INTRODUCTION:

Cellulose is a polymer component of cell walls that becomes an important natural resource because of its renewable resource¹ where its production potential reaches up to 1011-1012 tons/year². The main composition of cellulose is very simple, which consists of D-glucose residues associated with β-1,4-glycosidic bonds to form polymeric bond chains to reach 10,000 glucose residues³. Although it looks very simply, the morphological form of intermolecular bond is very complex. One of microorganisms that can degrade the complexity of cellulose bonds is Trichoderma⁴. Trichoderma is proven to be capable of producing cellulose enzyme which can be used to degrade cellulose⁵. In addition, Trichoderma can also produce protease and chitinase enzymes that protect plants from pathogens⁶.

Cellulolytic moulds from sugarcane bagasse are successfully isolated and identified conventionally by Surakhman⁷ among 13 mould isolates with different characteristics. Based on the morphological, microscopic, and physiological appearance, the best moulds in producing cellulose are Trichoderma, Botrytis, and Gliocladium. However, Trichoderma is indicated to have the highest cellulase activity compared to Gliocladium and Botrytis. The study can only identify moulds to the genus level, thus, further research is needed for identification to the species level.

Indonesia is a tropical country with very high biodiversity rate^8,9 including the diversity of microbes^10,11. Mould is one of the biological resources, which play important roles in human life¹¹. In addition, species identification plays an important role in taxonomy. Conventional species identification steps are more complex, subjective, require a long time and can cause identification errors. However, with the advanced technologies and the availability of cheaper molecular devices, mould identification to species level can be carried out molecularly^12,13. Ab Majid stated that the appearance of morphology cannot accurately identify species. Therefore, the sequencing of DNA region can support further identification⁶.

DNA region can be used as potential DNA barcoding for mould¹⁴. Furthermore, DNA barcoding has been used for taxonomy in recent decades. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identifications by using short, standardized gene regions as internal species marker¹⁵. Ko et al.¹⁶also stated that DNA barcoding is the best method that can be used to confirm the species identification. One of the DNA barcodings that has been widely used for the mould identification is internal transcribed spacer (ITS) region^17,18,19. ITS region has the highest probability for huge of identification, with barcode differences being the most obvious among species caused by inter and intraspecific variations of DNA¹⁴. The ITS region has been proven to be used to identify Trichoderma from various types of samples; such as compost²⁰, oil²¹, and oranges²². Identification of Trichoderma from sugarcane bagasse has not been carried out to species level. Therefore, we aimed to investigate Trichoderma from sugarcane bagasse based on internal transcribed spacer (ITS) rDNA.

MATERIAL AND METHODS:

Trichoderma Sample Preparation:

The sample used in this study was the result of isolation from indigenous Trichoderma of sugarcane bagasse. Morphological identification and enzyme production have been done by using the method of Surakhman⁷. Rejuvenation of moulds for DNA extraction was accomplished by using potato dextrose agar (PDA) purchased from Merck Millipore. 4.68 gr of PDA was dissolved in 120 mL of distilled water and homogenized with hotplate and stirrer. The process was carried out in laminar air flow. Then the mould was incubated at room temperature.

Isolation of the DNA of Trichoderma:

The DNA isolation process was completed by the modified CTAB method of Doyle and Doyle²³. The process of Trichoderma DNA isolation was propagated with the addition of 500 µL CTAB buffers and then vortexed. Next, it was closed with parafilm and incubated in a water bath 65 °C for 45 min, vortexed every 10 min, and centrifuged 13,000 rpm at 25 °C for 10 min. The supernatant was mixed with chloroform-isoamyl alcohol and centrifuged at 13,000 rpm at 25 °C for 5 min and repeated. The next step was to move the supernatant into a sterile 1.5 mL tube and added with ethanol absolute. Samples were incubated 24 h at -20 °C and centrifuged 13,000 rpm at 4 °C for 10 min. The results in the form of DNA pellets are mixed with 500 µL 70% ethanol and centrifuged at 13,000 rpm for 10 min at 4 °C. It was aerated pellets at a temperature of 55 °C and added with 50 µL TE buffers pH 7.6. Then, the DNA was obtained²⁴.

Quality and Quantity of Trichoderma:

A quantitative test of DNA purity and concentration was performed by using Nanodrop spectrophotometer and ND-1000 software. The PCR amplification reaction was accomplished in 30 µL mixture containing PCR mix, Primer, ddH₂O and DNA template. The cycle parameter included an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 sec, primer annealing at 55 °C for 1 min, the extension at 72 °C for 6 min and post extension at 72°C for 6 min. The primer used for ITS rDNA amplification were ITS-1 (5'-TCC GTA GGT GAA CCT G-3') and ITS-4 (5'-TCC TCC TGA TAT GC-3'). Meanwhile, the amplified products were separated on 1.5% agarose gel in TBE Buffer using horizontal electrophoresis in 50 V for 60 min. One kb ladder was used as a marker. The gel was visualized in UV transilluminator.

Sequencing and Phylogenetic Analysis:

The purified DNA was sequenced using automated DNA sequencer (ABI PRISM 3730 Genetic Analyzer, Applied Biosystem, USA), cycled sequencing using the Big Dye TerminatorR v3.1 kit at First Base Laboratories, Singapore. The sequencing results were read with Sequence Scanner 1.0 and continued with the BLAST program. Forward and reverse nucleotides were repaired with BioEdit 7.0.9.0 then the multiple alignments and phylogenetic construction between ITS sequences were made with MEGA 6.0.

RESULTS AND DISCUSSION:

The amplification of ITS rDNA sequence was successfully completed in this study. Amplicon was obtained at 600 bp. Suharjono also successfully amplified the complete ITS rDNA sequence Trichoderma from Purworejo, Brastagi, and Poncokusumo, Indonesia with the length of amplicon for 600 bp²³. ITS rDNA sequences in Trichoderma longibrachiatum are also 600 bp²⁵.

Amplicons are sequenced and visualized by color peaks in the top of the graph in chromatograms that characterized by a nucleotide base. Non-specific sequences are shown by red on the peak of chromatogram which indicates the quality of the sequencing results. Multiple peak chromatograms can be caused by more than one DNA template in one reaction. It means that there is more than one homologous sequence found in the genome²⁶.

The sequencing results are 589 bp for forward and 591 bp for reverse. According to Dewi, the forward and reverse sequences are combined and improved by the Contig method so that the complete sequence of ITS rDNA regions is obtained. The Contig method can be done using Bioedit. The process is done by removing the ends of forward and reverse sequences that have poor quality. The union of two sequences is done by reverse-complement in one sequence to minimize the mismatch between sequences. The result of the contig process is the Trichoderma sequence, which is 570 bp²⁷.

The nucleotide sequences from the Contig are beneficial for the construction of phylogenetic tree analysis. The complete sequence of ITS rDNA Trichoderma from sugarcane bagasse is compared to other mould species according to the NCBI database based on rDNA regions (ITS1, 5.8S, and ITS2 regions). Phylogenetic tree analysis uses 14 sequences, including Trichoderma from sugarcane bagasse, 11 sequences of Trichoderma or Hypocreae, Sphaerostilbella aureonitens, and Gliocladium. Phylogenetic tree construction using MEGA X software.

Fig. 1: The result of Trichoderma DNA amplification from the bagasse. M: Marker, K-: Negative Control, and A: Trichoderma.

Phylogenetic construction begins by looking at the homology between sequences with the alignment of all sequences using the crustal W program. The alignment process will show a gap marked by a dashed line caused by the varied nature of the ITS1 and ITS2 regions. The gap indicates the occurrence of mutation processes in the form of deletions or insertions²⁷. The results of alignment show that the ITS rDNA region is a high evolutionary area, this is indicated by the high differences in nucleotide sequences possessed by 14 species of mould.

The results of the alignment are utilized for the construction of phylogenetic trees. The method employed in the analysis is the neighbor-joining (NJ) method. According to Saitou and Nei, NJ can be applied to all types of data that have evolutionary differences and use the Kimura-2 parameter formula which is an algorithmic calculation model to determine evolutionary distances or nucleotide changes that occur so that they can form phylogenetic trees between species²⁸. To increase the value of confidence (accuracy) a statistical test was used using the 1000 replication bootstrap method²⁹.

The results of phylogenetic tree construction exhibit the separation of sessions into two large groups. The first group applied as outgroup consists of Gliocladium penicillioides and Sphaerostibella aureotines as different genus comparison species. The second group as ingroup iss divided into two groups. The first group is supported by a 78% bootstrap value occupied by Trichoderma viride while the second group is supported with a 99% bootstrap value consisting of two subgroups with a 100% bootstrap value are Hypocrea gelatinosa, Trichoderma gelatinosum, Trichoderma sp. from sugarcane bagasse isolation, Trichoderma harzianum, Trichoderma piluliferum, Trichoderma sp. SQR339, Hypocrea nigricans, Trichoderma sp. NFML CH12 BB. 15. Trichoderma aureoviride, Hypocrea lixii with 70% bootstrap value, and Trichoderma BAB-4585 with 100% bootstrap value.

Bootstrap values between 70 and 100 indicate that branching and phylogenetic trees will not change. Conversely, if the bootstrap value is less than 70, the probability of branching arrangement is very high. Thus, when a phylogenetic tree analysis is completed again the grouping of microorganisms can be differentt³⁰. Ubaidillah and Sutrisno explained that the bootstrap method is a method of reshaping data for the reconstruction of phylogenetic trees. The bootstrap method determines the trust level of phylogenetic tree; therefore, the greater bootstrap value is, the higher level of branch trust (topology) of the tree as the results from the reconstruction³¹.

Based on the bootstrap value, it can be concluded that the genus Trichoderma sp. monophyletic with 5 species includes Trichoderma harzianum, Trichoderma piluliferum, Trichoderma sp. SQR339, Hypocrea nigricans, Trichoderma sp. NFML CH12 BB. 15. Trichoderma aureoviride, Hypocrea lixii, Trichoderma BAB-4585. According to Topik, monophyletic groups are the groups whose members originate from one ancestor and have the same genetic or biochemical patterns or characteristics³².

The results of phylogenetic tree construction present the similarity between samples and genetic distance. The similarity value is inversely proportional to genetic distance. The smaller the genetic distance between species, the greater the similarity value. According to Saitou and Nei, genetic distance shows the level of gene differences between populations or species²⁸. According to Shamir, phylogenetic analysis can reveal the genetic distance between samples with each individual being a comparable species, thus, the closeness between species can be shown³³.

Samples of Trichoderma sp. has 100% similarity with Trichoderma harzianum, Trichoderma piluliferum, Trichoderma sp. SQR339, Hypocrea nigricans, Trichoderma sp. NFML CH12 BB. 15. based on ITS region with a genetic distance of 0.00. While the similarity value of Trichoderma sp. 99.78% with Trichoderma aureoviride, hypocrea lixii, and Trichoderma BAB-4585 with genetic distance values of 0.002.

Most microorganisms are producers of cellulolytic enzymes. Trichoderma reesei is one of the microorganisms that can degrade cellulose on plant cell walls. Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, and Trichoderma viride can also produce cellulolytic enzymes. In addition, the species is also called saprotroph which contributes to the degradation of lignocellulose plant material⁴.

CONCLUSION:

In sum, Trichoderma from sugarcane bagasse has been identified by molecular methods. Trichoderma DNA has a concentration of 92.56 mg with a purity of 1.91. Trichoderma has an amplicon of 600 bp. Phylogenetic analysis shows that Trichoderma samples are Trichoderma harzianum, Trichoderma piluliferum, Trichoderma sp. SQR339, Hypocrea nigricans, and Trichoderma sp. NFML CH12 BB. 15, Trichoderma aureoviride, Hypocrea lixii, or Trichoderma BAB-4585.

ACKNOWLEDGEMENT:

We thank to PMDSU Scholarship - Batch III by the Directorate General of Higher Education, Ministry of Education and Culture of the Republic of Indonesia (awarded to Arif Nur Muhammad Ansori and Muhammad Khaliim Jati Kusala). We thank EJA – Professional Translation Services for editing the manuscript.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 30.06.2019 Modified on 10.08.2019

Research J. Pharm. and Tech. 2020; 13(7): 3300-3304.

DOI: 10.5958/0974-360X.2020.00585.5