Abstract: The present study was undertaken to explore the effect of cyanobacterial filtrates against three sugarbeet pathogens Fusarium verticillioides, Rhizoctonia solani and Sclerotium rolfsii. Based on this study, it was concluded that Phormidium fragile and Nostoc muscorum filtrates have potential for the suppression of phytopathogenic fungi. In vitro and in vivo growth, sporulation and sclerotial production were significantly inhibited with the almost species of cyanobacteria.
INTRODUCTION
Microorganisms are proving to be rich sources of variety of bioactive natural products of scientific and commercial interest. Extensive screening programs of cyanobacteria have been conducted worldwide and led to the discovery of nevel compounds with antineoplastic, antimicrobial and antiviral properties (Caire et al., 1987; Patterson et al., 1991, 1993, 1994; Kulik, 1995; Falch et al., 1995; Moore, 1996; Jaki et al., 2001).
Fungi and bacteria are the chief biological agents that have been studied for the control of plant pathogens, particularly soil borne fungi. In addition viruses, amoebae, nematodes and arthropods have been mentioned as possible biocontrol agents (Whipps and McQuilken, 1993; Hanlon et al., 1994; Kulkarni et al., 1994).
A number of cyanobacteria and eukaryotic algae particularly macroalgae, produce various biologically active compounds, these include antibiotics which in laboratory tests inhibited bacteria and fungi that incite diseases of humans (Cano et al., 1990). Microalgae the best known for the production of toxins by certain species that live in fresh and saltwater. These toxins are associated with the mass growth (algal biomass) of these microorganisms and affect fish, birds and animals (Lawton and Cood, 1991). The marine algae Acrosiphonia coalita extract inhibited the growth of Bacillus subtilis, Candida albicans and Streptococcus aureus (Barnart et al., 1993).
Kulik (1995) reported that Chaetomium globosum, Cunninghamella blakesleeana, Aspergillus oryzae and the plant pathogen Sclerotinia sclerotiorum were inhibited in vitro by substances produced by various cyanobacteria.
The aim of this study was to investigate the influence of cyanobacterial filtrates as biocontrol agents against sugarbeet plant pathogens.
MATERIALS AND METHODS
Microorganisms and culture conditions: Fungi used in this investigation were isolated from saline solis or the rhizosphere of sugarbeet cultivars in Egypt; Sclerotium ralfsii, Rhizoctonia solani and Fusarium verticillioides (El-Abyad et al., 1988). These were maintained in culture on Czapek-Dox agar.
Cyanobacteria were grown on Bold basal medium for 21 days at room temperature. The cultures were homogenized and filtered through wathman No. 1 paper and the filtrate was filter-sterilized through 0.45 μm filter. Two concentrations of the filtrate (5 and 10% v/v) were prepared.
Agar diffusion method: This method was used to test the sensitivity of fungi against the cyanobacterial toxins (Odds, 1989; Hadacek and Greger, 2000). One milliliter of spore suspension of F. verticillioides or 10 sclerotia of each of S. rolfsii or R. solani poured into petridishes with Czapek-Dox agar medium and separate discs impregnated with algal filtrate were placed on the surface of the agar and incubated for 8 days at 27°C. The inhibition zone was recorded.
Growth activities of the pathogenic fungi were examined in the absence and presence of cyanobacterial filtrate concentrations (5 and 10%). Mycelial growth of fungi was determined by the soil tube method (El-Abyad and Saleh, 1971). The tubes were incubated at 27°C for 8 days. Growth was measured daily and compared with control. Three tubes were prepared for each treatment.
Dry weight yields were determined by mixing the filtrate of cyanobacteria with Czapek-Dox medium in 50 mL Erlenmeyer flask to produce the required concentration in a total vol 20 mL per flask. Control flasks without algal filtrate were also prepared. Each flask was inoculated with a 4 mm agar disk bearing mycelium of the fungus cut from young colonies and incubated at 27°C for 10 days. Three flasks were prepared for each treatment and filtered under suction, the mycelium produced was dried to constant weight at 80°C and the dry weight estimated.
Sporulation counts: Czapek-Dox agar was mixed aseptically with cyanobacterial filtrate to produce the required concentration and poured into petridishes. The petridishes were inoculated with a 4-mm disk of myceliym of F. verticillioides, incubated for 10 days at 27°C and the sporulation was counted by a haemocytometer.
Production of sclerotia by S. rolfsii was studied on Czapek-Dox agar medium. This was supplemented with two various amounts of algal filtrate as described earlier, inoculated with agar disk bearing mycelium of the fungus and incubated for 10 days at 27°C. The number of sclerotia was visually counted. Method of Manning et al. (1971) was used for production of sclorotia of R. solani.
Statistical analysis: The obtained data were carried out according to Snedecor and Cochran (1980), using LSD to compare the significance of the results.
RESULTS AND DISCUSSION
All selected cyanobacterial genera Anabaena, Nostoc and Phormidium were tested for their capability to exert inhibition effect.
The inhibition zone test revealed as shown in Table 1. The culture filtrates of cyanobacterial species have a remarkable inhibition affect on the tested pathogenic fungi of sugarbeet.
Mycelial dry weight yields were significantly suppressed by all selected cyanobacterial sp. (Table 2). In case of Fusarium verticillioides, the lowest growth appeared only with Phormidium fragile and Nostoc muscorum filtrates. It was noticed that, with increasing the concentration of filtrate, the fungal growth decreased. The maximum inhibition was 52% at Phormidium filtrate. The same trend was observed in case of S. rolfsii. Also, the greatest inhibition in the biomas yields of R. solani was 38 and 31% with Nostoc muscorum and Phormidium fragile, respectively.
| Table 1: | Inhibition zone of fungi (mm) F. verticilloides, S. rolfsii and R. solani under the effect of cyanobacterial filtrates |
| Table 2: | Dry weight yields of fungi (mg) in Czapek-Dox under the effect of two different concentrations of cyanobacterial filtrates (5 and 10%), incubation period 10 days at 27°C |
| Table 3: | Growth of Fusarium verticillioides, Selerotium rolfsii and Rhizoctonia solani in soil (mm) under the effect of two different concentrations of cyanobacterial filtrates (%), incubation period 8 days at 27°C |
| Table 4: | Numbers of spores produced (x104) of Fusarium verticillioides, Numbers of sclerotia/plate produced by Sclerotium rolfsii and Rhizoctonia solani under the effect of two different concentrations of cyanobacterial filtrates (%) on Czapek-Dox agar, incubation period 10 days at 27°C |
These results are in agreement with the results reported by Cano et al. (1990), Smitka et al. (1992), Caire et al. (1993), Fish and Cood (1994) and Borowitzka (1995), who reported that the extracts of Nostoc muscorum and Phormidium sp. significantly inhibited the growth of Candida albicans, Sclerotinia sclerotiorum and Staphylococcus aureus.
In connection, Adam (1999) studied the effect of nostoc muscorum on the growth of some crop plants. Growth parameters of wheats, sorghum, maize and lentil were significantly increased with addition of algal filtrate.
In vivo studies (Table 3) showed that F. verticillioides and S. rolfsii were very sensitive to almost cyanobacterial species. The maximum inhibition of Fusarium growth in soil was 81% with Anabaena flos-aquae and 60% incase of Sclerotium with Phormidium filtrate.
Most species of cyanobacteria studied, have no significant affect on growth of R. solani in soil. Kulik (1995) reported that the growth of R. solani on PDA was significantly inhibited by using Nostoc muscorum extract.
A new bioactive compound known as tanikolide was isolated from the marine cyanobacterium Lyngbya majuscula. This compound exhibited activity against Artemia salina, Biomphalaria glabrata and Candida albicans (Singh et al., 1999). The growth activities of Fusarium oxysporum betae, F. oxysporum lycopersici and F. oxysporum vasinfectum were inhibited with increasing the concentration of cyanobacterial extracts (Moussa and Shanab, 2001).
Number of spores produced by F. verticillioides was highly significantly decreased with all different filtrates of cyanobacteria. The lowest number of the fungal spores was (155 x104) compared to control by using Phormidium filtrate (Table 4).
Production of sclerotia by S. relfsii and R. solani was highly significantly suppressed with almost cyanobacteria (Table 4). The lowest number of sclerotia was appeared with Phormidium fragile. Nostoc muscorum has no affect on the production of sclerotia in both fungi. These results are in agreement with that obtained by Benjamin et al. (1999), Begum et al. (1999) and Moussa and Shanab (2001).
Finally, it may be worth noting that the sensitivity to cyanobacteria metabolites not only depend on the fungal genus but also its species and/or mode of growth.