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Molecular Identification of Trichoderma Isolated from the Rhizosphere of Cacao (Theobroma cacao L.) in Central Sulawesi, Indonesia

R
Ratnawati1
K
Kasman Jaya1
A
Arfan2
S
Sri Sudewi1,2,*
0000-0001-8562-5859
1Graduate Program of Agricultural Science, Postgraduate Program, University of Alkhairaat, Palu 94223, Central Sulawesi, Indonesia.
2Research Center for Food Crops, National Research and Innovation Agency (BRIN), Bogor 16911, West Java, Indonesia.

ABSTRACT

Background: Morphology-based identification methods, especially in cocoa, often have limitations, especially in similar overlapping symptoms. DNA-based molecular identification is required to ascertain effective fungal biocontrol species. This study aimed to identify and characterize Trichoderma isolates from the rhizosphere of cacao from three different altitudes in Central Sulawesi using a molecular DNA barcoding approach.

Methods: The method used in this study consists of various stages, namely sampling, isolation, purification of Trichoderma, DNA extraction, DNA amplification, electrophoresis, sequencing and phylogenetic analysis. Quick-DNA Magbead Plus Kit (Zymo Research, D4082) was used for genomic DNA extraction, primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCT TATTGATATGC-3') were used for PCR amplification and DNA sequence analysis using Basic Local Alignment and Search Tool (BLAST) on NCBI database.

Result: The results obtained showed that the amplified DNA electrophoresis had high quality with thick and clear bands with a size range of 575-578 bp. The five Trichoderma isolates based on sequencing analysis were identified as Trichoderma asperellum with different strains, namely T. asperellum strain NG125, T. asperellum strain NECC30406, T. asperellum strain IIPR-81, T. asperellum strain BHU203 and T. asperellum strain WZ-184, with a similarity rate (query cover and identity percentage) of 100%. The results of this study enrich the sequence data of Trichoderma asperellum in NCBI and can be a reference for the development of Trichoderma-based biological agents.

INTRODUCTION

Cocoa production in Indonesia, especially in Central Sulawesi, plays an important role in supporting the national economy. Central Sulawesi is known as one of the main cocoa producing regions, contributing the most to the total cocoa production in Indonesia. Favorable climatic conditions and fertile soil create an ideal environment for cocoa cultivation (Amponsah-Doku  et al., 2022).
       
Pest and disease attacks often cause declining cocoa productivity in this region (Delgado-Ospina  et al., 2021; Ratnawati et al., 2024). Similarly, the biocontrol agents inhabiting the cacoa plant also differs in their potential of biocontrol ability. Characterization of efficient Trichoderma species to be used as biocontrol agents is an eco-friendly approach. 
       
The morphological identification process alone is not enough to accurately prove a species (Mirta and Rasyid, 2023). Central Sulawesi, with its diverse agroecosystems, is a strategic area to explore Trichoderma diversity from the cocoa rhizosphere. Furthermore Khatun  et al. (2021) and Elita et al., (2022) observed that conventional methods of Trichoderma identification that rely on morphological characteristics are often difficult to distinguish species that have similar appearances in their morphology.
       
The approach through DNA sequence analysis of the Internal Transcribed Spacer and Translation Elongation Factor (TEF-1α) genes has now developed into the most reliable and accurate method used by various global researchers. Molecular-based identification methods have evolved into a more accurate and reliable approach. One such method involves DNA sequence analysis of the Internal Transcribed Spacer (ITS) and Translation Elongation Factor (TEF-1α) genes (Pakshir et al., 2020; Salehi et al., 2020; Hussein and Saadullah, 2023; Weaver et al., 2024). Genetic variation of Trichoderma using molecular approaches can be revealed with more depth and clarity, thus allowing for the identification of species and strains that are potentially superior as biocontrol agents.
       
The genetic diversity of Trichoderma isolated from the rhizosphere of cocoa plants has significant implications for the management of biological resources. These strains show certain advantages such as their ability to produce specific enzymes or bioactive compounds, which can be used as basic ingredients in the development of biofungicide and biofertilizer products (Abdul-Halim  et al., 2023; Xiao et al., 2023). This study aims to identify Trichoderma species isolated from the rhizosphere of cocoa plants at several different altitude locations in Central Sulawesi. It’s hoped that this research can provide in-depth insight into the potential of Trichoderma strains as biocontrol agents and biofertilizers and support effective management of local microorganisms that contribute positively to sustainable cocoa cultivation in Central Sulawesi.

MATERIALS AND METHODS

Location and time of research
 
This research was conducted in Sigi Regency, Central Sulawesi, on cocoa farms representing three different altitudes: lowland in Labuan Subdistrict, Donggala Regency 50 m asl (0.650627°, 119. 86215°), medium altitude in Sigi Biromaru Subdistrict of Sigi Regency 200 masl (-1.091568°, 119.906393°) and high altitude in Palolo Subdistrict of Sigi Regency 1,106 masl (-1.132666°, 120.037956°) Central Sulawesi Province, Indonesia (Fig 1). Trichoderma isolation was carried out at the Microbiology Laboratory of Alkhairaat University Palu Central Sulawesi Indonesia, while molecular identification was carried out at the Molecular Biotechnology Laboratory, Tangerang, Indonesia. This research lasted for 3 months, from July to September 2024.

Fig 1: DNA amplification results of Trichoderma fungus isolates using ITS-1 and ITS-4 primers.


 
Sampling, isolation, purification of Trichoderma
 
Soil samples from the cocoa rhizosphere were taken using the Stratified Random Sampling method (Susila et al., 2023). For each location, three replicate samples weighing 300 grams each were taken from the soil around the cocoa root at a depth of 20 cm (Ratnawati et al., 2022; Sudewi et al., 2024). Subsequently, soil samples were stored in sterile plastic clips and brought to the laboratory for fungal isolation. The plate culture technique and serial dilution method on Potato Dextrose Agar medium (enriched with chloramphenicol (100 mg/L) were applied to prevent contamination from bacteria. The diluted rhizosphere samples were placed into petri dishes containing PDA media and incubated for 5-7 days at 28°C. Trichoderma spp colonies with specific morphological characteristics having whitish green hyphae were purified on new PDA media.
       
A total of 5 Trichoderma isolates obtained from the screening were then cultured on PDA media and purified. Seven days, old purified isolate diluted to a density of 10 v  CFU mL-1 were plated onto PDA medium observed for 7 morphological characters such as color, elevation, edges (margins) of mycelium, coniodophores and conidia and growth rate of colonies for seven days (Widiati et al., 2022).
 
DNA extraction
 
Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, D6005) was used for Trichoderma spp genomic DNA extraction according to standard procedures. Bashing BeadTM buffer and bead-beating were used to lyse 50 mg fungal samples to break down their cell walls. After centrifugation, it was mixed with fungal DNA binder and loaded onto a Zymo-Spin™ II purification column (Gand et al., 2023; Li et al., 2022). Additionally, the DNA was washed twice to remove contaminants, after which the extracted product was stored at 200°C for use in further analysis (Dilhari et al., 2017).
 
DNA amplification and electrophoresis
 
Trichoderma fungi were identified through DNA amplification using 2X MyTaq HS red mix (Bioline, BIO-25048) using primers ITS-1 (5'-TCCGTAGG TGAAC CTGCGG - 3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC - 3') (Sukapiring et al., 2024). The DNA extract was used as a template in PCR. To obtain a total volume of 25 µL, mixed 12.5 µL MyTaq HS Red Mix (2X), 1 µL forward primer, 1 µL reverse primer, 1 µL template DNA and 9.5 µL nuclease-free water. Initial denaturation (1 cycle) for 1 minute at 95°C, followed by 35 cycles of denaturation at 95°C for 10 seconds and at 72°C for 5 minutes for final extension was done.
       
Electrophoresis was done at 75 volts for ± 90 minutes, then visualized with a UV Transilluminator and documented using a digital camera. PCR results were then confirmed through 0.8% agarose gel electrophoresis in 1X TBE buffer. Agarose gel solution of 50 mL was heated until dissolved, cooled and then add GelRed as a DNA dye (Lee et al., 2012). The results obtained were compared with marker DNA to confirm the success of amplification. The names and sample codes of fungal isolates are presented in Table 1.
 
Sequencing and phylogenetic analysis
 
Sequencing uses the two-way Sanger Capillary Electrophoresis method with several steps. Previous PCR results in the form of DNA chain samples were analyzed using Bio-edit software then aligned with ClustaIW to compare with DNA chain sequences available in the Gene Bank database through blast on the web https://www.ncbi.nlm.nih.gov/ which was accessed on August 26, 2024. Phylogenetic tree analysis constructed with 1000 bootstraps using MEGA 10 software combined with the NCBI Blast Tree neighbor joining (NJ) approach (An et al., 2022).

RESULTS AND DISCUSSION

DNA amplification and sequencing
 
The electrophoresis showed that the DNA obtained from PCR with ITS-1 and ITS-4 primers was of good quality as characterized clear visible bands on the electrophoregram for the five isolated samples (Fig 2). Leite et al., (2021) reported that ITS primers are able to amplify DNA optimally, making species identification easier. Mustafa and Rostam, (2021) suggested that fungal isolates that successfully amplify the ITS zone well are characterized by thick and sharp amplification results (Fig 1).

Fig 2: Phylogenetic tree based on DNA barcoding with ITS locus from cocoa rhizosphere fungi isolates (N05, A03, SA04, RAN16, NK3).


       
Based on the results of DNA amplification of Trichoderma fungus isolates with sample codes N05, A03, SA04, RAN16 and NK3 from 3 different altitudes showed very clear DNA bands with sizes of 575 bp, 578 bp, 577 bp, 576 bp and 577 bp respectively. DNA amplification of the 5 isolates using ITS-11 and ITS-4 primers, resulted in obtained amplicons in the range of 500-600 bp (Op De Beeck  et al., 2014). The DNA amplification looked very clear, stuck optimally and had, thick lines without the effect of shadows.
       
The sequence results of the fragments of Trichoderma fungus isolates based on the NCBI nucleotide database in Table 1 showed that the five fungal isolates were identified as Trichoderma asperellum strains (NG125, NECC30406, IIPR-81, BHU203 and WZ-184) with a similarity identity percentage and query cover of 100%. The ratio indicated the degree of similarity between sample DNA sequences and target DNA sequences as percentage identity (Weaver et al., 2024; Salini and Dhandapani, 2021). This result can be attributed to many factors, one of which is the conservative nature of the ITS (Internal Transcribed Spacer) gene. ITS is commonly used to identify fungi due to low variation within the same species (Bradshaw et al., 2023). The isolates, although originated from different strains, may have a small degree of genetic variation within the amplified ITS region, however identified as the same species with a high percentage of identity.

Table 1: Summary of sequence results from fragments of Trichoderma fungus isolates based on NCBI nucleotide database.


       
Another factor for getting 100% similarity could be the use of reference databases such as NCBI BLAST, which had many T. asperellum entries from various strains. This is likely since the isolates obtained from the same place/having a close evolutionary relationship so that genetic differences between strains are not large enough to affect identification results. The length and quality of DNA sequences can also help the matching with the database to be more accurate (Abedulridha and Al-Shamery, 2018). The similarity of the five fungal isolates belongeds to the same species, although differences between strains can only be found through further genetic analysis, using methods such as multilocus sequence typing (MLST) or whole genome sequencing.
 
Phylogenetic analysis
 
The know the relationship between the isolates the phylogenetic tree analysis of the five Trichoderma fungus isolates was done based on the NCBI database Fig 2. The phylogenetic tree analysis indicates that Trichoderma isolate 1 (N05) belongeds to the same cluster as several T. asperellum species and other related species. The sequence identity percentage reaching 100% strengthens the hypothesis that this isolate has a close genetic relationship with T. asperellum. This suggests that the isolate may share similar biological characteristics with species in its group, such as functioning as a biocontrol agent or playing a crucial role in soil ecosystems (Jaya et al., 2023) as reflected by its position in the phylogenetic tree (Malik and Verma, 2025). These findings reveal that Trichoderma isolate 2 (A03) is related to the T. asperellum species, known as a plant growth-promoting fungus and biocontrol agent. Similar findings were reported by Budi et al., (2023), T. asperellum, isolated from oil palm plantations, was found to inhibit Ganoderma boninense growth by 93.9% in a dual culture assay. Additionally, Youassi et al., (2024) reported that T. asperellum extract effectively inhibited the growth of Phytophthora, the cacao pod rot disease. All these findings confirm that T. asperellum has the potential to be used as a biocontrol agent, biofumog, or biopesticide.
       
The Trichoderma isolates 3 (SA04) and 4 (RAN16) were phylogenetically highly related with T. asperellum, T. atrovirides and other Trichoderma species (Baltzis et al., 2025). The similarity of the tested isolates to sequence data stored in the NCBI database confirms that molecular identification using ITS sequences will produce strain data with a high degree of accuracy and clarity (Pere et al., 2024; Kanthiya et al., 2025). The bootstrap with green dots indicates a high level of validity for the species identified  (Lemoine  et al., 2018), confirming the reliability of the molecular identification method in determining phylogenetic relationships between test isolates.
       
The same cluster is also seen in the Trichoderma 5 (NK3) isolate in Fig 2, which appears to be related to the T. asperellum isolate. The confidence level is indicated by the branches generated with green bootstrap dots. Meanwhile, the NK3 isolate is closely related to the T. asperellum strains WZ-186 and NG125, as seen in the phylogenetic tree structure, both of which are known to have similar potential as biocontrol agents.

CONCLUSION

Five isolates of the Trichoderma fungus (N05, A03, SA04, RAN16 and NK3) originating from the rhizosphere of cocoa plants at 3 different altitudes showed good amplification quality, a clear DNA band with a size of 575-578 bp based on the results of DNA electrophoresis. The use of primers ITS-1 and ITS-4 is recommended as good molecular markers for phylogenetic analysis and species recognition. Based on DNA sequence analysis supported by matching results to the NCBI nucleotide database with query cover of 100%, the five Trichoderma fungus isolates were identified as T. asperellum with different strains.
       
Phylogenetic analysis based on ITS sequences shows that the genetic closeness of these isolates has possibly the same biological and ecological characteristics. Strain variation within the same species can be caused by selection pressures, environmental factors and geographic conditions. Therefore, further research is needed to determine the physiological properties and ability of each strain to be used in various biotechnological applications, as a biocontrol agent or biofertilizer to promote plant growth.

ACKNOWLEDGEMENT

The present study was supported by Kementerian Pendidikan Tinggi, Sains dan Teknologi (Kemdiktisaintek) for funding under Grant No. 118/E5/PG.02.00PL/2024.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Molecular Identification of Trichoderma Isolated from the Rhizosphere of Cacao (Theobroma cacao L.) in Central Sulawesi, Indonesia

    R
    Ratnawati1
    K
    Kasman Jaya1
    A
    Arfan2
    S
    Sri Sudewi1,2,*
    0000-0001-8562-5859
    1Graduate Program of Agricultural Science, Postgraduate Program, University of Alkhairaat, Palu 94223, Central Sulawesi, Indonesia.
    2Research Center for Food Crops, National Research and Innovation Agency (BRIN), Bogor 16911, West Java, Indonesia.

    ABSTRACT

    Background: Morphology-based identification methods, especially in cocoa, often have limitations, especially in similar overlapping symptoms. DNA-based molecular identification is required to ascertain effective fungal biocontrol species. This study aimed to identify and characterize Trichoderma isolates from the rhizosphere of cacao from three different altitudes in Central Sulawesi using a molecular DNA barcoding approach.

    Methods: The method used in this study consists of various stages, namely sampling, isolation, purification of Trichoderma, DNA extraction, DNA amplification, electrophoresis, sequencing and phylogenetic analysis. Quick-DNA Magbead Plus Kit (Zymo Research, D4082) was used for genomic DNA extraction, primers ITS-1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS-4 (5'-TCCTCCGCT TATTGATATGC-3') were used for PCR amplification and DNA sequence analysis using Basic Local Alignment and Search Tool (BLAST) on NCBI database.

    Result: The results obtained showed that the amplified DNA electrophoresis had high quality with thick and clear bands with a size range of 575-578 bp. The five Trichoderma isolates based on sequencing analysis were identified as Trichoderma asperellum with different strains, namely T. asperellum strain NG125, T. asperellum strain NECC30406, T. asperellum strain IIPR-81, T. asperellum strain BHU203 and T. asperellum strain WZ-184, with a similarity rate (query cover and identity percentage) of 100%. The results of this study enrich the sequence data of Trichoderma asperellum in NCBI and can be a reference for the development of Trichoderma-based biological agents.

    INTRODUCTION

    Cocoa production in Indonesia, especially in Central Sulawesi, plays an important role in supporting the national economy. Central Sulawesi is known as one of the main cocoa producing regions, contributing the most to the total cocoa production in Indonesia. Favorable climatic conditions and fertile soil create an ideal environment for cocoa cultivation (Amponsah-Doku  et al., 2022).
           
    Pest and disease attacks often cause declining cocoa productivity in this region (Delgado-Ospina  et al., 2021; Ratnawati et al., 2024). Similarly, the biocontrol agents inhabiting the cacoa plant also differs in their potential of biocontrol ability. Characterization of efficient Trichoderma species to be used as biocontrol agents is an eco-friendly approach. 
           
    The morphological identification process alone is not enough to accurately prove a species (Mirta and Rasyid, 2023). Central Sulawesi, with its diverse agroecosystems, is a strategic area to explore Trichoderma diversity from the cocoa rhizosphere. Furthermore Khatun  et al. (2021) and Elita et al., (2022) observed that conventional methods of Trichoderma identification that rely on morphological characteristics are often difficult to distinguish species that have similar appearances in their morphology.
           
    The approach through DNA sequence analysis of the Internal Transcribed Spacer and Translation Elongation Factor (TEF-1α) genes has now developed into the most reliable and accurate method used by various global researchers. Molecular-based identification methods have evolved into a more accurate and reliable approach. One such method involves DNA sequence analysis of the Internal Transcribed Spacer (ITS) and Translation Elongation Factor (TEF-1α) genes (Pakshir et al., 2020; Salehi et al., 2020; Hussein and Saadullah, 2023; Weaver et al., 2024). Genetic variation of Trichoderma using molecular approaches can be revealed with more depth and clarity, thus allowing for the identification of species and strains that are potentially superior as biocontrol agents.
           
    The genetic diversity of Trichoderma isolated from the rhizosphere of cocoa plants has significant implications for the management of biological resources. These strains show certain advantages such as their ability to produce specific enzymes or bioactive compounds, which can be used as basic ingredients in the development of biofungicide and biofertilizer products (Abdul-Halim  et al., 2023; Xiao et al., 2023). This study aims to identify Trichoderma species isolated from the rhizosphere of cocoa plants at several different altitude locations in Central Sulawesi. It’s hoped that this research can provide in-depth insight into the potential of Trichoderma strains as biocontrol agents and biofertilizers and support effective management of local microorganisms that contribute positively to sustainable cocoa cultivation in Central Sulawesi.

    MATERIALS AND METHODS

    Location and time of research
     
    This research was conducted in Sigi Regency, Central Sulawesi, on cocoa farms representing three different altitudes: lowland in Labuan Subdistrict, Donggala Regency 50 m asl (0.650627°, 119. 86215°), medium altitude in Sigi Biromaru Subdistrict of Sigi Regency 200 masl (-1.091568°, 119.906393°) and high altitude in Palolo Subdistrict of Sigi Regency 1,106 masl (-1.132666°, 120.037956°) Central Sulawesi Province, Indonesia (Fig 1). Trichoderma isolation was carried out at the Microbiology Laboratory of Alkhairaat University Palu Central Sulawesi Indonesia, while molecular identification was carried out at the Molecular Biotechnology Laboratory, Tangerang, Indonesia. This research lasted for 3 months, from July to September 2024.

    Fig 1: DNA amplification results of Trichoderma fungus isolates using ITS-1 and ITS-4 primers.


     
    Sampling, isolation, purification of Trichoderma
     
    Soil samples from the cocoa rhizosphere were taken using the Stratified Random Sampling method (Susila et al., 2023). For each location, three replicate samples weighing 300 grams each were taken from the soil around the cocoa root at a depth of 20 cm (Ratnawati et al., 2022; Sudewi et al., 2024). Subsequently, soil samples were stored in sterile plastic clips and brought to the laboratory for fungal isolation. The plate culture technique and serial dilution method on Potato Dextrose Agar medium (enriched with chloramphenicol (100 mg/L) were applied to prevent contamination from bacteria. The diluted rhizosphere samples were placed into petri dishes containing PDA media and incubated for 5-7 days at 28°C. Trichoderma spp colonies with specific morphological characteristics having whitish green hyphae were purified on new PDA media.
           
    A total of 5 Trichoderma isolates obtained from the screening were then cultured on PDA media and purified. Seven days, old purified isolate diluted to a density of 10 v  CFU mL-1 were plated onto PDA medium observed for 7 morphological characters such as color, elevation, edges (margins) of mycelium, coniodophores and conidia and growth rate of colonies for seven days (Widiati et al., 2022).
     
    DNA extraction
     
    Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research, D6005) was used for Trichoderma spp genomic DNA extraction according to standard procedures. Bashing BeadTM buffer and bead-beating were used to lyse 50 mg fungal samples to break down their cell walls. After centrifugation, it was mixed with fungal DNA binder and loaded onto a Zymo-Spin™ II purification column (Gand et al., 2023; Li et al., 2022). Additionally, the DNA was washed twice to remove contaminants, after which the extracted product was stored at 200°C for use in further analysis (Dilhari et al., 2017).
     
    DNA amplification and electrophoresis
     
    Trichoderma fungi were identified through DNA amplification using 2X MyTaq HS red mix (Bioline, BIO-25048) using primers ITS-1 (5'-TCCGTAGG TGAAC CTGCGG - 3') and ITS-4 (5'-TCCTCCGCTTATTGATATGC - 3') (Sukapiring et al., 2024). The DNA extract was used as a template in PCR. To obtain a total volume of 25 µL, mixed 12.5 µL MyTaq HS Red Mix (2X), 1 µL forward primer, 1 µL reverse primer, 1 µL template DNA and 9.5 µL nuclease-free water. Initial denaturation (1 cycle) for 1 minute at 95°C, followed by 35 cycles of denaturation at 95°C for 10 seconds and at 72°C for 5 minutes for final extension was done.
           
    Electrophoresis was done at 75 volts for ± 90 minutes, then visualized with a UV Transilluminator and documented using a digital camera. PCR results were then confirmed through 0.8% agarose gel electrophoresis in 1X TBE buffer. Agarose gel solution of 50 mL was heated until dissolved, cooled and then add GelRed as a DNA dye (Lee et al., 2012). The results obtained were compared with marker DNA to confirm the success of amplification. The names and sample codes of fungal isolates are presented in Table 1.
     
    Sequencing and phylogenetic analysis
     
    Sequencing uses the two-way Sanger Capillary Electrophoresis method with several steps. Previous PCR results in the form of DNA chain samples were analyzed using Bio-edit software then aligned with ClustaIW to compare with DNA chain sequences available in the Gene Bank database through blast on the web https://www.ncbi.nlm.nih.gov/ which was accessed on August 26, 2024. Phylogenetic tree analysis constructed with 1000 bootstraps using MEGA 10 software combined with the NCBI Blast Tree neighbor joining (NJ) approach (An et al., 2022).

    RESULTS AND DISCUSSION

    DNA amplification and sequencing
     
    The electrophoresis showed that the DNA obtained from PCR with ITS-1 and ITS-4 primers was of good quality as characterized clear visible bands on the electrophoregram for the five isolated samples (Fig 2). Leite et al., (2021) reported that ITS primers are able to amplify DNA optimally, making species identification easier. Mustafa and Rostam, (2021) suggested that fungal isolates that successfully amplify the ITS zone well are characterized by thick and sharp amplification results (Fig 1).

    Fig 2: Phylogenetic tree based on DNA barcoding with ITS locus from cocoa rhizosphere fungi isolates (N05, A03, SA04, RAN16, NK3).


           
    Based on the results of DNA amplification of Trichoderma fungus isolates with sample codes N05, A03, SA04, RAN16 and NK3 from 3 different altitudes showed very clear DNA bands with sizes of 575 bp, 578 bp, 577 bp, 576 bp and 577 bp respectively. DNA amplification of the 5 isolates using ITS-11 and ITS-4 primers, resulted in obtained amplicons in the range of 500-600 bp (Op De Beeck  et al., 2014). The DNA amplification looked very clear, stuck optimally and had, thick lines without the effect of shadows.
           
    The sequence results of the fragments of Trichoderma fungus isolates based on the NCBI nucleotide database in Table 1 showed that the five fungal isolates were identified as Trichoderma asperellum strains (NG125, NECC30406, IIPR-81, BHU203 and WZ-184) with a similarity identity percentage and query cover of 100%. The ratio indicated the degree of similarity between sample DNA sequences and target DNA sequences as percentage identity (Weaver et al., 2024; Salini and Dhandapani, 2021). This result can be attributed to many factors, one of which is the conservative nature of the ITS (Internal Transcribed Spacer) gene. ITS is commonly used to identify fungi due to low variation within the same species (Bradshaw et al., 2023). The isolates, although originated from different strains, may have a small degree of genetic variation within the amplified ITS region, however identified as the same species with a high percentage of identity.

    Table 1: Summary of sequence results from fragments of Trichoderma fungus isolates based on NCBI nucleotide database.


           
    Another factor for getting 100% similarity could be the use of reference databases such as NCBI BLAST, which had many T. asperellum entries from various strains. This is likely since the isolates obtained from the same place/having a close evolutionary relationship so that genetic differences between strains are not large enough to affect identification results. The length and quality of DNA sequences can also help the matching with the database to be more accurate (Abedulridha and Al-Shamery, 2018). The similarity of the five fungal isolates belongeds to the same species, although differences between strains can only be found through further genetic analysis, using methods such as multilocus sequence typing (MLST) or whole genome sequencing.
     
    Phylogenetic analysis
     
    The know the relationship between the isolates the phylogenetic tree analysis of the five Trichoderma fungus isolates was done based on the NCBI database Fig 2. The phylogenetic tree analysis indicates that Trichoderma isolate 1 (N05) belongeds to the same cluster as several T. asperellum species and other related species. The sequence identity percentage reaching 100% strengthens the hypothesis that this isolate has a close genetic relationship with T. asperellum. This suggests that the isolate may share similar biological characteristics with species in its group, such as functioning as a biocontrol agent or playing a crucial role in soil ecosystems (Jaya et al., 2023) as reflected by its position in the phylogenetic tree (Malik and Verma, 2025). These findings reveal that Trichoderma isolate 2 (A03) is related to the T. asperellum species, known as a plant growth-promoting fungus and biocontrol agent. Similar findings were reported by Budi et al., (2023), T. asperellum, isolated from oil palm plantations, was found to inhibit Ganoderma boninense growth by 93.9% in a dual culture assay. Additionally, Youassi et al., (2024) reported that T. asperellum extract effectively inhibited the growth of Phytophthora, the cacao pod rot disease. All these findings confirm that T. asperellum has the potential to be used as a biocontrol agent, biofumog, or biopesticide.
           
    The Trichoderma isolates 3 (SA04) and 4 (RAN16) were phylogenetically highly related with T. asperellum, T. atrovirides and other Trichoderma species (Baltzis et al., 2025). The similarity of the tested isolates to sequence data stored in the NCBI database confirms that molecular identification using ITS sequences will produce strain data with a high degree of accuracy and clarity (Pere et al., 2024; Kanthiya et al., 2025). The bootstrap with green dots indicates a high level of validity for the species identified  (Lemoine  et al., 2018), confirming the reliability of the molecular identification method in determining phylogenetic relationships between test isolates.
           
    The same cluster is also seen in the Trichoderma 5 (NK3) isolate in Fig 2, which appears to be related to the T. asperellum isolate. The confidence level is indicated by the branches generated with green bootstrap dots. Meanwhile, the NK3 isolate is closely related to the T. asperellum strains WZ-186 and NG125, as seen in the phylogenetic tree structure, both of which are known to have similar potential as biocontrol agents.

    CONCLUSION

    Five isolates of the Trichoderma fungus (N05, A03, SA04, RAN16 and NK3) originating from the rhizosphere of cocoa plants at 3 different altitudes showed good amplification quality, a clear DNA band with a size of 575-578 bp based on the results of DNA electrophoresis. The use of primers ITS-1 and ITS-4 is recommended as good molecular markers for phylogenetic analysis and species recognition. Based on DNA sequence analysis supported by matching results to the NCBI nucleotide database with query cover of 100%, the five Trichoderma fungus isolates were identified as T. asperellum with different strains.
           
    Phylogenetic analysis based on ITS sequences shows that the genetic closeness of these isolates has possibly the same biological and ecological characteristics. Strain variation within the same species can be caused by selection pressures, environmental factors and geographic conditions. Therefore, further research is needed to determine the physiological properties and ability of each strain to be used in various biotechnological applications, as a biocontrol agent or biofertilizer to promote plant growth.

    ACKNOWLEDGEMENT

    The present study was supported by Kementerian Pendidikan Tinggi, Sains dan Teknologi (Kemdiktisaintek) for funding under Grant No. 118/E5/PG.02.00PL/2024.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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