Occurrence and Microbiological Characteristics of Trichoderma in Al-Jabal Al-Akhdar Region, Libya
Idress Hamad Attitalla,
Suliman S. Abdelrawaf,
Khawila Saeed Omar,
The fungal flora with special attention to Trichoderma
in 23 soil samples collected from Al-Jabal Al-Akhdar Region, Libya was studied
using different culture media. Trichoderma Selective Medium (TSM), Martin's
medium (MT) and Potato Dextrose Agar (PDA) were the superior media for isolating
Trichoderma. Martin's medium (MT) and Malt Extract medium (ME) were the
most valuable for isolating the greatest number of total fungal count. Trichoderma
Selective Medium (TSM) supplemented with 100 μg mL-1 PCNB was
the most effective medium for counting Trichoderma and recorded 120-140%
efficacy of re-isolation. Trichoderma occurred in moderate frequency
in the tested soil and was isolated from 5 soil samples. Trichoderma counted
0.5-1x103 CFU g-1 dry soil and five Trichoderma isolates
were identified as Trichoderma harzianum. Aspergillus and
Penicillium sp. were the most frequent fungi isolated from the tested soil
and were averaged 8.3-5.5 CFU mg-1 soil, respectively. Further studies
are needed to clarify the distribution of fungal flora especially Trichoderma
sp. in the Libyan soil.
Received: April 02, 2012;
Accepted: June 12, 2012;
Published: July 04, 2012
Trichoderma is a genus of the hypomycetes which gained specific attention
during the past few years and some excellent reviews were published on this
subject (Papavizas, 1985; Samuels,
1996; Chet, 1987; Lumsden, 1992).
The genus Trichoderma was introduced into mycological literature by Papavizas
(1985) to accommodate four species of fungi now commonly considered to be
unrelated to one another, namely Trichoderma viride Pers. Ex S. F. Gray,
Xylohypha nigrescens (Pres. Ex Fr) Mason, Sporotrichum aureum
Pers. Ex Fr. and Trichothecium roseum (Pers.) Link ex S. F. Gray. The
most frequently recorded green Trichoderma species found on fallen branches
and other substrate has been regarded as the most typical representative of
this genus. Therefore, it is not surprising that Trichoderma had been
classified in the Gasteromycetes or the Myxomycetes and that many totally unrelated
forms have been assigned to it. However, the hyphomycetous nature and the concept
of this genus have been well established.
Trichoderma sp. are widely distributed all over the world (Domsch
et al., 1980a, b; Attitalla
and Salleh, 2010) and occur in nearly all soils and other natural habitats,
especially in those containing organic matter. Individual aggregates may be
restricted in their geographical distribution (Danielson
and Davey, 1973a). Trichoderma is a secondary colonizer since it
is usually isolated from well decomposed organic matter. Studies with Trichoderma
prior to the work of Rifai (1969) were hampered by taxonomic
uncertainties and thereafter also by the lack of precise techniques for culturing,
isolation and enumeration of these fungi. Samuels (1996)
provided detailed observations and comments on the utility of morphological
characters to define species in However, molecular techniques allow rapid and
reliable identification of Trichoderma sp. and strains (Moubasher,
1993; El-Naghy et al., 1998; Gherbawy
et al., 2004).
Trichoderma species were isolated from forest humus layer (Wardle
et al., 1993). Individual species were reported to exhibit some restriction
in their geographic distribution and were also formed to show preference to
certain soil temperature and moisture content (Danielson
and Davey, 1973a). T . viride and T. polysporum for example,
were reported to be restricted to areas where low temperature prevail and T.
harzianum were mostly found in warm climatic regions whereas, T. hamatum
and T. koningii occur widely under diverse climatic conditions (Samuels,
1996). T. hamatum and T. pseudokoningii were reported to
be adapted to conditions of excessive soil moisture (Danielson
and Davey, 1973b). Additional factors which were reported to influence the
distribution of Trichoderma members in different soils include; soil
pH, soil chemical properties, salt and organic matter content and presence or
absence of microorganisms in soil (Samuels, 1996; Kredics
et al., 2003).
Trichoderma sp. have been reported from Congo, New Zealand, Australia,
Germany, Norway, Italy, Spain, Turkey, Chad, Pakistan, Nepal, China, Peru, Canada,
UK, India and the USA (Domsch et al., 1980a, b;
Barooah and Borthakur, 1994). In Arabic-countries, Trichoderma
spp. were recovered also from Libya (Youssef, 1974),
Kuwait (Halwagy et al., 1982), Saudi Arabia (Abdel-Hafez,
1982) and Syria (Abdel-Kader et al., 1983).
The aim of the this study was to identify and characterized the fungal flora
in Al-Jabal Al-Akhdar soil, Libya and focusing on the distribution and recognition
of the genus Trichoderma. This study is the first that demonstrated the
occurrence and the distribution of fungal flora in Al-Jabal Al-Akhdar region,
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,
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-Czapeks agar medium (CZ) (2) Martins
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
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
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.
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-Czapeks
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).
|| Map of Al-Jabal Al-Akhdar region shown different places of
collection of soil samples (1-23 place)
|| Characteristics of the soil samples and plant used for isolation
|| Total fungal counts (colonies /mg dry soil) isolated from
different soil samples on different media
|*Trichoderma was slated
||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.
||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
|Fig. 2(a, b):
|| Growth of Trichoderma harzianum (T7) on (a) TSM+PCNB
and (b) without PCNB
|| Growth of Trichoderma harzianum (T7) on different
culture media, (a) T7-PDA, (b) T7-CZ, (c) T7-TSM and (d) T7-ME
|| 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.
||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
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,
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.
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.
The study made possible through the financial support from the Omar Al-Mukhtar
University, Al-Bayda, Libya.
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