MATERIALS AND METHODS

Collection of soil samples: Twenty three soil samples were collected from different localities, during 2008 and 2009, collections have been made in all seasons (autumn, winter, spring and summer times) (Fig. 1) which represented both cultivated and non cultivated soils in Al-Jabal Al-Akhdar. For each soil sample the top surface soil was removed (about 3 cm) and 5 subsamples were taken at random to a depth of 15 cm for each site using a sterile auger. The soil was transferred to the laboratory in sterile polyethylene bags under aseptic conditions the subsamples of each site were bulked to yield one composite sample representing the area. The soil was allowed to dry by exposure to ambient temperature. When adequate moisture content was reached the samples were sieved through 2 mm mesh and soil characters were determined.

Determination of soil texture: The soil type was determined by the hydrometer method, as described by Piper (1955). Bouyoucos hydrometer was calibrated to read directly in percentage of soil remaining in suspension.

Soil chemical analysis
Total soluble salts: For the determination of total soluble salts, a known weight of each soil sample was shaken in a volume of distilled water for about 30 min and the mixture was left overnight to settle. The soil extract was then filtered and a known volume was evaporated in an oven at 105°C. The dry residue was then weighed and the amount of total soluble salts per one g oven-dry soil was calculated.

Organic matter content: It was determined according to Walky and Black method (Jackson, 1958).

pH value: A Beckman pH meter was used for the determination of soil pH. The electrodes were immersed in the soil paste made with water to a ratio of 1:1 to avoid the error arising through higher dilutions (Jackson, 1958).

Determination of soil fungi: Using the dilution plating method modified by Johnson et al. (1959). After incubation at 28°C, usually from five to seven days, the resulting colonies were counted. The average number of colonies per dish was multiplied by the dilution factor to obtain the number per gram in the original soil sample.

Isolation media: Five different types of media were compared for isolation soil fungi including Trichoderma from the tested soil. These culture media include: (1) glucose-Czapek’s agar medium (CZ) (2) Martin’s medium (Martin, 1950), Rose Bengal was added at a concentration of 1/15000 and streptomycin 50 ppm after autoclaving. (3) Malt Extract medium (ME); (4) Trichoderma Selective Medium (TSM) (Elad et al., 1981), Chloromycetin, 0.25 and Rose-Bengal, 0.15 were added after autoclaving.

Potato Dextrose Agar (PDA): For determination of the medium efficiency for isolation of Trichoderma from soil, conidial suspension was prepared from Trichoderma slants growing on PDA medium and added to the soil to give a concentration of 10⁴ Conidia/g soil and inoculated soil was incubated for 24 h at 28°C. After which Trichoderma count was determined on different-media by the plate count method.

Identification of fungi: The identification of fungal genera other than Trichoderma was made through the help of the following references: Ainsworth (1971), as dictionary of the fungi; Bernett (1960), for the genera of imperfect fungi; Domsch and Gams (1972), for fungi in general; Trichoderma species were identified using the key of Rifai (1969).

Some culture characteristics of Trichoderma isolates
Microscopic characters: Trichoderma isolates of grown on malt extract from PDA slants for 4 days at 28°C in the dark then exposed to light before examination were obtained. Cultures were examined under a light microscope (Olympus, Cx21, Japan). Mode of branching Conidiophores, shape of Conidia, phialides characters were observed for each isolate.

Measurement of growth at 28°C: Czapek's agar medium was inoculated in the center with a mycelial disc (0.5 cm diameter) of each isolate obtained from fresh culture of the fungus grown on PDA and incubated at 28°C for 4 days. The radial growth (colony diameter) of the fungus colony was measured daily from the reverse side (three replicates were used and the results were averaged).

Colony colour and reverse side: Three agar plates of each medium for each isolate were prepared from fresh culture on PDA and incubated for 2 days in the dark and allowed 2 days in the light at 28°C to sporulate and the colony colour as well as the reverse side was recorded.

Odour observation: The growth experiment was also used to record the fungus of specific odour by the fungus particularly the aromatic or the coconut odour of each isolate on each media.

RESULTS

For studying the distribution of Trichoderma in different areas on Al-Jabal Al-Akhdar region, 23 soil samples were collected from different localities which represented both cultivated and non cultivated soils.

Table 1 show that the soil organic matter content was ranging between 0.8% (cultivated sandy soil in El-Hania region) and 4.3 (cultivated sandy loam soil in Al-Byda region). Total soluble salts were ranging between (E.C.) 0.13-0.51 dS m^-1 with exception of sample No. 23 collected form El-Hania region which recorded 0.92 dS m^-1. The pH values of the soil samples were mostly alkaline, ranging between 7.6-8.4.

Quantitative and qualitative estimation of Trichoderma sp. in soil is often difficult due to the relatively rapid growth of some soil fungi in conventional agar media. Five different types of media were compared for their suitability for isolation of Trichoderma from soil. These include: 1: glucose-Czapek’s agar medium (CZ), 2: Martin's medium (MT), 3: Malt extract medium (ME), 4: Trichoderma selective medium (TSM), 5: Potato dextrose agar (PDA), Comparing the Trichoderma selective medium with 4 other media, results presented in Table 3, 4 indicated that both of Martin's medium and Potato dextrose agar were superior to the others for isolating and counting of Trichoderma and gave the highest number (1.0x10³ CFU g^-1 dry soil) in soils numbers 5 and 8.

Table 2 shows that Martin's medium and Malt extract medium were the most efficient in isolation. They gave the highest fungal numbers for soil numbers: 2, 6, 9, 10, 12, 20, 22 and 23 for Martins medium and soil number: 1, 3, 4, 7, 8, 12, 13 and 21 for Malt extract medium. However, Glucose Czapek's and Potato Dextrose Agar and Trichoderma selective medium recorded the lowest total fungal population except in case of soil numbers: 11, 16, 18 and 19 for CZ medium and soil numbers 5 and 17 for PDA and soil number 15 for Trichoderma selective medium. Results also indicate the Trichoderma were recovered on 3 out of 5 media (MT, TSM and PDA).

Fig. 1:	Map of Al-Jabal Al-Akhdar region shown different places of collection of soil samples (1-23 place)

Table 1:	Characteristics of the soil samples and plant used for isolation of Trichoderma

Table 2:	Total fungal counts (colonies /mg dry soil) isolated from different soil samples on different media
*Trichoderma was slated

Table 3:	Counts of Trichoderma (colonies/mg dry soil) isolated from different soil samples* on different media
*The total soil samples were 23 and Trichoderma was only present in 5 soil samples

The growth of Trichoderma isolates on different culture media (Fig. 3, 4) were studied and were identified according to the key of Rifai (1969) as T. harzianum. Trichoderma was isolated only from 5 out of 23 soil samples (Table 2).

In an attempt to evaluate the efficiency of the five media for isolation of Trichoderma added to the soil, Table 4 shows that both Trichoderma Selective Medium (TSM) and Martin's medium (MT) were efficient for isolation of Trichoderma from the tested soil.

Table 4:	Evaluation of medium efficiency for isolation of Trichoderma harzianum (T7 and T8) from soil

TSM was superior compared with other media and this medium recorded an efficiency ranging from 120-140%, indicating the favorability of TSM for further studies with Trichoderma to determine its survival and proliferation in Al-Jabal Al-Akhdar soils.

Fig. 2(a, b):	Growth of Trichoderma harzianum (T7) on (a) TSM+PCNB and (b) without PCNB

Fig. 3(a-d):	Growth of Trichoderma harzianum (T7) on different culture media, (a) T7-PDA, (b) T7-CZ, (c) T7-TSM and (d) T7-ME

Fig. 4(a-b):	Conidia, conidiophore branching and phialospores in Trichoderma harzianum isolates, (a) T8 and (b) T15

Results presented in Fig. 2a and b also show the effect of addition of the fungicide pentachloronitro benzene (PCNB) at the concentration of 100 μg mL^-1 on total fugal counts on different culture media. Results showed that the addition of the PCNB clearly reduced the total fugal counts isolated on all the five culture media. Moreover, the addition of PCNB restricted the colony size (especially with Trichoderma) to be easily for counting.

Results presented in Table 5 shows that Aspergillus was the most frequent fungus. It was collected from 20 samples out of 23 on Martin's medium with average total count 8.3 colony mg dry soil and it was isolated from 19 soil samples out of 23 on Malt extract medium with count 13.3 colony mg dry soil. The second most common genus was Penicillium which was collected from 20 soil samples out of 23 on Martin's medium with total count 22.3 colony mg dry soil and it was isolated from 19 soil samples out of 23 on malt extract medium with average total count 5-4 colony mg dry soil.

Members of mucorales (such as Mucor and Rhizopus sp.) and class deuteromycetes; (Fusarium, Alternaria, Curvularia, Humicola and Cephalosporium sp.) were also recorded in moderate to low frequency of occurrence.

Table 5:	Percentage of tatal Aspergillus, Penicillium and other genera counts in soil-samples (1-23) collected from Al-Jabal Al-Akhdar region
Asp.: Aspergillus, Pen.: Penicillum

DISCUSSION

Counting and estimation of Trichoderma in soil is difficult because of the relatively rapid growth of other fungi on agar medium. The results of the experiments which carried out in this study to find the most favourable medium for isolation and enumeration of Trichoderma from the test soil samples showed that, besides Trichoderma Selective Medium (TSM), Martin's medium (MT) and Potato Dextrose Agar (PDA) were suitable for isolation of Trichoderma. Results also indicated that despite of the negative effect of the addition of the fungicide, pentachloronitro benzene (PCNB), at the concentration of 100 μg mL^-1 on total fungal counts, the fungicide restricts the colony size and making it easy to be count. Results also indicated that TSM recorded higher efficiency for isolation of Trichoderma, which ranged from 120-140% compare with other tested media.

The suitability of the addition of PCNB to the culture media for counting of Trichoderma sp. was noticed and recommended by Papavizas and Lumsden (1982), El-Naghy et al. (1998) and El-Komy (2001).

PCNB is an effective agent in restricting the colony size of fast spreading fungi such as Mucorales as well as Trichoderma thus, facilitating the enumeration of the soil fungi (El-Katatny et al., 2004). The suitability of TSM with PCNB for counting Trichoderma is related to the fact that Trichoderma sp. are relatively tolerant to high levels of PCNB and rose Bengal (Papavizas, 1981; Elad et al., 1981) and to the capacity of Trichoderma to grow and sporulate on media containing low levels of glucose (Elad et al., 1983; El-Naghy et al., 1998). PCNB also reduced the number-forming units (CFU) of soil-fungi (Papavizas, 1981; Papavizas and Lumsden, 1982).

Thus, PCNB has been previously recommended to be included as an ingredient in the culture media used for isolation and counting Trichoderma sp. from soil (Elad et al., 1981) for example, the performance of V-8 medium containing PCNB as a fungal inhibitory agent (referred to as TME medium) which was suggested for the direct isolation and enumeration Trichoderma sp. from soil by Papavizas (1981) has been found among the various media tested to be the most satisfactory for inhibiting the rapidly growing fungi such as mucorales and encouraging and allowing the enumeration of Trichoderma from soil.

Results of the survey of Trichoderma in different localities in Al-Jabal Al-Akhdar region indicated that Trichoderma was isolated from 5 out 23 soil samples and its numbers per gram soil was ranging from 0.5-1x10³ CFU g^-1 soil. The natural levels of Trichoderma sp. in soil are between 10² and 10⁴ CFU g^-1 of soil, with the lower level being most common (Green, 2003). The population level of Trichoderma in soil depends on the abiotic and biotic factors of the environment. Generally, high levels of organic matter and clay and low pH values make higher population levels (Alabouvette and Steinberg, 1995; El-Naghy et al., 1998).

Literatures concerning the survey of Trichoderma in Libyan soil are very rare, Youssef (1974) reported that sixty three fungal species in twenty genera were isolated from sixteen different localities in Libya. Of these species four were Phycomycetes, ten were Ascomycetes and forty nine were Deuteromycetes. In Egypt, Shaban (1986) reported that Trichoderma fungi were isolated from 13 soil samples out of 20, pointed out that non-cultivated soils with high content of soluble salts and very low organic matter are not favourable for the development of Trichoderma. Generally, previous studies on mycoflora of Egyptian soil revealed that Trichoderma occur in moderate or low frequencies (Mazen and Shaban, 1983).

Results of this investigation showed that the 5 isolates of Trichoderma were identified as T. harzianum according to the key of Rifai (1969). The latter author have discussed the morphological characters used to characterise and differentiate species of Trichoderma.

Trichoderma can be identified by distinctive morphological characters such as, rapid growth, bright green or white conidial pigments and a repetitively branched, but otherwise, poorly defined condiophores structure. Reverse side uncoloured or variously buff, yellow, amber dull reddish, or yellow green. Characteristic aromatic odours resembling coconut are produced by some strains of T. viride, T. atroviride and T. harzianum. Conidation effuse or tufted or forming compact pustules typically in green shades or less often white, grey or brown conidia one celled, typically green, or otherwise colourless, greyish or brownish, smooth walled, to distinctly roughened or with sinuate, bullate, or wing-like projections, from outer wall, subglobose, obovoid, ellipsoid, oblong, or short cylindrical. Chlamydospores usually present and often abundant especially in submerged mycelium. Vegetative hyphae usually hyaline, smooth walled (Samuels, 1996). Recently molecular techniques allow a rapid and reliable identification of Trichoderma sp. and strains. Moreover, the molecular data confirm the morphological classification of Trichoderma (Papavizas, 1985; Gherbawy et al., 2004).

Results of this study also indicated that Aspergillus and Penicillium sp. were the most frequent fungi isolated from the studied soil-samples. Several other genera (Mucor, Fusarium, Alternaria, Curvularia; etc.) were also isolated in moderate, low or rare frequencies on the studied culture media. These results are in accordance with many investigations reported everywhere. In Egypt, Abdel-Hafez (1974), Moubasher et al. (1977), Mazen and Shaban (1983), Shaban (1986) and Yaser (1999) reported that Aspergillus. Penicillium and Fusarium species were the most common genera recorded in their studies in Egyptian cultivation and desert soils.

CONCLUSION

Such Trichoderma species can be found in different regions in Libya, where the importance of such species as biocontrol agent should be tested for its capacity to control plant diseases. However, many research needed using molecular techniques to record and to identify fungal flora in Libya, this report is considered to the first which showed the distribution and identification of Trichoderma sp. in Al-Jabal Al-Akhdar, Libya.

ACKNOWLEDGMENTS

The study made possible through the financial support from the Omar Al-Mukhtar University, Al-Bayda, Libya.