Species diversity of indigenous Trichoderma from alkaline pistachio soils in Iran

Document Type: Original Article

Authors

Department of Plant Protection, Faculty of Agriculture, of Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran

Abstract

The diversity of Trichoderma spp. was investigated in alkaline soils of pistachio orchards at different geographic areas in Kerman province, Iran. A total of 161 Trichoderma isolates were obtained and identified at the species level by their morphological characters and sequence analysis of their internal transcribed spacer (ITS) and tef1-α genomic regions. Totally, five species of Trichoderma were identified, including T. harzianum, Trichoderma sp., T. virens, T. brevicompactum and T. longibrachiatum. ITS nucleotide sequences could not find molecular differences between T. harzianum and Trichoderma sp. To identify and differentiate these two species, the sequences of the translation-elongation factor 1-α (tef1-int 4 (large)) were determined for five representative isolates of T. harzianum and Trichoderma sp. The TrichoBLAST similarity search using the tef1-α sequences of the T. harzianum and Trichoderma sp. isolates determined in this study, revealed as T. harzianum. However, there was 34 nucleotides difference in tef1-α sequences, between the two groups of isolates. According to the results, more than 80% of the isolates belonged to two species T. harzianum and Trichoderma sp. T. harzianum was introduced as the dominant species in soil of pistachio orchards. Logistic regression analysis showed no relationship between the soil properties (pH, EC) and presence of Trichoderma spp. (R2 = 0.26, Pr = 0.74, 0.26 > 0.05).

 

Keywords

Main Subjects


INTRODUCTION

 

The fungal genus Trichoderma (Hypocreales, Ascomycetes) includes cosmopolitan soil-borne species that are frequently found in decaying wood, compost and other organic matters (Harman et al. 2004, Samuels 2006). Several Trichoderma species are significant biocontrol agents against fungal plant pathogens, either through direct parasitism, competition with pathogens for nutrients, stimulation of plant health, or inducing plant systemic resistance to pathogens (Bailey et al. 2006, Harman et al. 2004, Hjeljord & Tronsmo 1998). The ability for mycoparasitism in some species also has a negative economic impact on the commercial production of Agaricus bisporus and Pleurotus ostreatus Rolland mushrooms (Hatvani et al. 2007, Krupke et al. 2003, Samuels et al. 2002). On the other hand, Trichoderma species produce a wide diversity of metabolites, most notably commercially important cellulase and hemicellulases, antibiotics, peptaibiotics, as well as toxins, such as trichodermamides and trichothecenes that display in vitro cytotoxicity (Degenkolb et al. 2008, Kubicek & Penttila 1998, Liu et al. 2005). Because of the great effect of Trichoderma species on human activity, there is a great need for their accurate identification.

The initial approach to understand the diversity and relationship between Trichoderma species based on morphological observation was made by Rifai (1969) and later by other researchers (Bissett 1984, 1991a, 1991b, 1992, Hoyos-Carvajal et al. 2009). However, accurate species identification only based on morphology is difficult, because of the paucity and similarity of useful morphological characters (De Respinis et al. 2010, Druzhinina et al. 2005). Bissett et al. (2003) stated that precise resolution of Trichoderma species is possible only through combination of morphological and molecular methods, and increasing number of morphologically cryptic species that can be distinguished through their DNA characters are being described (Samuels et al. 2010). This has already resulted in incorrect identification and the propagation of errors for strains associated with the production of secondary metabolites (Humphris et al. 2002), human diseases (Gautheret et al. 1995), and biological control (Kullnig et al. 2001). However, with the advent of molecular methods and identification tools which are based on sequence analysis of multiple genes, including rDNA (the nuclear ribosomal internal transcribed spacers (ITS) and 28S rDNA gene (LSU)), and genes encoding actin, calmodulin, endochitinase, RNA polymerase II and translation-elongation factor 1-alpha (tef1-α), it is now possible to identify every Trichoderma isolate and/or recognize it as a putative new species (Druzhinina & Kubicek 2005, Kubicek et al. 2008, Samuels 2006). For the identification of new species in the genus Trichoderma, most authors have used the combination of ITS and tef1-α (Bissett et al. 2003, Kraus et al. 2004, Kubicek et al. 2003, Lu et al. 2004). At present, the International Subcommission on Trichoderma and Hypocrea Taxonomy (ISTH) lists more than 100 species, all of which have been characterized at the molecular level, using TrichOKey program as a molecular identification tool for Trichoderma species (http://www.isth.info). Many studies have been carried out on the taxonomy of this genus in the world. Nevertheless, the information about the diversity of Trichoderma/Hypocrea in Iran is scarce. A preliminary checklist of micromycetes in Iran reported 25 Trichoderma species (Ershad 2009).

However, all of these species were identified based on morphological and in some cases molecular characters through amplification of rDNA-ITS region. Due to the mentioned reasons, the establishment of biocontrol agents seems to be the first and the most important step in their use in biological control. Thus, identifying the effective native biocontrol agents to be used against plant pathogens in an area have received considerable interest. As the latter application implies the introduction of Trichoderma into the rhizosphere of a given ecosystem, knowledge of the indigenous Trichoderma taxa in different soils and climates will contribute to criteria influencing the choice of strains to be applied. The occurrence and species diversity of Trichoderma in soil and many other different substrata has been the subject of several investigations in different areas, including South-East Asia (Kubicek et al. 2003), Austria (Wuczkowski et al. 2003), on alkaline agricultural soil in the Nile valley, Egypt (Gherbawy et al. 2004), South America (Druzhinina et al. 2005), China (Zhang et al. 2005, Sun et al. 2012), Sardinia (Migheli et al. 2009), neotropical regions such as Colombia, Mexico, Guatemala, Panama, Peru, Ecuador and Brazil (Hoyos-Carvajal et al. 2009) and Poland (Blaszczyk et al. 2011).

There are no reports for biodiversity of Trichoderma species on saline and alkaline soils of pistachio orchards in Iran and the world. Because of the alkaline and saline nature of Iranian pistachio soils and the importance and applications of Trichoderma species in biological control of plant pathogens, the objective of the present study was to evaluate the occurrence and species diversity of Trichoderma isolates recovered from alkaline soils of pistachio orchards based on morphological and molecular analyses.

 

MATERIALS AND METHODS

 

Sampling and isolation of Trichoderma isolates

Soil samples were collected from a depth of 0 – 60 cm of alkaline pistachio soils and the rhizosphere of pistachio trees in Kerman province, Iran, during 2010 – 2012. The soil samples were then transferred into sterile polyethylene bags and transported to the laboratory. Trichoderma isolates were isolated from soil samples using dilution plate technique (Johnson 1959), on a selective Trichoderma medium (Elad & Chet 1983). The plates were incubated at most for 10 days in dark at room temperature (25°C ± 2). Regularly, after 48 hours, the colonies were counted and transferred to Petri dishes containing PDA (potato dextrose agar, Merck, Germany)-in case of suitable growth- for strain purification, using single spore method. Pure cultures were transferred to the tubes containing PDA and stored at 4°C for further studies. All of the Trichoderma isolates are available from mycological collections of Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan. Representative isolates of each of the identified species have also been deposited at Iranian Fungal Culture Collection in Iranian Research Institute of Plant Protection, Tehran, Iran.

 

Determination of chemical properties of soil

To determine the relationship between the abundance of Trichoderma population and values of electrical conductivity (EC) and pH, the soil samples were dried at room temperature and processed according to the applicable protocol (Ehyaie & Behbahanizade 1993). Five hundred mg of soil samples were passed through 2 mm sieve and sterile distilled water was added to prepare a saturated paste. The samples were stirred briefly and allowed to equilibrate for 24h at room temperature. The pH of each saturated soil sample was then measured using a digital pH meter (744 Metrohm, Sweden). The EC of each saturated paste extract was measured using a digital EC TDS analyzer (WTW 3310 model, Germany).

 

Morphological identification of Trichoderma isolates

The preliminary identification of Trichoderma isolates was done based on morphological observation and comparison with morphological identification keys from Gams & Bissett (1998) as well as Samuels et al. (2009). Colony appearance was described on PDA at 25°C. The growth rate of colony, formation and shape of tufts or pustules, occurrence of diffusing pigment in agar plate and sporulation model were used for macroscopic observation. For microscopic criteria structure, morphology, size and shape of conidiophores, phialides, conidia and chlamydospores were measured on PDA and CMD media (corn meal agar, Merck, Germany, with 0.5% w/v dextrose) at 25°C under ambient daylight conditions during approximately one week. The following characters were measured: phialide width at the widest point, phialide length and length/width ratio (L/W), conidium length, width and length/width ratio (L/W), presence of chlamydospores and chlamydospore width. Fifty units of each character were measured for each isolate.

 

DNA extraction and RAPD PCR amplification

Each isolate was grown on 100 ml of liquid potato dextrose medium. Cultures were maintained at 25±1°C with shaking (150 rpm) for three days. Mycelial mats were harvested by filtration, washed three times with sterile distilled water and powdered with liquid nitrogen using a mortar and pestle. The powdered mycelia were kept at -20°C. Genomic DNA was extracted from the pulverized mycelium, using a modification of the cetyltrimethylammonium bromide (CTAB) extraction procedure, described by Alaei et al. (2009). A volume of 400μl of extraction buffer (1.4 M NaCl, 50mM Tris-Hcl pH 8, 0.01 M Na-EDTA, 1% β-mercaptoethanol and 2% CTAB) was added to 100 mg of each sample. The reaction mixture was briefly vortexed and incubated at 65°C in a water bath for 30 min. Then, 400 μl of chloroform: isoamyl alcohol (24:1 (vol:vol)) was added to the sample. The mixture was emulsified using a vortex and subsequently centrifuged at 7378 g for 15 min. The clear supernatant was transferred to a new tube and the nucleic acids were precipitated with 200 μl isopropanol and centrifuged at 10625 g for 5 min. The pellet was washed in 70% ethanol and re-centrifuged. Finally, the pellets were dried at room temperature, suspended in 50 μl of Tris-EDTA buffer (10mM Tris-HCl, 1mM EDTA), and stored at -20°C. The DNA concentrations were determined using Nanodrop (BioRad). The DNA was amplified with the random amplified polymorphic (RAPD) technique, using the primer A-5 (5'-AGGGGTCTTG-3') (Chakraborty et al. 2010). Amplification was carried out in a C-1000 Touch™ thermal cycler (Bio Rad, USA) in a volume of 25 µl, containing 75 ng of template genomic DNA, PCR reaction buffer (10 mM Tris-HCl, 50 mM of KCl; pH 9.0), 2.5 mM of MgCl2, 0.2 mM of dNTPs, 0.2 µM of oligonucleotide primers and 1.25 unit Taq DNA polymerase (Bioflux biotech). The amplification conditions were as described by Chakraborty et al. (2010) with some modification: an initial denaturation step of 5 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 36°C and 90 s at 72°C, and a final extension step of 10 min at 72°C. Twenty microliters of RAPD-PCR products were separated in a 1.5% agarose gel for electrophoresis with 1X TAE buffer, followed by staining with ethidium bromide and the bands were visualized under UV light. The image of the gel electrophoresis was documented through Bio-Profile Bio-1D gel documentation system. All reproducible polymorphic bands were scored and RAPD profile patterns of different isolates of Trichoderma were obtained by primer A-5.

 

Representative isolates of Trichoderma and DNA sequencing

To confirm the morphological identification of Trichoderma species, molecular identification of Trichoderma isolates was carried out based on DNA sequencing of the ITS (ITS1-5.8S-ITS2). In cases where ITS1 and ITS2 did not provide unambiguous identification, a 0.3 kb fragment of tef1-α, containing the large intron was amplified using the primer pair EF1-728F (5'-CATCGAGAAGTTCGAGAAGG-3') and EF1-986R (5'-TACTTGAAGGAACCCTTACC-3') (Druzhinina et al. 2004). Trichoderma isolates were grouped in preliminary experiments based on morphological characteristics and the data from RAPD molecular marker using the primer A-5 (Chakraborty et al. 2010). PCR amplification of the representative isolates of each group was achieved with a C-1000 Touch™ thermocycler (Bio Rad, USA). The ITS region was amplified, using the primer pair ITS1F (5'-CTTGGTCATTTAGAGGAA GTAA-3') (Gardes & Bruns 1993) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (White et al. 1990), in a final volume of 50 µl by mixing 5 µl of diluted (1:10) DNA extract (containing 85-300 ng/µl of template genomic DNA) with 0.2 µM of each of the primers, 0.2 mM of dNTPs, 2.5 mM of MgCl2, PCR reaction buffer (10 mM Tris-HCl, 50 mM KCl; pH 9.0) and 1.25 unit Taq DNA polymerase. The cycle parameters were as follows: an initial denaturation for 5 min at 95°C, followed by 40 cycles of 1 min denaturation at 94°C, 1 min primer annealing at 45°C, 1 min extension at 72°C and a final extension of 10 min at 72°C. PCR amplification of a 0.3 kb fragment of tef1-α, containing the large intron was done in a final volume of 25 µl under the same PCR conditions as described above with the exception of the annealing temperature of 49°C. Aliquots of 5 µl of PCR products were analyzed by electrophoresis in a 1.5% agarose gel and stained with ethidium bromide for visualization under UV light. The PCR products were cut from agarose gel and purified using a commercial Axyprep PCR Cleanup Kit according to the manufacturer's protocol and sequenced in both directions by Bioneer Company (Daejeon, South Korea). Sequences were analyzed using BioEdit ver. 7.1.3 (Thomas Hall, Ibis Biociences an Abbott Company) and Chromas Pro ver. 1.7.1 (Technelysium, Australia) and verified manually. The resulting consensus sequences were submitted to the BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) against the GenBank database of the National Center for Biotechnology Information (NCBI) and the TrichoBLAST interface (http://www.isth.info/tools/ blast/index.php) (Druzhinina et al. 2005).The ITS and tef1 gene nucleotide sequences determined in this study have been submitted to the GenBank database.

 

Phylogenetic analysis

DNA sequences were aligned using the multiple sequence alignment program Clustal X 1.81 (Thompson et al. 1997) and then visually adjusted. The alignment of sequence data was performed comprising complete ITS1, 5.8S and ITS2 sequences of 21 representative isolates along with the sequences of the identified species of sections Pachybasium B, Hypocreanum, Lone Lineage and Longibrachiatum obtained from Genebank, including T. harzianum (HQ259312, AF19
4009, AF194008, AF194011, AF194014, AF194019, AY605733), T. aureoviride (AF362108, AF362109, JQ040329, JQ040330), T. virens (EU280073, AF099
006, AF099008, HQ608079), T. brevicompactum (EU280087, EU280088, JQ040334, JQ040333) and T. longibrachiatum (JQ040374, JQ040373, JQ040376, EU280095). Single gaps were treated as missing data. Phylogenetic analysis was performed in MEGA 4.0 (Tamura et al. 2004). Trichoderma viride AY665699 (section Trichoderma) and T. longibrachiatum AY93
7420 were used as outgroups for ITS and tef1-α analyses, respectively. Phylogenic trees for both rDNA ITS and tef1-α sequences were generated using Maximum Parsimony (MP) and Neighbor Joining (NJ), respectively. The NJ tree was constructed using the Kimura2 parameter model. The MP analysis was performed using a heuristic search, with a starting tree obtained via step-wise addition. Stability of the clades was assessed with 1000 bootstrap replications.

 

RESULTS

 

Morphological identification

A total of 200 alkaline soil samples were collected from pistachio orchards at different geographic areas in Kerman province, Iran. Measurement of pH and EC are shown in Table1. The soils in the present study were near neutral to alkaline with a pH range of 7.0 to 8.3. The EC ranged from 1.5 to12.3 dSm–1. One hundred and sixty one isolates of Trichoderma were obtained and purified. Isolates were identified to the species level by a combination of morphological and genotypic characters. Before molecular identification, the Trichoderma isolates were grouped based on the examination of their morphology on PDA and CMD, using macroscopic as well as microscopic characteristics. Identification and origin of the 161 isolates are listed in Table 1. Five species were identified in this study: T. harzianum Rifai, Trichoderma sp., T. virens (J.H. Miller, Giddens & A.A. Foster) von Arx, T. brevicompactum (G.F. Kraus, C.P. Kubicek & W. Gams) and
T.
longibrachiatum Rifai. However, based on macroscopic and microscopic morphological criteria, 87 and 44 isolates were identified as T. harzianum and Trichoderma sp., respectively. Isolates of Trichoderma sp.were differentiated from isolates of T. harzianum with clavate to ellipsoidal or subglobose conidia, and phialides with 2 or 3–verticillate, narrow ampulliform or lageniform, dirty yellow to brownish yellow pigmentation with the development of needle shape, golden yellow crystals in agar plate and their colonies typically gave an olive green to brownish green color (Fig. 1 and Fig. 2). Nineteen isolates were identified as T. virens. Only two isolates were identified as T. longibrachiatum. Nine isolates were identified as T. brevicompactum accordingto description of Kraus et al. (2004).

 

RAPD PCR analysis

Trichoderma species were grouped based on the morphological characteristics. All of the isolates of each group were analyzed by RAPD molecular marker using the primer A-5 (Chakraborty et al. 2010). Detailed analyses of the RAPD-PCR profiles were revealed no intra-specific variability and genetic diversity among Trichoderma isolatesobtained from one soil. Even in some cases, the isolates obtained from various soil samples that were similar in morphology, had genetic similarity. The RAPD-PCR profiles analysis showed 21 different band patterns. The representative isolates of each profile were selected for DNA sequencing. 

 

DNA sequencing

PCR amplification of the rDNA ITS sequences of 21 representative isolates of Trichoderma (Table 2), using ITS1F-ITS4 primer pair was successful. Direct sequencing of the gel-purified PCR fragments from theisolates was successful and consistently produced good sequencing reads. All sequences were submitted to GenBank. Their accession numbers are listed in Table 2. The similarity search using the sequences of the isolates determined in this study showed that the majority of the sequenced strains (12 isolates of T. harzianum out of 21 repre sentative isolates) were closely similar to T. harzianum isolates in TrichOKey and GeneBank (Table 2). The ITS sequence of five isolates out of 21 representative isolates in which were identified as Trichoderma sp. based on morphological studies were closely similar to T. harzianum(CPK2656) and Trichoderma aureviridea (JQ040330) inTrichOKey and GeneBank respectively (Table 2). In fact, the rDNA ITS genomic region of T. harzianum as well as Trichoderma sp. isolates showed high sequence similarity and were identified as T. harzianum. Detailed analyses of the rDNA ITS sequences revealed no intra-isolates variability among T. harzianum and Trichoderma sp. obtained from one alkaline soil sample. All of the T. harzianum rDNA ITS fragments had only 4 bp difference in ITS1 and 7 bp difference in ITS2 (Fig. 3).On the other hand, RAPD-PCR analysis with A-5 primer confirmed that no genetic diversity was found within isolates obtained from one soil. Due to the identical ITS sequences for isolates of T. harzianum and Trichoderma sp.isolates, the final identification of these isolates were done by sequenceing of tef1 for representative isolates (Table 2). However, 34 nucleotide differences were observed between the two groups of isolates of T. harzianum and Trichoderma sp. The BLAST similarity search in TrichoBLAST using the sequences of the T. harzianum (tef1-α) revealed the similarity to H. Lixii/T.harzianum entries CBS227.95 (E-value’s: e – 146) and for Trichoderma sp.(tef1-α) was similar to H. Lixii/T.harzianum entries GJS97.264 and E-value’s of e – 152.  

 

Phylogenetic analysis

Phylogenetic analyses aimed to determine the phylogenetic position and relationship of the obtained isolates among the identified species of Trichoderma. The phylogenetic analyses were done based on Maximum Parsimony (MP) analyses. In the ITS tree, the Harzianum clade (Clade A), including T. harzianum and Trichoderma sp., the Virens clade with T. virens (Clade B), the Longibrachiatum clade

with T. longibrachiatum (Clade C) and the Lutea clade with T. brevicompactum (Clade D) were distinguished with bootstrap support of 75%. Among

the 161 isolates, 93.2% (150 isolates), 5.6% (9 isolates) and 1.2% (2 isolates) were located in Trichoderma sections Pachybasium, Lone Lineages and Longibrachiatum, respectively. The separation of these sections was supported by bootstrap values of 94 and 99%, respectively. Seventeen isolates located within Trichoderma sect. Pachybasium were grouped in a complex including T. harzianum and Trichodermasp. (Fig. 4). Five isolates out of these isolates (Ta3-90, Ta9-117, Ta1-41, Ta2-43 and Ta7-116) formed a separate subclade with a bootstrap value 79%. Seven (Th22-45, Th55-147, Th26-62, Th27-65, Th38-127, Th24-61, Th52-134) and five isolates (Th19-43, Th1-1, Th33-113, Th23-53, Th4-11) were grouped in two distinct subclades with poor resolution.

 

 

 

 

 

Fig. 1. Trichoderma sp.: a-b. Phialides, C. Chlamydospore, d-e. Conidia, f-G.  Needle shaped and yellow crystals, h. Colony on PDA after 10 days, i. Reverse colony on PDA after 10 days.

 

Fig. 2. Trichoderma harzianum: a. Phialide, b. Conidia, c. Chlamydospore, d-e. Colony on PDA after 10 days, f. Reverse colony on PDA after 10 days.

 


Table 1. Soil properties and morphological identification of strains of Trichoderma isolated at different geographical areas in Kerman province, Iran.

location

soil sample code

number of isolates

isolate code(s)

soil properties

species

pH

EC(dS.m-1)

Rafsanjan-Nogh

R 01

3

Th01:Th03

7.91

6.1

T. harzianum

Bardsir

B 11

2

Th04:Th05

7.72

7.99

T. harzianum

Rafsanjan-HematAbad

R 41

1

Ts01

-

-

Trichoderma sp.

Rafsanjan-HematAbad

R 43

15

Th06:Th20

8.07

9.1

T. harzianum

Rafsanjan-HematAbad

R 43

1

Ts02

8.07

9.1

Trichoderma sp.

Rafsanjan-RaeesAbad

R 45

2

Th 21:Th 22

7.87

4.02

T. harzianum

Rafsanjan

R 53

1

Th 23

-

-

T. harzianum

Rafsanjan-AliAbad

R 61

2

Th 24:Th 25

-

-

T. harzianum

Rafsanjan-EsmaeelAbad

R 62

1

Th 26-62

7.65

5.57

T. harzianum

Rafsanjan-EsmaeelAbad

R 65

1

Th 27-65

7.68

4.99

T. harzianum

Rafsanjan-EsmaeelAbad

R 67

3

Th28:Th30

7.99

6.72

T. harzianum

Rafsanjan-GhaderAbad

R 69

1

Th31

7.72

12.25

T. harzianum

Rafsanjan-Ahmadieh

R 77

4

Tb01:Tb04

 

 

T. brevicompactum

Anar

A90

1

Ts03

7.5

4.39

Trichoderma sp.

Sirjan

S 111

1

Th 32

8.2

2.38

T. harzianum

Sirjan

S113

2

Th 33:Th 34

8.24

3.73

T. harzianum

Sirjan

S 115

1

Ts04

7.94

12.26

Trichoderma sp.

Sirjan

S 116

3

Ts05:Ts7

8.13

2.77

Trichoderma sp.

Sirjan

S117

30

Ts08:Ts39

7.61

7.2

Trichoderma sp.

Sirjan

S 118

5

Ts38:Ts42

8.28

3.97

Trichoderma sp.

Rafsanjan-JalalAbad

R127

17

Th35:Th51

7.97

4.05

T. harzianum

Sirjan-FakhrAbad

S 132

6

Tv01:Tv06

7.83

3.59

T. virens

Sirjan-Khafriz

S 133

1

Tv07

8.3

2.41

T. virens

Sirjan- Khafriz

S 133

1

Tb05

8.3

2.41

T. brevicompactum

Sirjan-JafarAbad

S134

2

Th52: Th53

8.12

2.31

T. harzianum

Sirjan-AliAbad

S 136

2

Tb06:Tb07

7.67

2.22

T. brevicompactum

Sirjan-AliAbad

S 136

5

Tv08:Tv12

7.67

2.22

T. virens

Rafsanjan-GhavamAbad

R142

2

Ts43:Ts44

8

6.8

Trichoderma sp.

Sirjan- Khafriz

S 145

7

Tv13:Tv19

7.96

1.82

T. virens

Sirjan

S 146

2

Tb08:Tb09

8.12

1.48

T. brevicompactum

Sirjan-NosratAbad

S147

4

Th54:Th57

7.92

4.05

T. harzianum

Anar

A189

29

Th58: Th87

7.97

1.86

T. harzianum

Rafsanjan

R54

2

Tl01: Tl02

-

-

T. longibrachiatum

 

 

 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

Fig. 3. Alignment of the complete nucleotide sequence of the internal transcribed spacer (ITS1 and ITS2) region of the nuclear ribosomal RNA genes of Trichoderma harzianum and Trichoderma sp. isolates, including the 5.8S subunit. The sequences are written 5' to 3'. Identical nucleotides are indicated by dots. The ITS1 and ITS2 regions are marked with arrows.

 

Table 2. Representative Trichoderma isolates included in molecular study and their highest similarities with TrichOKey and NCBI GenBank species (for ITS rDNA and tef1)

species

strain code

accession number

GenBank strains identification/ Identities

ITS rDNA

tef1

(ITS-TrichoKEY)

(ITS-NCBI)

(tef1-TrichoBLAST)

Trichoderma sp.

Ts 1-41

KJ000318

-

H. lixii CPK2656 (100%)

T.aureoviride JQ040330

 

Trichoderma sp.

Ts 2-43

KJ000315

-

H. lixii CPK1085 (99%)

T.aureoviride JQ040329

 

Trichoderma sp.

Ts 3-90

KJ000319

-

H. lixii CPK2656 (100%)

T.aureoviride JQ040330

 

Trichoderma sp.

Ts 7-116

KJ000316

KJ206536

H. lixii CPK1085 (99%)

T.aureoviride JQ040330

T.harzianumAY605845

Trichoderma sp.

Ts 9-117

KJ000317

-

H. lixii CPK2656 (100%)

T.aureoviride JQ040330

 

Trichoderma sp.

Ts39-118

-

KJ206537

 

 

T.harzianumAY605845

T. brevicompactum

Tb 2-77

KJ000306

-

CPK723 (99%)

EU280087

 

T. harzianum

Th 1-1

KJ000311

KJ206538

CPK2649 (99%)

T.harzianum HQ259312

T.harzianumEU918165

T. harzianum

Th 4-11

KJ000312

-

CPK2649 (99%)

T.harzianum HQ259312

 

T. harzianum

Th 19-43

KJ000313

-

CPK2649 (99%)

T.harzianum HQ259312

 

T. harzianum

Th 22-45

KJ000323

-

CPK2660 (99%)

T.harzianum AY605733

 

T. harzianum

Th 23-53

KJ000310

-

CPK2649 (99%)

T.harzianum HQ259312

 

T. harzianum

Th 24-61

KJ000320

-

CPK2660 (100%)

T.harzianum AY605733

 

T. harzianum

Th 26-62

KJ000326

-

CPK1102 (100%)

T.harzianum AY605733

 

T. harzianum

Th 27-65

KJ000324

-

CPK1102 (100%)

T.harzianum AY605733

 

T. harzianum

Th 33-113

KJ000314

-

CPK2649 (99%)

T.harzianum HQ259312

 

T. harzianum

Th 38-127

KJ000321

KJ206535

CPK2660 (100%)

T.harzianum AY605733

T.harzianumAY605832

T. harzianum

Th 52-134

KJ000325

-

CPK1102 (100%)

T.harzianum AY605733

 

T. harzianum

Th 55-147

KJ000322

KJ206534

CPK2660 (100%)

T.harzianum AY605733

T.harzianumAY605832

T. longibrachiatum

Tl 1-54

KJ000309

-

CPK1692 (99%)

JQ040374

 

T. virens

Tv 3-132

KJ000307

-

CPK2941 (100%)

T.virens AF099008

 

T. virens

Tv 17-142

KJ000308

-

CPK2939 (100%)

T.virens AF099008

 

 

 


131 Trichoderma isolates were identified as
T. harzianum (H. lixii), but this species is known to include several ITS alleles (Hermosa et al. 2004, Migheli et al. 2009, Karimian et al. 2014) and is considered to be a species complex (Chaverri et al. 2003, Karimian et al. 2014). Two out of 20 strains (Tv3-132 and Tv17-142) included within Trichoderma sect. Pachybasium were grouped with T. virens which formed a separate clade with a bootstrap value of 98%, isolate Tb2-77 was grouped with T. brevicompactum with a bootstrap value of 99% and Tl1-54 was the only isolate in this study which belonged to section Longibrachiatumand was grouped with T.longibrachiatum with a bootstrap value of 99% (Fig. 4). A phylogenetic analysis based on tef1 sequences was also performed for the representative isolates of T. harzianum and Trichoderma sp. (Fig. 5).

 

 

 

 

 

 

 

 

 

 

 

Fig. 4. Phylogenetic tree of representative isolates of Trichoderma belonging to sections Pachybasium, Lone Lineages and Longibrachiatum, inferred by Maximum Parsimony analysis of ITS1, 5.8s and ITS2 sequences in MEGA 4.0. T. viride (Accession No. AY665699) was used as outgroup. The bootstrap support from 1000 replication is indicated on the branches. Isolates of related to Trichoderma sp.:Ts1-41, Ts2-43, Ts3-90, Ts7-116, Ts9-117; T. harzianum: Th19-43, Th23-53, Th1-1, Th4-11, Th33-113, Th26-62, Th52-134, Th27-65, Th22-45, Th55-147, Th38-127 and Th24-61; T. virens:Tv3-132, Tv17-142, T. brevicompactum: Tb2-77 and T. longibrachiatum: Tl1-54.

 

 

As a result, the isolates related to two species T. harzianum and Trichoderma sp.were located within three separate clades. Two isolates (Ts116 and Ts118) were identified as Trichoderma sp. based on morphological characteristics, forming a separate clade with a bootstrap value of 94%. Isolate Th1 was grouped in a distinct clade with a bootstrap value of 82% and two isolates (Th127 and Th147 which were identified as T. harzianum ) were located in separate clades that were resolved with high bootstrap support

100% (Fig 5).

The phylogenetic position of species based on tef1-α had correlation with their morphological characteristics. In fact, the isolates related to two species T. harzianum and Trichoderma sp. were differentiated based on morphological characteristics, as well as RAPD-PCR analysis using four random primers OPA4, OPA3, A-5, Pr3. Based on the results, isolates are grouped into two major clusters which shared 0.47% similarity (Mirkhani et al. unpublished).

 

 

Fig. 5. Phylogenetic tree of the Trichoderma isolates inferred by Nieghbour Joining analysis of tef1-α sequences in MEGA 4.0. The bootstrap support from 1000 replication is indicated on the branches.

 


DISCUSSION

 

Trichoderma strains have biotechnological potential as biological agents for the control of soil-borne plant pathogens and for their ability to increase root growth and development, crop productivity, resistance to abiotic stresses, and uptake and use of nutrients. However, the choice of active Trichoderma strains is important in designing the effective and safe biocontrol strategies. In fact, acidity and alkaline conditions are factors that can affect Trichoderma population, such as its presence, density, longevity, as well as production of enzymes (Kredics et al. 2003, Michel-Aceves et al. 2001, Samaniego 2008).

Regarding the above-mentioned points, we aimed to isolate and identify indigenous Trichoderma strains from alkaline soils which can be potentially used as biocontrol agents to control many plant pathogenic fungi and nematodes. The present study is a preliminary assessment of Trichoderma diversity in alkaline soils of pistachio orchards in Iran. Trichoderma isolates were identified at the species level by analysis of their morphological characters and sequence analysis of their ITS and tef1-α genomic regions as the phylogenetic markers. A low degree of biodiversity of Trichoderma isolates was found and five species were identified, including: T. harzianum, Trichoderma sp., T. virens, T. brevicompactum and T. longibrachiatum. Molecular identification of Trichoderma species confirmed their morphological identification except for Trichoderma sp.that its rDNA ITS sequences was closely resembled those described for isolates of T. harzianum. Isolates related to this two species were not differentiated by their ITS sequences, because sequences of the two species are very similar.  Although the results of genetic diversity and phylogeny studies of T. harzianum, even using ITS rDNA gene sequence analysis, was shown a complex speciation within H. lixii/
T. harzianum species group (Druzhinina et al., 2010, Karimian et al., 2014) but our data based on morphological criteria on some strains was different, so we identified them as Trichoderma sp. and need to do further investigation of gene sequences.

Based on the morphological criteria, production of the brownish yellow colony with the development of needle shape, golden yellow crystals are observed as the characteristics of Trichoderma sp.isolates,when incubated at 25°C on PDA, whereas a pale yellow colony without crystals is associated with all
T. harzianum isolates. All of the isolates assigned to T. harzianum and Trichoderma sp. grow fast at 25, 30 and 35°C on PDA. Jaklitsch (2009) reported that the anamorph strains related to H. lixii obtained from Europe grew at 35°C and had optimum growth at 30°C on all media. They had often pigment appearing like yellow crystals in the colony, but often dissolving again and unstable as well as not having a clear shape. In our study, any of the T. harzianum isolates did not produce crystals, whereas all Trichoderma sp. isolates produced needle shaped golden yellow crystal bodies which were stable in the medium.

Studies on biodiversity of Trichoderma were carried out in Russia, Siberia and Himalaya (Kullnig-Gradinger et al. 2000), South-East Asia (Kubicek et al. 2003), Austria (Wuczkowski et al. 2003), on alkaline agricultural soil in the Nile valley, Egypt (Gherbawy et al. 2004), South America (Druzhinina et al. 2005), China (Zhang et al. 2005, Sun et al. 2012), Sardinia (Migheli et al. 2009), neotropical regions such as Colombia, Mexico, Guatemala, Panama, Peru, Ecuador and Brazil (Hoyos-Carvajal et al. 2009) and Poland (Blaszczyk et al. 2011).

In Iran, more than 25 species of Trichoderma have been identified from different substrates such as soil, wood and plant material samples across the country, including Southern coast of the Caspian Sea (Nazmi Rodsari et al. 2007, Zafari et al. 2002, 2004, Naeimi et al. 2014). In comparison with these studies, the regions with alkaline soils appear to be with a relatively low biodiversity of Trichoderma, in which T. harzianum and Trichoderma sp. are the predominant taxa. In this study, T. harzianum was the most frequently isolated species (54.4%) and Trichoderma sp.(27.3%) was the second most common species. T. harzianum was the predominant taxon in many locations and habitats(Druzhinina et al. 2005, Druzhinina et al. 2010, Kubicek et al. 2003, Migheli et al. 2009, Wuczkowski et al. 2003, Zhang et al. 2005).

T. harzianum is the most commonly reported species in the genus, occurring in diverse ecosystems and ecological niches. T. harzianum sensu stricto is also a species with a broad north temperate distribution, including at least North America, Europe and Asia (Zhang et al. 2005). T. harzianum, which is commonly associated with the rhizosphere of cultivated plants, is frequently used as a biocontrol agent against phytopathogenic fungi. The predominance of T. harzianum in many different environments might be explained by its ability to assimilate a comparatively wide array of carbon substrates (Zhang et al. 2005). The concept of
T. harzianum as a genetically variable complex, comprised by one morphological species and possibly several phylogenetic species (Chaverri et al. 2003, Druzhinina et al. 2010, Gams & Bissett 1998, Karimian et al. 2014) is coherent with the adaptive range of this taxon. Another striking result from this study was that only one soil sample yielded two isolates as T. longibrachiatum. According to its phylogenetic position, it is unlikely that this is due to the use of biased cultivation conditions, because this method readily isolated T. longibrachiatum and T. citrinoviride from soils of India, eastern USA and Iran (Kullnig-Gradinger et al. 2000, Zafari et al. 2002). Furthermore, the basic medium used in these studies can be used to grow virtually all recognized Trichoderma species. It is, therefore, concluded that members of this section are in low abundance or almost absent in the soils investigated.

Trichoderma strains were expected to be found in more than 50% of the sampled plots, because of their cosmopolitan character and the fact that it is a natural inhabitant of soils, but Trichoderma strains were found in just 15% of the plots (30 samples from 200 soil samples). These results correspond to those reported by Campos et al. (2012), in which Trichoderma strains were found in just 22% of the sampled sites. However, they differ from the results reported by Michel-Aceves et al. (2001), that native strains of Trichoderma were found in 88% of the sampled sites. Moreover, in the research conducted by Kubicek et al. (2003), strains of Trichoderma from all soil samples were separated. The results show that soils of pistachio orchards of Kerman province are not rich in terms of number of isolates and species diversity of Trichoderma. Furthermore, strains of this important genus are not compatible with soil ecological conditions of pistachio orchards. In fact, the environmental parameters, such as soil temperature, moisture, pH, organic matter (OM), nutrient content which affect the growth and proliferation of fungal genus and plant types are key factors  affecting soil colonization by Trichoderma species (Gherbawy et al. 2004).

Soil acidity is one of the important factors for the establishment of Trichoderma species. Thus, the reason for this low degree of biodiversity of Trichoderma in the soil of pistachio orchards may be related to its alkaline pH value. Danielson & Davey (1973) stressed that the soil pH is one of the most critical parameters for Trichoderma propagation. Kredics et al. (2003) reported that species of Trichoderma grow optimally at around pH 4.0-5.0, and exhibit little or no growth below pH 2.0 and above 6.0. Acidity is a factor which affects presence, density and longevity of this fungal genus (Michel-Aceves et al. 2001). Okoth et al. (2007) and Campus et al. (2012) reported that Trichoderma is abundant in acid soils. Our results therefore showed that Trichoderma could also be isolated from such adverse habitats, but with a lower frequency than the other soils, while it is possible that the five species found have a general tolerance to high pH and EC. On the other hand, we could not detect any correlation between pH and EC of the soil and the Trichoderma species recovered. Correlation between any of these characters and the five taxa were essentially random, and it is thus believed that the populations of Trichoderma species detected in this study are generally indigenous components of the soil in pistachio orchards in Iran. The results of the present study correspond to the findings reported by Gherbawy et al. (2004), that only T. harzianum and the anamorph of Hypocrea orientalis were found in the soils of Nile valley in Egypt.

Present results confirm the ecological plasticity of genus Trichoderma, as pointed out by Samuels (2006) and Infante et al. (2009). Although Trichoderma species could be found in all altitudes and all types of soils, but geographic distribution of Trichoderma species are quite different. Some species such as
T. pseudokoningii and T. harzianum are broadly spread, while others like T. viride have a limited geographic distribution and are not commonly found in the colder northern regions. Another example is
T. aureoviride, whose distribution is limited to the United Kingdom and northern Europe (Samuels 2006). Thus, the set of factors such as soil moisture, temperature, texture and structure, organic matter (OM), nutrient content, plants type, specie type and the existence of other organisms in soil will affect the adaptation of a species in a region. In fact, the soil is a very complex environment and it is generally the interaction of several factors which can affect the number of soil microorganisms, diversity or activity (Bourguignon 2008).

Alaei H, Baeyen S, Maes M, Hofte M, Heungens K. 2009. Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR. Journal of Microbiological Methods 76: 136-145. doi: 10.1016/j.mimet.2008.10.001.

Bailey BA, Bae H, Strem MD, Roberts DP, Thomas SE, Crozier J, Samuels GJ, Choi IY, Holmes KA. 2006. Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species. Planta 224: 1449-1464.

Bissett J. 1984. A revision of the genus Trichoderma. I. Section Longibrachiatum sect. nov. Canadian Journal of Botany 62: 924-931. doi: 10.1139/b84-131.

Bissett J. 1991a. A revision of the genus Trichoderma. II. Infrageneric classification. Canadian Journal of Botany 69: 2357-2372.

Bissett J. 1991b. A revision of the genus Trichoderma. III. Section Pachybasium. Canadian Journal of Botany 69: 2373- 2417. doi: 10.1139/b91-298.

Bissett J. 1992. Trichoderma atroviride. Canadian Journal of Botany 70: 639-641. doi: 10.1139/b92-082.

Bissett J, Szakacs G, Nolan CA, Druzhinina I, Gradinger C, Kubicek CP. 2003. New species of Trichoderma from Asia. Canadian Journal of Botany 81: 570-586.

Błaszczyk L, Popiel D, Chełkowski J, Koczyk G, Samuels GJ, Sobieralski K, Siwulski M. 2011. Species diversity of Trichodermain Poland. Applied Genetics 52: 233-243. doi: 10.1007/s13353-011-0039-z.

Bourguignon E. 2008. Ecology and diversity of indigenous Trichoderma species in vegetable cropping systems. PhD Thesis, Lincoln University Canterbury, New Zealand.

Campos ARM. 2012. Isolation native strain of Trichoderma spp from soilhorticulture tiluca valley with potentail of biocontrol on  Sclerotinia. Tropical and Subtropical Agroecosystem 15: 357-365.

Chakraborty BN, Chakraborty U, Saha A, Dey PL, Sunar K. 2010. Molecular characterization of Trichoderma viride and Trichoderma harzianum isolated from soils of North Bengal based on rDNA markers and analysis of their PCR-RAPD profiles. Global Journal of Biotechnology and Biochemistry 5: 55-61.

Chaverri P, Castlebury LA, Samuels GJ, Geiser DM. 2003. Multilocus phylogenetic structure within the  Trichoderma harzianum/Hypocrea lixii  complex. Molecular Phylogenetics and Evolution 27: 302-313.

Danielson RM, Davey CB. 1973. The abundance of Trichoderma propagules and the distribution of species in forest soils. Soil Biology and Biochemistry 5: 485-494. doi: 10.1016/0038-0717(73)90038-2.

De Respinis S, Vogel G, Benagli C, Tonolla M, Petrini O, Samuels GJ. 2010. MALDI-TOF MS of Trichoderma: A model system for the identification of microfungi. Mycological Progress 9: 79-100. doi:10.1007/s11557-009-0621-5.

Degenkolb T, Dieckmann R, Nielsen KF, Gräfenhan T, Theis C, Zafari D, Chaverri P, Ismaiel A, Brückner H, von Döhren H. 2008. The Trichoderma brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins. Mycological Progress 7: 177-219. doi: 10.1007/s11557-008-0563-3.

Druzhinina I, Kubicek CP. 2005. Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters? Journal of Zhejiang University Science B 6: 100-112. doi: 10.1631/jzus.2005.B0100.

Druzhinina IS, Chaverri P, Fallah P, Kubicek CP, Samuels GJ. 2004. Hypocrea flaviconidia, a new species from Costa Rica with yellow conidia. Studies in Mycology 50: 401-407.

Druzhinina IS, Kopchinskiy AG, Komon M, Bissett J, Szakacs G, Kubicek CP. 2005. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal Genetics and Biology 42: 813-828.

Druzhinina IS, Kubicek CP, Komoń-Zelazowska M, Mulaw TB, Bissett J. 2010. The Trichoderma harzianum demon: complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages. BMC Evolutionary Biology 10: 94- 105.

Elad Y, Chet I. 1983. Improved selective media for isolation of Trichoderma spp. or Fusarium spp. Phytoparasitica 11: 55-58. doi: 10.1007/bf02980712.

Ehyaie A, Behbahanizade AA. 1993. The methods of soil chemical analysis. Soil and Water Research Institute, Technical Bulletin, No. 893.

Ershad D. 2009. Fungi of Iran. Iranian Research Institute of Plant Protection, Tehran, Iran. 531 pp.

Gams W, Bissett J. 1998. Morphology and identification of Trichoderma. In: Trichoderma and Gliocladium, Basic Biology, Taxonomy and Genetics. (CP Kubicek & GE. Harman, eds): 3-34. Taylor and Francis, London.

Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts.  Molecular Ecology 2: 113-118. doi: 10.1111/j.1365-294X.1993.tb00005.x.

Gautheret A, Dromer F, Bourhis JH, Andremont A. 1995. Trichoderma pseudokoningii as a cause of fatal infection in a bone marrow transplant recipient. Clinical Infectious Diseases 20: 1063-1064. doi: 10.1093/clinids/20.4.1063.

Gherbawy Y, Druzhinina I, Shaban GM, Wuczkowsky M, Yaser M, El-Naghy MA, Prillinger HJ, Kubicek CP. 2004. Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species. Mycological Progress 3: 211-218.

Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. 2004. Trichoderma species-opportunistic, avirulent plant symbionts. Nature Review Microbiology 2: 43-56. doi:10.1038/nrmicro797.

Hatvani L, Antal Z, Manczinger L, Szekeres A, Druzhinina IS, Kubicek CP, Nagy A, Nagy E, Vagvolgyi C, Kredics L. 2007. Green mold diseases of Agaricus and Pleurotus spp. are caused by related but phylogenetically different Trichoderma species. Phytopathology 97: 532-537. doi: org/10.
1094/PHYTO-97-4-0532.

Hermosa MR, Keck E, Chamorro I, Rubio B, Sanz L, Vizcaino JA, Grondona I, Monte E. 2004. Genetic diversity shown in Trichoderma biocontrol isolates. Mycological Research 108: 897-906. doi: 10.1017/S0953756204000358.

Hjeljord LG, Tronsmo A. 1998. Trichoderma and Gliocladium in biological control: an overview. In: Trichoderma and Gliocladium, vol. 2, enzymes, biological control and commercial application. (GE Harman & CP Kubicek, eds): 131-151.Taylor and Francis, London. 

Hoyos-Carvajal L, Orduz S, Bissett J. 2009. Genetic and metabolic biodiversity of Trichoderma from Colombia and adjacent neotropic regions. Fungal Genetics and Biology 46: 615-631. doi: 10.1016/j.fgb.2009.04.006.

Humphris SN, Bruce A, Buultjens E, Wheatly RE. 2002. The effects of volatile microbial secondary metabolites on protein synthesis in Serpula lacrymans. FEMS Microbiology Letters 210: 215-219. doi: 10.1111/j.1574-6968.2002.tb11183.x.

Infante D, Martinez B, González N, Reyes Y. 2009. Mecanismos de accion de Trichoderma frente a hongos fitopatogenos. Revista de Proteccion Vegetal 24: 14-21.

Johnson LF. 1959. Methods for Studying Soil Microflora Plant Disease Relationships. Burgess Publishing Company, Minneapolis.

Jaklitsch WM. 2009. European species of Hypocrea part I. the green-spored species. Studies in Mycology63: 1-91. doi: 10.3114/sim.2009.63.01.

Karimian B, Javan-Nikkhah M, Zafari D, Fotouhifar Kh –B, Naeimi S. 2014. Phylogenetic structure of Trichoderma harzianum s. l. from some climates of Iran. Iranian Journal of Plant Protection Science 45: 59-69.

Kraus GF, Druzhinina I, Gams W, Bissett J, Zafari D, Szakacs G, Koptchinski A, Prillinger H, Zare R, Kubicek CP. 2004. Trichoderma brevicompactum sp. nov. Mycologia 96: 1059-1073.

Kredics L, Antal Z, Manczinger L, Szekeres A, Kevei F, Nagy E. 2003. Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Technology and Biotechnology 41: 37-42.

Krupke OA, Castle AJ, Rinker DL. 2003. The North American mushroom competitor, Trichoderma aggressivum f. aggressivum, produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroonm Agaricus bisporus. Mycological Research 107: 1467-1475.

Kubicek CP, Bissett J, Druzhinina I, Kullnig-Gradinger C, Szakacs G. 2003. Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates. Fungal Genetics and Biology 38: 310-319. doi: 10.1016/s1087-1845(02)00583-2.

Kubicek CP, Komon-Zelazowska M, Druzhinina IS. 2008. Fungal genus Hypocrea/Trichoderma: from barcodes to biodiversity. Journal of Zhejiang University Science B 9: 753-763. doi: 10.1631/
jzus.B0860015

Kubicek CP, Penttila ME. 1998. Regulation of production of plant polysaccharide degrading enzymes by Trichoderma. In: Trichoderma and Gliocladium, Basic Biology, Taxonomy and Genetics. (GE Harman & CP Kubicek, eds): 49-71.Taylor and Francis, London.

Kullnig-Gradinger CM, Szakacs G, Kubicek CP. 2002. Phylogeny and evolution of the genus  Trichoderma: a multigene approach. Mycological Research 106: 757-767. doi: 10.1017/S0953756202006172.

Kullnig-Gradinger CM, Szakacs G, Kubicek CP. 2000. Molecular identification of Trichoderma species from Russia, Siberia and the Himalaya. Mycological Research 104: 1117-1125.

Kullnig-Gradinger CM, Krupica T, Woo SL, Mach RL, Rey M, Benitez T, Lorito M, Kubicek CP. 2001. Confusion abounds over identities of Trichoderma biocontrol isolates. Mycological Research 105: 769-772.

Lieckfeldt E, Kullnig CM, Kubicek CP, Samuels GJ, Börner T. 2001. Trichoderma aureoviride: phylo-genetic position and characterization. Mycological Research 105: 313-322.

Liu R, Gu QQ, Zhu WM, Cui CB, Fan GT. 2005. Trichodermamide A and aspergillazine A, two cytotoxic modified dipeptides from a marinederived fungus Spicaria elegans. Archives of Pharmacal Research 28: 1042-1046. doi: 10.1007/BF02977399.

Lu B, Druzhinina IS, Fallah P, Chaverri P., Gradinger C, Kubicek CP, Samuels GJ. 2004. Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T. minutisporum and T. polysporum and their newly discovered relatives. Mycologia 96: 310-342.

Michel-Aceves A, Rebolledo-Dominguez O, Lezama-Gutierrez R, Ochoa-Moreno ME, Samuels GJ. 2001. Especies de Trichoderma en suelos cultivados con mangoafectador por Escoba de bruja y su potencial inhibitorio sobre Fusarium oxysporum y F. subglutinans. Revista Mexicana de Fitopatologia 19: 154-160.

Migheli Q, Balmas V, Komoñ‐Zelazowska M, Scherm B, Fiori S, Kopchinskiy AG, Kubicek CP, Druzhinina IS. 2009. Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/ Trichoderma. Environmental Microbiology 11: 35-46. doi: 10.1111/j.1462-2920.2008.01736.x.

Naeimi S, Khodaparast SA, Javan Nikkhah M, Vagvolgyi Cs, Kredics L. 2014. Phylogenetic relationship of Trichoderma isolates from paddy fields of Mazandaran province based on sequence analysis of tef1 geneIranian journal of Plant Pathology 50: 65-68.

Nazmi Roudsari F, Zafari D, Khodaparast SA, Rouhani H. 2007. Introducing some new species of Trichoderma for Iran. Rostaniha 8: 67-83. 

Okoth SA, Roimen H, Mutsotso B, Muya E, Kahindi J, Owino JO, Okoth P. 2007. Land use systems and distribution of Trichoderma species in Embu region, Kenya tropical and subtropical  agroecosy-
stems. Tropical and Subtropical Agroecosystem 7: 105-122.

Rifai MA. 1969. A revision of the genus Trichoderma. Mycological Papers 116: 1-116.

Samaniego RM. 2008. Entry, exit and business cycles in a general equilibrium model. Review of Economic Dynamics11: 529–541.

Samuels GJ. 2006. Trichoderma: Systematics, the Sexual State, and Ecology. Phytopathology 96: 195-206. doi: 10.1094/phyto-96-0195.

Samuels GJ, Chaverri P, Farr DF, McCray EB. 2009. Trichoderma online. Systematic Mycology and Microbiology Laboratory. Home page at <http://nt.arsgrin.gov/taxadescriptions/keys/TrichodermaIndex.cfm>.

Samuels GJ, Dodd SL, Gams W, Castlebury LA, Petrini O. 2002. Trichoderma species associated with the green mold epidemic of commercially grown Agaricus bisporus. Mycologia 94: 146-170.

Samuels GJ, Ismaiel A, Bon MC, De Respinis S, Petrini O. 2010. Trichoderma asperellum sensu lato consists of two cryptic species. Mycologia 102: 944-966. doi: 10.3852/09-243.

Sun RY, Liu ZC, Fu K, Fan L. 2012. Trichoderma biodiversity in China. Applied Genetics 53: 343-354. doi: 10.1007/s13353.

Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbour joining method. Proceedings of the National Academy Sciences USA 101: 11030-11035.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882. doi: 10.1093/nar/25.24.4876.

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications. (MA Innis, DH Gelfand, JJ Shinsky & TJ White, eds): 315-322. Academic Press, San Diego.

Wuczkowski M, Druzhinina I, Gherbawy Y, Klug B, Prillinger H, Kubicek CP. 2003. Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiol Research 158: 125-133. doi: 10.1078/0944-5013-00193.

Xia X, Lie TK, Qian X, Zheng Z, Huang Y, Shen Y. 2011. Species diversity, distribution, and genetic structure of endophytic and epiphytic Trichoderma associated with banana roots. Microbial Ecology 61: 619-625. doi: 10.1007/s00248-010-9770-y.

Zafari D, Ershad D, Zare R, Alizadeh A. 2002. A contribution to the identification of Trichoderma species in Iran. Iranian Journal of Plant Pathology 38: 21-45.

Zafari D, Ershad D, Zare R, Alizadeh A. 2004. Three new species of Trichoderma for the mycoflora of Iran. Rostaniha 5: 159-170.

Zhang C, Druzhinina IS, Kubicek CP, Xu T. 2005. Trichoderma biodiversity in China: evidence for a North to South distribution of species in East Asia. FEMS Microbiology Letters 251: 251-257. doi:10.
1016/j.femsle.2005.08.034.