Abstract: The purpose of the present study was to establish the species composition of fungi living on rhizosphere soils of maize and to establish the antagonistic activity of these fungi. In this study, 15 fungal species have been identified. Fusarium verticillioides, Fusarium oxysporum, Fusarium lateritium, Fusarium flocciferum, Penicillium bilaiae, Penicillium solitum, Aspergillus versicolor, Aspergillus ustus, Trichoderma harzianum, Cladosporium herbarum, Trichoderma viride, Gliocladium virens, Penicillum verrucosum, Fusarium sp. and Curvularia lunata are the fungal species. Interactions between the fungi were also investigated. A. versicolor, Aspergillus ustus and Gliocladium viride were able to dominant almost all species tested.
INTRODUCTION
Soil is perhaps the most complex and heterogenous of natural systems, being composed of a solid, liquid and gaseous phase of changing proportions in time and space. Soil is also a medium for plant growth and a habitat for myriads of microorganisms (Sorensen, 1997). Concerning fungi, the mycelial networks of soil are extensive, several hundred meters per gram of soil (Thorn, 1997; Gomes et al., 2003).
Although the total biomass represents only about 0.02-0.1% of the soil mass, the mycelial Networks of soil have an important function in binding the soil particles together, helping to maintain a good soil structure for plant growth (Graystone et al., 1998; Soonthornpoct et al., 2001).
Fungi play an important role in nutrient cycling and plant health and development (Chiarini et al., 1998; El-Mehalawy et al., 2004; Sorensen, 1997). While some fungi are well known to cause a range of plant diseases and in some cases to devastate agricultural crops (Rheeder et al., 1990; Palumbo et al., 2008), others are known to antagonist plant pathogens (Whipps, 2001; Reyes et al., 2002). Maize (Zea mays L.) is one of major cereal in Turkey (Sahin, 2001).
The aim of this observation was to study fungi distribution in maize rhizospheres and to determine interactions and enzyme activities of isolated cultures.
MATERIALS AND METHODS
Soil: Soil were sampled from agricultural site of Eskiþehir, Turkey. Eskiþehir is located in central part of Anatolia. The characteristics of the soil are shown in Table 1.
Isolation: Fungi were isolated from the rhizosphere of 120 maize plants grown in field site of the Eskiþehir region in Turkey. Soil samples were diluted (Bateman, 1993). An aliquot of 1 mL of each suspension was pipeted into each of five replicate petri dishes containing Creatine Sucrose Agar (KCl, 5 g; MgSO4, 5 g; FeSO4.7H2O, 0.1 g; ZnSO4.7H2O, 0.1 g; CuSO4.5H2O, 0.05 g; agar, 15 g; distilled water, 100 mL), 25 % Glycerol Nitrate agar (K2HPO4, 1 g; Chapex concentration, 7.5 g; yeast extract, 3.7 g; glycerol 250 g; adar, 12 g; distilled water 750 mL), Neutral creatine sucrose agar (Chapex concentration 10 mL; sucrose, 10 g; creatine, 5 g; KH2PO4, 1 g; bromocresol purple, 0.06 g; agar, 15 g; distilled water, 1000 mL), Malt Extract Agar (MEA, MERCK), Chapex Yeast Agar (K2HPO4, 1 g; Chapex concentration 10 mL; yeast extract, 5 g; sucrose, 200 g; agar, 15 g; distilled water 1000 mL) and Potato Dextrose Agar (PDA, MERCK).
The dishes were kept in darkness at 20°C for 7 days and then in an illuminated incubator with 16 h length and day/night temperatures 18/13°C for 7 days. Fungi isolated were identified using of Biolog commercial identification systems.
Interaction: In vitro comparisons consisted of removing 5 mm diameter disks from the edge of colonies grown on PDA (Potato Dextrose Agar) in petri dishes. The paired fungi were placed on opposite sides of petri containing PDA. Paired cultures were incubated for 7 days then scored for degree of antagonism on a scale of classes 1-5, 1: antagonist completely overgrew the pathogen and covered the entire medium surface, 2: antagonist overgrew at least two-thirds of the medium surface, 3: antagonist and the pathogen each colonized approximately one-half of the medium surface and either organism appeared to dominate the other, 4: the pathogen colonized at least two-thirds of the medium surface and appeared to withstand encroachment by antagonist, 5: the pathogen completely overgrew the antagonist and occupied the entire medium surface (Bell et al., 1982).
Cellulase activity: The plates containing cellulase the medium (NH4H2PO4, 1.0 g; MgSO4.7H2O, 0.2 g; KCl, 0.2 g; CaCI2, 0.2 g; 4% ball milled cellulose 250 mL; agar, 12 g; distilled water, 750 mL) for testing cellulase activity were inoculated either centrally and 25°C incubated. Cellulase activity is then visualized as clear zones around fungal colonies (Paterson and Bridge, 1994).
Chitinase activity: The plates containing synthetic agar medium (glucose, 15 g; MgSO4.7H2O, 0.2 g; KH2PO4, 0.9 g; Kcl, 0.2 g; NH4NO3, 1.0 g; Fe2+, 0.002 g; Zn2+, 0.002 g and colloidal chitin, 3 g; agar, 12 g; distilled water 1000 mL) were inoculated and 25°C incubated and then clear zones were observed indicating chitinase activity (Rosato et al., 1981).
Metabolite analysis: The tested fungi were inoculated on Yeast Extract Sucrose Agar (YES) (which contained per litre; yeast extract, 20 g; sucrose, 150 g; agar, 20 g and MgSO4.7H2O, 0.5 g) incubated for 7 days at 25°C. To extract extracellular metabolites remove agar plugs from the colonies grown on YES using a 5 mm cork borer and plugs were extracted with ethylacetate. The extracts (15 μL) were spotted onto TLC plates. The plates were developed with chloroform:methanol (2:1 v/v) and bands were observed under UV light (254 nm) for spots with Rf values (Zelenkova et al., 2003).
| Table 1: | Characteristics of soil used in this study |
RESULTS AND DISCUSSION
Fungal species from rhizosphere soil of maize were obtained. From 15 fungal species isolated were identified at least to the genus level, however, one of them did not sporulate and could not be identified. The one non-sporulating mycelial fungus as assigned as sterile fungus. Fifteen fungal species were identified from maize rhizosphere soil (Table 2). The genus Fusarium seems to be the most abundant genus for representing 78.8% of the total fungal genus. Curvularia lunata is considered to be the least abundant organisms for representing 4.2% of the total fungi. Similar results have reported by Chiarini et al. (1998), Gomes et al. (2003) and Marin et al. (1998).
Fungi are of biotechnological interest due to their potential use as guetic vectors (Paterson and Bridge, 1994) as a source of secondary metabolites (Herbert, 1989; Miller, 1995) and as biological control agents (Magan and Lacey, 1985). The release of organic substances by plant roots has an interesting ecological aspect, since it influences the nutrient availability in the rhizosphere and indirectly acts on the soil microorganisms that in turn influence plant growth (Graystone et al., 1998). It is clear that some of the most isolated form maize rhizosphere soil may benefit the host and further studies will be carried out to evaluate the potential use of endophytes in biological control. In our work, a large number of species could be isolated from maize but only very few species were present in significant amount as cited by McLean and Berjak (1987).
In this study, the interactions of the between fungi were categorized according to their behaviour on the dual assay plates as described in Table 3. For example, interactions between Fusarium species were mutually antagonistic upon contact. This results is similar to that by Marin et al. (1998). A. versicolor, Aspergillus ustus and Gliocladium viride were able to dominant almost all species tested (Table 3).
| Table 2: | Fungi isolated from soil and their percentage of occurrence and Rf of metabolites producted by fungi |
| Table 3: | Interaction types between isolated fungal strains |
| Vers: A. versicolor, ust: A. ustus, vert: F. verticillioides, soli: P. solitum, viri: G. viride, bil: P. bilaiae, oxy: F. oxysporum, lat: F. lateritium, lun: C. lunata, har: T. harzianum, floc: F. flocciferum, vire: T. virens, verr: P. verrucosum, herb: Cladosporium herbarum, Fus: Fusarium sp., Sf: Sterile fungus. Scores are applied at left interacting with those listed above | |
Aspergillus ustus, Gliocladium viride, Trichoderma harzianum and T.virens were able to grow faster than the other isolated fungi. Fusarium verticillioides, F. lateritium, F. flocciferum, Penicillium bilaiae and Sterile fungus were mutually antagonistic either on contact or at a distance (Table 3).
Sterile fungus and Cladosporium herbarum grew more slowly than all the other fungi. The present work gives a general impression of how a range of fungal colonies from maize grown soil interact with each other and their potential competitiveness. There are many reports that note the interaction between fungi (Rheeder et al., 1990; Chiarini et al., 1998; Hussein et al., 2002). The enhanced competitiveness due to metabolite production in agar medium needs to be treated with caution as in grain substrates the production patterns and role may be very different (Magan and Lacey, 1985; Marin et al., 1998).
Marin et al. (1998) reported some Aspergillus species were able to inhibit the growth of some Fusarium species. In conrast, the dominance of Fusarium and Trichoderma species could be predominantly due to their ability to grow rapidly and invasively. Although the Pencillium species are known to export antifungal metabolites they did not have high levels under tested conditions and grew significantly more slowly than the Trichoderma, Fusarium and Aspergillus spp.
Wicklow et al. (1988) indicated that common fungal colonists of maize kernels interfere with the ability of A. flavus to infect preharvest maize; F. moniliforme was particularly effective in inhibiting kernel infection by A. flavus. The study of fungal interactions has attracted much interest due to the fact that mycotoxin production by toxigenic species which are present in stored grain can be both enhanced or inhibited by the other species which are colonizing the grain.
Wicklow et al. (1988) showed how aflatoxin production by A. flavus was stimulated by Bacillus amyloliquefacins and Hyphopichia burtonii when growing together on irradiated maize and rice grains under certain environmental conditions.
The enzymic activity tests gave more encouraging results in respect of differentiating fungal species. All cultures tested showed different level of cellulase and chitinase activity and only four (Penicillium solitum, Cladosporium herbarum, Curvularia lunata and Penicillium verrucosum) showed no reducing ability.
In this study, examination of thin layer chromatograms of fungi showed that several different compounds were being produced by the isolated fungal species (Table 2). Toxins produced by Fusarium, Penicillium and Aspergillus species have been reported among the general phytotoxins being able to result in leaf chlorosis and could inhibit seed germination and growth (Visconti et al., 1992; D’Mello, 1997). If the fungi isolated in this assay were pathogens in latent infection phase, their early detection in the field would influence the recognition of disease spread within the maize crop. On the other hand, these fungi could be either avirulent or hypovirulent strains. These fungi could be adapted to this host and be antagonists of soilborne pathogens and depending on their antagonistic capacity, they would be able to displace reduce, suppress or induce resistance against them.
Conslusively the results shown a strong warning for the toxicant productivities by soil fungi and rhizosphere is a useful potential source for isolation of fungi producers of biological active products. Further work will be carried out to identify those metabolites produced by soil fungi.