# Volatile Organic Compound from _Trichoderma asperelloides_ TSUI: Impact on Plant Pathogenic Fungi
## 2 Materials and Methods
### Molecular Identification  
_Trichoderma_ sp. TSU1 was cultured on PDA for 3 days and subjected to DNA extraction by DNA Extraction Mini Kit (FAVORGEN, Ping-Tung, Taiwan) following the manufacturer's protocol. A polymerase chain reaction (PCR) was performed in 20 uL volume containing 1.0 uL DNA template, 1.0 uL of each primer, 10.0 uL 2\(\times\) Quick TaqMan(r) HS DyeMix (TOYOBO, Saitama, Japan), and 7 uL deionized water. PCR amplification of internal transcribed spacer (ITS), part of RNA polymerase 2 (_rpb2_), and translation elongation factor 1-\(\alpha\) (_left_-_a_) genes were amplified by using ITS1/ITS4 [29], RFPB2-5F/RFPB2-7 cr [30], and Tef1-728F [31]/Tef1-986R [32] primer pairs, respectively, in the following thermal conditions: 94 \(\lx@math@degree\)C for 2 min, followed by 35 cycles of 94 \(\lx@math@degree\)C for 2 min, annealing at a temperature dependent on the amplified gene (Table 2) for 60 s and 72 \(\lx@math@degree\)C for 1 min, and a final 72 \(\lx@math@degree\)C for 10 min in a peqSTAR thermal cycler (PEQLAB Ltd., Fareham, UK). PCR products were observed on 1% agarose gels stained with ethidium bromide under UV light. PCR products were purified using a PCR clean-up Gel Extraction Nucleopin(r) Gel and PCR Clean-Up Kit (Macherey-Nagel, Duren, Germany). The purified PCR products were directly sequenced. Sanger sequencing was carried out by 1 st Base Company (Kembangan, Selangor, Malaysia) using the PCR primers mentioned above. Sequences were used to query the GenBank gene sequence database via BLAST ([[http://blast.ddbj.nig.ac.jp/top-e.html](http://blast.ddbj.nig.ac.jp/top-e.html)]([http://blast.ddbj.nig.ac.jp/top-e.html](http://blast.ddbj.nig.ac.jp/top-e.html)), accessed on 6 December 2020). Multiple-sequence alignment was carried out using MUSCLE [33]. A phylogenetic tree was constructed using maximum-likelihood (ML) and Bayesian inference (BI) methods. The best substitution models for ML and BI analyses were estimated by the Akaike information criterion (AIC) in jModeltest v2.1.10 [34]. ML analysis was carried out on RAxML v7.0.3 under the GTR + I + G model with 1000 bootstrap replications [35; 36]. BI analysis was conducted with MrBayes v3.2.6 [37] to evaluate the posterior probabilities (PPs) by Markov chain Monte Carlo (MCMC) sampling. Markov chains were run for 1 million generations; trees were sampled every 100 th generation, and 10,000 trees were obtained. The first 2000 trees representing the burning phase of the analyses were discarded. The remaining 8000 trees were then used to calculate PP in the majority-rule consensus tree. Bootstrap support (BS) and PP values greater than or equal to 70% and 0.95 of each branch, respectively, were considered to be significantly supported [38; 39]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Pathogen** & **Disease** & **Host** & **Sources** \\ \hline _Corynespora cassiicola_ & Leaf spot & _Lactuca sativa_ & [25] \\ _Fusarium incarnatum_ & Fruit rot & _Cucumis melo_ & [28] \\ _Neopestalotiopsis clavispora_ & Flower blight & _Antthurium andraenum_ & [26] \\ _N. cubuna_ & Leaf fall & _Hewea brasiliensis_ & [27] \\ _Sclerotium rolfsii_ & Stem rot & _Helianthus tuberosus_ & CCPM * \\ \hline \hline \end{tabular}  
* Culture Collection of Pest Management (PSU-HT01), Faculty of Natural Resources, Prince of Songkla University.  
\end{table} 
Table 1: List of fungal pathogens used for bioassay in this study. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Region** & **Primer** & **Orientation** &
\begin{tabular}{c} **Annealing** \\ **Temperature (\(\lx@math@degree\)C)** \\ \end{tabular} & **Reference** \\ \hline ITS * & ITS5 & Forward & 50 & [29] \\  & ITS4 & Reverse & 50 & [29] \\ _rpb2_ & RFPB2-5F & Forward & 54 & [30] \\ _tef1_-\(\alpha\) & Tef1-728F & Forward & 52 & [31] \\  & Tef1-986R & Reverse & 52 & [32] \\ \hline \hline \end{tabular}  
* ITS = internal transcribed spacer; _rpb2_ = RNA polymerase II; _left_-_a_ = translation elongation factor 1-\(\alpha\).  
\end{table}