Species diversity of indigenous Trichoderma from alkaline pistachio soils in Iran

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 MgCl_2, 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.

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)

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.