## 2 Materials and Methods
### Polymerase Chain Reaction (PCR) Test  
The infected seedlings were confirmed using a polymerase chain reaction (PCR) test. Polymerase chain reaction (PCR) is a well-established molecular technique to detect and identify pathogen by amplifying the specific or targeted parts of a DNA sequence. The part needed for the test was the roots because the _G. boninense_ infection starts at the roots of oil palm. DNA was extracted from the root of inoculated oil palm seedlings where 2 g of the infected root was grounded to a fine powder with liquid nitrogen and then transferred into a 1.5 mL centrifuge tube. In the tube, 800 \(\mu\)L of extraction buffer, 80 \(\mu\)L 2% sodium dodecyl sulfate (SDS), and 20 \(\upmu\)L \(\beta\)-mercaptoethanol were added and subsequently vortexed. The tube was incubated for 30 min at 65 \({}^{\circ}\)C. After that, 300 \(\upmu\)L of phenol and chloroform were added. The tube was vortexed again for 1 min, then centrifuged at 11,000 rpm for 10 min. The supernatant was then transferred into a new centrifuge tube, and phenol and chloroform were added again. An equal volume of ice-cold propanol was added into the supernatant and incubated at \(-\)20 \({}^{\circ}\)C for overnight. Later, the tube was centrifuged at 11,000 rpm for 10 min. The supernatant was removed, and 70% ethanol was used to wash DNA pellet. The pellet was dried in laminar flow for 10 min and dissolved in sterile distilled water then incubated at 65 \({}^{\circ}\)C to ensure complete dissolution of the pellet. The DNA was kept at \(-\)20 \({}^{\circ}\)C for long term storage. Pure culture of _G. boninense_ was extracted and used as a positive control, whereas the distilled water was used as a negative control. Specific primer of _G. boninense_ designed by [PERSON] and [PERSON] (2000) Gan1: 5\({}^{\prime}\)-TTG ACT GGG TTG TAG CTG-3\({}^{\prime}\) and Gan2: 5\({}^{\prime}\)-GCG TTA CAT CGC AAT ACA-3\({}^{\prime}\)was used. The PCR thermal cycler protocol was performed with four steps. The first step was denaturation for 5 min at 95 \({}^{\circ}\)C. The second step was 35 cycles of denaturation for 1 min at 95 \({}^{\circ}\)C, annealing for 1 min at 56 \({}^{\circ}\)C and elongation for 2 min at 72 \({}^{\circ}\)C. The third step was an extension for 10 min at 72 \({}^{\circ}\)C. The last step was storage at 4 \({}^{\circ}\)C. Gel electrophoresis was conducted using standard protocol to detect the specific band produced. Finally, PCR products were sent to the private lab for gene sequencing analysis for verification of the specific band produced. The sequences were aligned and edited using bio-edit software. The consensus sequences were then blasted in the GenBank to confirm the sequences identity to _G. boninense_.  
Figure 1 shows an example of an infected seedling and the associated PCR result. As shown in Figure 1a, the infected seedling did not show any visible symptoms related to _G. boninense_ infection such as fungal mass or foliar symptoms such as yellowing of leaves despite testing positive with the _G. boninense_ pathogen. The positive result indicated that _G. boninense_ pathogen had penetrated and infected the tested roots. The specific band size was approximately 160 to 170 bp (Figure 1b) that were obtained from the roots, which authenticated the presence of _G. boninense_ infection. Further confirmation was conducted using gene sequencing analysis between the specific bands and _G. boninense_ (taken from the GenBank dataset). The result showed 99.5-100% similarity index.