferred to 10 l BCPs containing the optimal medium with 0.2 vvm flow-rate-aeration and 5% CO₂ gas and 95% air under a constant continuous light intensity of 40 µE/m²/s. The temperature and pH were kept constant at 25℃ and 7.0 ± 0.5, respectively, during the period of cultivation.

Preparation of algal extracts

The freeze-dried algal cells (40 g) were extracted twice with 80 ml of methanol for 24 h at room temperature. The extract was separated from the cell residue by filtration (Whatman filter paper no. 3). The filtrate was concentrated at 40℃ under reduced pressure in a rotary evaporator (EYELA N-1000S, Tokyo RikakiKai Co. Ltd., Japan). The 285 mg of pale brown extract was collected, suspended in 80% ethanol, and kept at 4℃.

Determination of algal cell growth and antifungal compound production

Cell growth of N. commune FA-103 was estimated using cell dry weight. Cells were harvested by centrifugation, washed twice with distilled water, and dried to constant weight at 85℃ for 12 h. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone formed around the disk. The experiment was performed in triplicate, and the mean of the diameter of the inhibition zones was calculated. Quantitative bioassay of the antifungal compound was confirmed by comparing with a standard curve of commercial antifungal compound concentration, which was modified from Loo's method (Loo et al., 1945).

Microalgal methanol extract (500 µg) was applied to sterile paper disks (8 mm in diameter) for antifungal assay. Paper disks containing only methanol were used as control. Fusarium oxysporium f. sp. lycopersici (1.0 × 10⁶ spores/ml) were added to molten potato dextrose agar at 40℃. The seeded agar was poured into 9-cm Petri dishes. The algal extract treated paper disks were placed in the center of the seeded agar plates. Plates were incubated for 3 days at 28℃, and then inhibition zones of mycelial growth around the paper disks were measured. The commercial fungicide mancozeb, a polymeric complex of zinc and manganese salts of ethylene bisthiocarbamate (EBDC), (Sungbo Chemical Co. Ltd., Seoul, Korea) was used as reference standard of antifungal activity. The inhibition zone produced by the crude extract was converted to the concentration of mancozeb with the standard curve. Antifungal activity and antifungal compound production are presented as mg equivalent to mancozeb/g of algal dry weight and mg equivalent to mancozeb/l of medium, respectively.

Effects of algal extracts on the inhibition of F. oxysporum f. sp. lycopersici infection in tomato seeds

Experiments to determine the effect of algal extract concentrations on the inhibition of infection of tomato seeds were carried out in Petri dishes. The surface of tomato seeds was sterilized with 10% sodium hypochloride (v/v) then coated with algal extract. The coated seed were incubated at 25℃ for 24 h, and then the fungal spores were spread on the seeds. The inoculated seeds were laid on moist seed-cultivating paper in Petri dishes, and were incubated at 30℃ in dark conditions. After 7 days-incubation, seeds or seedling were observed.

The influence of algal extract concentration on F. oxysporum f. sp. lycopersici cell growth in tomato seeds was investigated at several different concentrations, ranging from 100 to 500 µg/seed. The algal extract was coated with methanol base, zeolite mixing, and dissolving in Tween 20 (10%, v/v) to enhance the antifungal activity. The same vehicles without the addition of the algal extract were used as controls. The fungal inoculum concentrations used in these experiments were 10, 100, and 500 spores/seed. Each experiment was performed in 10 replicates.

In vivo anti-Fusarium wilt activity by microalgal extract

The anti-fungal activity of algal extract in tomato plant was evaluated in a growth room. Tomato seeds (Lycopersicon esculentum cv. Pink charm) were prepared as previously described. We compared two different inoculation methods: (i) seeds were directly inoculated by fungal spores with the concentration of 500 spores/seed before they were sown in plastic pots (5 × 15 × 10 cm) containing steam-sterilized soil mixture (peat moss, perlite and vermiculite, 5 : 3 : 2, v/v/v), sand, and loam soil (1 : 1 : 1, v/v/v). It is representative model of direct inoculation (seed inoculation). (ii) In indirect inoculation, soil mixture was inoculated with the fungal spores (1.0 × 10³ spores/g of steam-sterilized soil mixture) before the seed were sown. The tested tomato seed were cultivated as representative model of mixing with soil mixture inoculated pathogen (soil inoculation). Tomato plants were raised in a growth room at 30℃ with 80 µE/m²/s and 60~90% relative humidity. Plants were watered regularly. After 14 days of sowing, infection was determined.

Results and Discussion

Optimization of culture medium to produce antifungal compound

BG-11+N medium (addition of 3.0 g/l NaNO₃) showed the best results among the media tested (data not shown). However, the optimal culture condition for producing antifungal substances from N. communes FA-103 was further investigated by using BG-11+N medium as basal medium. The optimal medium for production of antifungal compounds was prepared by adding NaNO₃ as N source to the concentration of 3.0 g/l, increasing K₂HPO₄ concentration to 0.15 g/l and reducing CaCl₂ concentration to 0.015 g/l with the initial pH 7.0.

The cell growth of N. commune FA-103 in N-free BG-11 and BG11+N media in 10 l bubble column photobioreactors sparged with 5% CO₂ and incubated at the controlled conditions were compared. Algal cell growth, antifungal activity, and antifungal compound production of the strain FA103 in BG11+N media could be increased 2.7-fold, 4.1-fold, and 13.4-fold, respectively (Table 1).

Table 1.

Growth, antifungal activity, and antifungal compound production by Nostoc commune FA-103 in different media.

^aTo determine significant differences among the treatments, one-way ANOVA tests were performed. Correlation coefficients among the experiments were analysed using SAS Systems v. 8.12 (SAS Institute Inc., Cary, NC).

^bAntifungal activity by Nostoc commune FA-103 against Fusarium oxysporum f. sp. lycopersici in vitro

Inhibition of F. oxysporum f. sp. lycospersici in tomato by crude algal extracts

The algal extract concentration of 150 µg/seed is the minimal concentration (Table 2) that has an effect similar to mancozeb. The antifungal activity of the crude algal extract could be maintained at 50℃ for more than 2 h, and the optimal pH ranges were from 5 to 8 (data not shown). These results are similar to those reported by Cano et al. (1990), Caire et al. (1993), Fish and Cood (1994), Stratmann et al. (1994), and Borowitzka (1995), who reported that the extracts of Nostoc muscorum significantly inhibited the growth of Candida albicans and Sclerotinia sclerotiorum.

Table 2.

Effect of crude algal extract concentration on the inhibition of Fusarium wilt by F. oxysporum f. sp. lycospersici in tomato seeds.

^aMeans from 20 replicates, each replicate containing 20 seeds

One-way ANOVA tests were performed to determine significant differences among the treatments. Correlation coefficients among the experiments were analyzed using SAS Systems v. 8.12 (SAS Institute Inc., Cary, NC).

^bFusarium oxysporum f. sp. lycospersici at the concentration of 500 spores/seed

^cAlgal extract formulated using surfactant (0.1% Tween 20; v/v).

^dDose of mancozeb as recommended by Sungbo Chemical Company

^eAny two means are not significantly different at the 5% level of significance by Duncans's multiple rang test (Gomez and Gomez, 1984)

Cyanobacteria produce exopolysaccharides that can function as energy sources for fungi and produce plant growth regulators, which are abscisic acid, ethylene, jasmonic acid, auxin, and cytokinin-like substances, the cytokinin isopentenyl adenine. These substances can influence fungal growth (Ördög and Pulz, 1996; Stirk et al., 1999).

Effect of formulation of algal extract on F. oxysporum f. sp. lycospersici in tomato

The crude algal extract by methanol coated with Tween 20 (0.1%; v/v) inhibited F. oxysporum f. sp. lycospersici in tomato seeds at the same rate as mancozeb. The length of roots, the number of hairy roots and the stem length were significantly increased. These results suggest that the algal extract coated with Tween 20 (0.1%; v/v) showed the best results not only in control of the pathogen but also in stimulating tomato seedling growth.

The effects of pathogen inoculum and algal extract on the inhibition of fungal infection in tomato seed were shown in Table 4. The infection of tomato seed increased with increasing concentration of fungal pathogen inoculum, and the tomato seeds were completely infected at the inoculum concentration of 500 spores/seed. The treatment of algal extract at a concentration lower than the minimum inhibitory concentration (150 µg/seed) showed inhibitory effect on fungal growth in tomato seeds, but no statistical differences were observed when inhibition was compared at higher inoculum concentrations. These results indicate that the application of algal extract concentration lower than the minimum inhibitory concentration (150 µg/seed) could mitigate the infection of F. oxysporum f. sp. lycospersici in tomato seeds to certain level.

Table 4.

Effect of fungal pathogen inoculum and algal extract concentration on the inhibition of Fusarium oxysporum f. sp. lycopersici in tomato seeds.

^aMeans from 20 replicates, each replicate containing 20 seeds

One-way ANOVA tests were applied to determine significant differences among the treatments. Correlation coefficients among the experiments were analyzed using SAS Systems v. 8.12 (SAS Institute Inc., Cary, NC).

^bFusarium oxysporum f. sp. lycopersici

^cAlgal extract formulated by surfactant (0.1% Tween 20, v/v)

Control of seed-borne and soil-borne F. oxysporum f. sp. lycospersici-diseases by microalgal extract

In the direct inoculation of F. oxysporum f. sp. lycospersici with the concentration of 500 spores/seed on tomato to seeds (seed inoculation), the same degree of infection was observed when the seeds were inoculated indirectly (soil inoculation) by a mixture of fungal inoculum (1.0 × 10³ spores/g of soil) and steam-sterilized soil mixture. However, the crude algal extract of 200 µg/seed had the same controlling effect as mancozeb at the recommended dose in both the seed and soil inoculation. This suggests that the crude algal extract of N. commune FA-103 is a potential natural fungicide that could effectively control the infection of F. oxysporum f. sp. lycospersici in tomato at a concentration similar to the commercial fungicide, mancozeb. Zulpa et al. (2006) studied the effect of Nostoc muscorm on the growth of some plant pathogens. Abo-Shady et al. (2007) also reported that cyanobacteria filtrates strongly inhibited the phytopathogenic fungi isolated from leaves, stems, and roots of Faba bean. Moreover, mycelial growth of several phytopathogenic fungi such as Fusarium oxysporum, Penicillium expansum, Phytophthora cinnamomi, Rhizoctonia solani, Sclerotinia sclerotiorum, and Verticillium albo-atrum were inhibited by the methanol extracts of the cyanobacterium Nostoc strain ATCC 53789 (Biondi et al., 2004).

Nostoc muscorum filtrates have potential for the suppression of phytopathogenic fungi such as the sugarbeet pathogens Fusarium verticillioides, Rhizoctonia solani and Sclerotium rolfsii (Rizk, 2006). In vitro and in vivo growth, sporulation, and sclerotial production were significantly inhibited with Nostoc muscorum. In vivo studies showed that F. oxysporum was very sensitive to cyanobacteria species (Rizk, 2006). The maximum inhibition of Fusarium growth in soil was 81% with Anabaena flosaquae. In addition, the growth activities of F. oxysporum f. sp. betae, F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. vasinfectum were strongly inhibited with increasing concentration of cyanobacterial extracts (Moussa and Shanab, 2001). Tomato Fusarium wilts due to F. oxysporum f. sp. lycopersici were reduced significantly by all formulation of algal extracts (Tables 4 and 5). Tomato seeds treated by 200 µg/seed algal extract showed the best control effect comparing with the control inoculated by the fungal pathogen only (Table 5). However, most concentrations of algal extracts have a significant effect on the growth of Rhizoctonia solani in soil (data not shown). This is in agreement with results obtained by Pushparaj et al. (1999), Tahmida Begum et al. (1999), and Rizk (2006). Nonetheless, Kulik (1995) reported that the growth of R. solani on PDA was significantly inhibited by using N. muscorm extract.

Table 5.

Effect of algal extract on tomato wilt pathogen control in tomato seeds cultivated in soil.

^aMeans from 20 replicates, each replicate containing 20 seeds

One-way ANOVA tests were applied to determine significant differences among the treatments. Correlation coefficients among the experiments were analyzed using SAS Systems v. 8.12 (SAS Institute Inc., Cary, NC).

^bFusarium oxysporum f. sp. lycopersici

^cAlgal extract formulated by surfactant (0.1% Tween 20, v/v)

^dMancozeb at the recommended concentration of 200 µg/seed

In conclusion, the sensitivity to cyanobacteria metabolites not only depends on the fungal genus but also on the species and on the mode of growth. Therefore, elucidation of chemical structure of the antifungal compounds produced by N. commune FA-103 is required and the mode of action of algal extracts to inhibit the mycelial growth of F. oxysporum f. sp. lycopersici should be investigated.

Table 3.

Effect of algal extract formulation on the inhibition of F. oxysporum f. sp. lycospersici in tomato seeds 14 days after treatment.

^aMeans from 20 replicates, each replicate containing 20 seeds.

One-way ANOVA tests were applied to determine significant differences among the treatments. Correlation coefficients among the experiments were analyzed using SAS Systems v. 8.12 (SAS Institute Inc., Cary, NC).

^bFusarium oxysporum f. sp. lycospersici at the concentration of 500 spores/seed

^cAlgal extract at the concentration of 150 µg/seed formulated using methanol, zeolite, or surfactant (0.1% Tween 20, v/v) as vehicles.

^dMancozeb at the recommended concentration of 200 µg/seed.