Ecological and physiological studies on soil fungi at Western region, libya.

Sixty three species and 5 varieties belonging to 30 fungal genera were collected from 75 soil samples. Cultivated (29 genera and 58 species + 5 var.), desert (22 and 35 + 2 var.) and saline soil (21 and 41 + 1 var.) fungi were recovered on glucose-, cellulose- and 50% sucrose-Czapek's agar at 28℃. The most common genera were Alternaria, Aspergillus, Emericella, Fusarium, Mycosphaerella, Nectria and Penicillium. The most prevalent species from the three types of soils on the three types of media were Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Emericella nidulans, Fusarium oxysporum, Mycosphaerella tassiana, Nectria haematococca and Penicillium chrysogenum. Chaetomium globosum was in the top of fungi in producing endo-β-1,4-glucanases among the 42 tested isolates obtained from soils on cellulose-Czapek's agar. Maximum production of this enzyme by C. globosum obtained after 6 days of incubation at 30℃ with culture medium containing maltose as a carbon source and ammonium nitrate as a nitrogen source and pH initially adjusted to 6.

Mycoflora of soils have been paid a considerable attention during the last forty years. The subject has been investigated from various points of view, but most of the work in this respect has been concerned with those fungi inhabiting cultivated, desert and saline soils in many parts of the world (Abdel-Hafez et al., 1991; Abdel-Hafez, 1994; El-Said, 1994; Ozerskaya et al., 2004; Lalley and Viles, 2005).

Cellulose, a major polysaccharide constituent of plant cell walls, is a -1,4 linked linear polymer of 8000~12000 glucose units. Three major enzymes are involved in the degradation of cellulose to glucose are endoglucanase (endo-1,4-d-glucanase, EG), cellobiohydrolase (exo-1,4-d-glucanase, CBH), and β-glucosidase (1,4-d-glucosidase, BG). EG acts in random fashion, cleaving linked bonds within the cellulose molecule; CBH removes cellobiose units from the nonreducing ends of the cellulose chain and BG degrades cellobiose and cellooligosaccharides to glucose (Saha, 2004). Several fungi such as members of Aspergillus, Penicillium, Trichoderma, Chaetomium and some other moulds of Mucors and dematiaceous hyphomycetes produced cellulolytic enzymes as reported by several researchers (Nelly, 1991; Abdel-Hafez, et al., 1995, 2003; Moharram et al., 2004; El-Said et al., 2005, 2006; Vasil'chenko et al., 2005). This investigation aimed to study the distribution and occurrence of various groups of fungi in cultivated, desert and saline soils as well as the ability of fungal isolates to produce cellulase enzyme under different environmental and nutritional conditions.

Materials and Methods

Cultures

Twenty-five soil samples of each of cultivated (Nos. 1-25), desert (Nos. 26-50) and saline (Nos. 51-75) were collected from different localities in Western region in Libya according to the method described by Johnson and Curl (1972). The geographical feature of El-Gaffara plain is refer that it is a big region in Libya, as it is covers more than 17,000 km2. It takes a triangle shape with apex at the east near Al-khums town. The north is parallel with the Mediteranean sea coast and about 275 km long. The western side forming the western borders of the Republic and about 150 km long (Fig. 1).

Fig. 1

A map showing the different sites Western region in Libya from which the soil samples were collected. *Dr. Ali Ayad Ben-Hamed. Geographic department. National Atlas of Jamahiriya. 1977, pp. 33-34.

Chemical analysis of soil samples

Organic matter content (OM) was determined by Walkely and Black method (Jackson, 1958). The amount of total soluble salts per one g oven-dry soil (TSS) was calculated according to Jackson (1958). A pH meter (Orior Research model 601T/digitalionalyzer) was used for the determination of soil pH according to Jackson (1958). Carbonate (CO3--) and bicarbonate (HCO3-) were determined directly in the soil by back-titration (Hydrochloric acid digestion) according to the method described by Jackson (1958). Soluble chloride (Cl-) was estimated by applying the silver nitrate titration method using potassium chromate as an indicator (Jackson, 1958). Calcium (Ca2+) and magnesium (Mg2+) were determined by titration method (Schwarzenbach and Biederman, 1948). The cations such as sodium (Na+) and potassium (K+) were determined by using Carl Zeiss flame photometer method (Williams and Twine 1960).

Estimation of soil fungi

The dilution-plate method as described by Johnson and Curl (1972) was used for estimation of soil fungi. Modified Czapek's Dox agar medium was employed (g/l: glucose 10.0 or cellulose powder 20.0 or sucrose 500.0, sodium nitrate 3.0, magnesium sulphate 0.5, potasium chloride 0.5, potassium dihydrogen phosphate 1.0, ferrous sulphate 0.01, agar 15), in which glucose or powdered cellulose or sucrose were used for the isolation of glucophilic, cellulose-decomposing and osmophilic (or osmotolerant) fungi, respectively. These media were supplemented with rose bengal (0.1 mg/ml) and chloramphenicol (0.05 mg/ml) in order to suppress bacterial growth. The plates were incubated at 28℃ for 5~10 days during which the developing fungi were counted, identified (purely morphologically, based on macro- and microscopic characters) and calculated per g dry soil.

Screening of fungal isolates for cellulase production

Forty-one species and 1 species variety representing 26 genera were screened for their abilities to produce endo-β-1,4-glucanase (Cx enzyme). Isolates were cultured on Eggins and Pugh medium (1962) and pH was adjusted to 5.4 using acetate buffer. Cultures were incubated at 28℃ for 7 days. Using a sterile cork borer (10 mm diameter) discs were cut to inoculate 50 ml sterile liquid medium (in 250 ml Erlnmeyer conical flasks) of Prasad and Verma medium (1979) for endo-β-1,4-glucanase. After 7 days incubation at 28℃ the cultures were filtered and the filtrates were used to detect the activity of Cx enzyme.

Detection of endo-β-1,4-glucanase (Cx enzyme)

Using a sterile cork borer three cavities (10 mm diameter) were made in plates containing solid medium of Dingle et al. (1953) for detection of endo-β-1,4-glucanase. 0.1 ml of culture filtrate was dropped in each of these cavities followed by incubation at 28℃ for 24 hours, then the plates were flooded with chloroiodide of zinc solution and the clear zone gave a measure for cellulolytic power of isolates.

Factors affecting cellulase production

The effect of different ecological and nutritional factors on production of Cx enzyme by Chaetomium globosum was studied, since this species was found to be highly active in endoglucanase production.

Effect of temperature and time course

Inoculated flasks were incubated at 20, 30 and 40℃ for 14 days and harvested at 48 hours intervals. Culture fluids were filtered and centrifuged at 5,000 rpm for 10 min. The clear supernatents were assayed for Cx enzyme activity.

Effect of pH value

C. globosum was grown on the basal medium of Deacon (1985). The initial medium was adjusted with 0.1 N NaOH or 0.1 N HCl to different values of pH ranging from 2 to 12. After inoculation, cultures were incubated at 30℃ (the best temperature for endoglucanase activity) for 6 days (the best incubation period), then filtered, centrifuged at 5000 rpm for 10 min and the clear supernatents were assayed for Cx enzyme activity.

Effect of different carbon sources

For estimation the effect of different carbon materials on endoglucanase activity, the carboxymethylcellulose (CMC) in cellulose-Czapek's medium was replaced with the same weight of different carbon sources such as: clover straw, filter paper, maltose, powdered cellulose, wheat bran, wheat straw and yeast extract. The inoculated flasks were incubated at 30℃ for 6 days and the cultures were filtered. After centrifugation the clear filtrate was used to detect the Cx enzyme activity.

Effect of different nitrogen sources

To determine the effect of nitrogen source on Cx enzyme activity, sodium nitrate (3 g/l) in cellulose-Czapek's medium was replaced with the same amount of various nitrogen compounds such as; NaNO2, KNO3, NH4NO3, (NH4)2SO4 and CaNO3. Cultures in flasks were incubated at 30℃ for 6 days, then filtered, centrifuged and the clear filtrate was used for the detection of endoglucanase activity using the method described by Naguib (1964).

Results and Discussion

The moisture contents of the soil samples tested varied a low value (0.1~3.6%), a moderate value (0.3~6.4%) and a high value (1.29~15.5%). The highest value (15.5%) occurred in the cultivated soil sample No. 22 collected from Sabratah under Solanum lycopersicum. Abdel-Sater (1987) found that the water content of 25 soil samples collected from different habitats of each of cultivated, desert and saline soils in Egypt fluctuated between a low value (2.4~9.9%), a moderate value (10~15.2%) and a high value (15.3~21.9%). The moisture contents of soil samples collected from Bahreen ranged between 0.1~1.1% (El-Said, 1994).

The soil samples were generally poor in their organic matter content, but the cultivated soil was the richest (0.18~1.71% of dry soil) followed by desert (0.1~0.57%) and saline soils (0.01~0.54%). The present observations almost agree with the result obtained from different types of soil in some Arab countries (Moubasher et al., 1985; Abdel-Sater, 1987; Abdel-Hafez et al., 1989a, b, 1990a, b, 1991, 1995; El-Said, 1994).

The highest value of total soluble salts was detected in saline soil (1.02~8.89%). These high amounts of salts were not found in the cultivated (0.06~0.85%) and desert (0.01~0.48%) soils. Similar results were recorded by Abdel-Sater (1987) who found that the total soluble salts in the samples of cultivated, desert and saline soil collected from Egypt ranged between 0.13~1.69%, 0.03~1.6% and 6.62~18.63%, respectively. Also, Abdel-Hafez et al. (1991, 1995) recorded that the total soluble salts collected from Egypt fluctuated between 2.2~4.7% and 0.18~0.30%. El-Said (1994) reported that the amount of total soluble salts fluctuated between 2.3~4.7% in cultivated soils of Bahreen.

The amount of carbonates, bicarbonates and chlorides in the samples tested fluctuated markedly from 2.01~7.60%, 0.23~2.04% and 0.02~0.24%; 3.21~7.75%, 0.32~2.02% and 0.001~0.32%; and 6.09~7.49%, 0.18~1.72% and 0.02~1.31% in cultivated, desert and saline soils, respectively. Abdel-Sater (1987) recorded that the amount of carbonate, bicarbonate and chlorides in the samples of cultivated, desert and saline soils collected from Egypt ranged between 2.26~5.4%, 0.36~1.5% and 0.07~0.68%; 1.65~5.88%, 0.23~1.02% and 0.14~3.9%; and 4.2~5.94%, 0.18~1.93% and 0.36~4.14%, respectively.

The amount of elements in cultivated, desert and saline soils were: Ca: 0.03~0.73, 0.05~0.2 and 0.09~2.85 mg; Mg: 0.03~0.19, 0.02~0.35 and 0.03~1.0 mg; K: 0.07~0.27, 0.11~0.53 and 0.10~0.89 mg and Na: 0.03~0.09, 0.02~0.46 and 0.1~0.79 mg/g dry soil, respectively. Abdel-Sater (1987) found that the amount of elements in cultivated, desert and saline soils collected from Egypt were: Ca: 0.3~0.75, 0.03~2.67 and 0.07~3.75; Mg: 0.13~0.54, 0.02~0.54 and 0.013~1.23; K: 0.02~0.27, 0.02~0.51 and 0.05~0.88; and Na: 0.16~4.8, 0.12~8.05 and 2.35~39 mg/g dry soil, respectively.

pH values of cultivated, desert and saline soils were ranged between 4.5~7, 6.4~7.2 and 6.4~7.3, respectively. Abdel-Sater (1987) found that the pH values of cultivated, desert and saline soils gathered from Egypt fluctuated between 7.2~8.9, 6.9~7.4 and 7.2~8.8, respectively. Similar observations were obtained by Abdel-Hafez et al. (1989b, 1991, 1995) and by El-Said (1994).

Sixty three species and 5 varieties belonging to 30 genera were collected from 75 soil samples. These fungi recovered from cultivated (29 genera and 58 species + 5 var.), desert (22 and 35 + 2 var.) and saline (21 and 41 + 1 var.) soils on glucose-, cellulose- and 50% sucrose-Czapek's agar at 28℃ (Tables 1, 2 and 3). The most common genera were: Alternaria (2 species), Aspergillus (11 + 4 var.), Emericella (1+2), Fusarium (4), Mycosphaerella (1), Nectria (1) and Penicillium (7). The most prevalent species from the three types of soils on the three types of media were: Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Emericella nidulans, Fusarium oxysporum, Mycosphaerella tassiana, Nectria haematococca and Penicillium chrysogenum. The above species were isolated with different numbers and frequencies from various soils in many places of the world by several workers (Abdel-Hafez et al., 1990a, b; Moubasher and Mazen, 1991; Abdel-Hafez, 1994; Karl and Iain, 2004; Lalley and Viles, 2005 and several others). Abdel-Hafez et al. (1991) found that the most common species in the Egyptian soils on glucose-, cellulose- and 50% sucrose-Czapek's agar were: A. flavus, A. fumigatus, A. niger, A. sydowii, A. terreus, E. nidulans var. dentata, E. nidulans var. lata, P. chrysogenum, P. puberulum and Rhizopus stolonifer. On the other hand, the most frequently encountered species in Bahreen soils recovered on glucose-, cellulose- and 50% Sucrose-Czapek's agar were A. alternata, A. flavus, A. fumigatus, A. niger, A. sydowii, A. terreus, E. nidulans, E. nidualns var. dentata, F. oxysporum, N. haematococca, P. chrysogenum and P. corylophilum (El-Said, 1994).

Table 1

Average total count (calculated per g dry soil in every sample), number of cases of isolation (NCI, out of 25 cases) and occurrence remarks (OR) for fungal genera and species recovered from 25 cultivated soil samples on glucose, cellulose and 50% sucrose-Czapek's agar at 28℃

ATC = Average total count (per g dry soil); NCI = Number of cases of isolation (out of 25); OR = Occurrence remarks: H = High occurrence, from 13~25 (out of 25); M =Moderate occurrence, from 6-12 cases; L = Low occurrence, from 3-5 cases; R = Rare occurrence, from 1 or 2 cases.

Table 2

Average total count (calculated per g dry soil in every sample), number of cases of isolation (NCI, out of 25 cases) and occurrence remarks (OR) for fungal genera and species recovered from 25 desert soil samples on glucose, cellulose and 50% sucrose-Czapek's agar at 28℃

ATC = Average total count (per g dry soil); NCI = Number of cases of isolation (out of 25); OR = Occurrence remarks: H = High occurrence, from 13-25 (out of 25); M =Moderate occurrence, from 6-12 cases; L = Low occurrence, from 3-5 cases; R = Rare occurrence, from 1 or 2 cases.

Table 3

Average total count (calculated per g dry soil in every sample), number of cases of isolation (NCI, out of 25 cases) and occurrence remarks (OR) for fungal genera and species recovered from 25 saline soil samples on glucose, cellulose and 50% sucrose-Czapek's agar at 28℃

ATC = Average total count (per g dry soil); NCI = Number of cases of isolation (out of 25); OR = Occurrence remarks: H = High occurrence, from 13-25 (out of 25); M =Moderate occurrence, from 6-12 cases; L = Low occurrence, from 3-5 cases; R = Rare occurrence, from 1 or 2 cases.

Eurotium was recovered from the three types of soils on plates of 50% sucrose-Czapek's agar and these were: Eurotium amestelodami, E. chevalieri and E. rubrum. Members of Eurotium are well known osmophilies as reported by some workers (Abdel-Hafez et al., 1989a, b, 1990a, 1991, 1995; El-Said, 1994; El-Said et al., 2005).

Cellulolytic activities of fungal isolates

All fungal isolates (42 isolates) were screened for their abilities to produce endo 1,4-β-glucanase (CMase or Cx enzyme) on solid medium proved to be active to utilize cellulose, but with different degrees (Table 4). Ten isolates (23.80% of total isolates) showed high cellulolytic activity for endo-β-1,4-glucanase and these were A. alternata, A. flavus, A. fumigatus, C. globosum, Cladosporium cladosporioides, F. oxysporum, Mucor racemosus, Papulaspora immersa, R. stolonifer and Sterile mycelia. The moderately cellulolytic isolates included 16 isolates (38.09% of total isolates) and the most important isolates being: A. niger, A. sydowii, E. nidulans, F. poae, P. chrysogenum, P. puberulum and Phoma glomerata. While, sixteen isolates (38.09% of total isolates) were found to be weak cellulolytic activities which comprised for examples: Aspergillus ochraceus, Cochliobolus spicifer, Myrothecium verrucaria, N. haematococca, Setosphaeria rostrata and Ulocladium botrytis and others. Most of the above fungal isolates were reported as cellulase producers, but with variable capabilities by several workers (Abraha and Gashe, 1992; Abdel-Hafez et al., 1995; Moharram et al., 1995, 2004; El-Said, 2001; Berlin et al., 2005).

Table 4

Activity of carboxymethyl cellulase (Cx) of different fungal species isolated on cellulose-Czapek's agar at 28℃

Degree of Cx activity; High activity, H = from 20~28 mm; Moderate activity, M= 16~19 mm; and Weak activity, W = 11~15 mm.

C. globosum was in the top of fungi in producing of endo 1,4-β-glucanase (Cx enzyme) in this investigation. Maximum production of the enzyme by C. globosum was achieved 6 days after incubation at 30℃ with the incorporation of maltose as carbon sucrce and NH4NO3 as nitrogen source in the culture medium which is initially adjusted to pH 6 (Figs. 2 and 3). These findings are almost in agreement with those reported by Sandhu and Kalra (1985) and Kalra and Sandhu (1986). They noticed that the maximum production of C1 and Cx enzymes produced by Trichoderma longibrachiatum and T. harzianum was achieved after 5~7 days of incubation at 27℃ but with the incorporation of 1% lactose in culture medium which initially adjusted to pH 5. They also found that CMC and malt extract were favourable for the enzyme production. The maximum production of exo- and endo-β-1,4-glucanase by C. globosum and Trichoderma viride were after 6 and 8 days of incubation at 25℃ with culture medium containing wheat bran as a carbon source and peptone as nitrogen source and initially adjusted to pH 6 (Abdel-Hafez et al., 1995; El-Said, 2001). Recently, El-Said et al. (2006) found that maximum production of endo-β-1,4-glucanase by F. oxysporum could be achieved after 8 days of incubation at 30℃ with the incoporation of carboxymethylcellulose as a carbon source and peptone as nitrogen source in the culture medium which initially adjusted to pH 6.

Fig. 2

Cellulase production by C. globosum in cultures incubated at different temperatures for different periods (A) and in cultures initially adjusted to different pH values (B).

Fig. 3

Cellulase production by C. globosum in culture media containing different carbon (A) and nitrogen (B) sources.