Isolation of the causal organism, identification and characterization
The study was conducted at Merapi Volcano National Park (MVNP), which is located in two Indonesian provinces (Yogyakarta and Central Java Provinces) and with geographic coordinates between 110°15ʹ00ʺ – 110°37ʹ30ʺ E and 07°22ʹ30ʺ - 07°52ʹ30ʺ S, within a 8.4 ha restoration plot. The study area has been occupied and dominated by A. decurrens and the trees were approx. 4 yrs old when the study was conducted.
Ten A. decurrens trees with gummosis symptoms on the stem were selected randomly and sampled from the study area. The discolored wood and bark samples were drawn from the leading edge of the gummosis after clearing the gummosis. These excised sections were then wrapped in newspaper to maintain moisture, and they were subsequently transferred to the laboratory for further processing. Carrot slices were used as a bait to isolateCeratocystis from the disease samples (Moller and de Vay 1968). Isolations from stem samples and pathogenicity tests were conducted using standard methodology (Waller et al., 2002). All isolates were maintained on potato dextrose agar (PDA) in the Laboratory of Forests Health and Protection, Faculty of Forestry, University Gadjah Mada, Indonesia. Morphological characteristics of the isolates were studied to identify the species.
DNA extraction from the fungus and amplification of rDNA ITS were conducted in the Molecular Genetics Laboratory, Ministry of Environment and Forestry, Yogyakarta. Sequencing of ITS fragments was conducted in 1st Base Singapore. DNA were extracted using SDS buffer (200mM Tris HCl pH 8.5; 250mM NaCl; 25mM EDTA and 0.5% SDS) (Raeder and Broda 1985) as modified by Glen et al. (2002). The primer pairs used to amplify the rDNA ITS are ITS4 (TCC TCC GCT TAT TGA TAT GC) (White et al. 1990) and ITS1-F (CTT GGT CAT TTA GAG GAA GTA A) (Gardes and Bruns 1993). PCR products were sent to 1st Base (Singapore) for sequencing. DNA sequence chromatograms were viewed in Chromas version 2.6.5 (Technelysium Pty Ltd) software and edited to remove poor quality sequences at each end. Searching of public DNA databases, GenBank (Benson et al. 2017) was conducted to retrieved sequences of high similarity using BLAST (Basic Local Alignment Search Tool) (Altschul et al. 1997). Phylogenetic analysis to confirm the identification of the isolates was conducted using Mega7 (Kumar et al. 2016). Sequences were aligned using Clustal W (Larkin et al. 2007) in BioEdit 7.0.9.0 (Hall 1999) with full multiple alignments prior to phylogenetic analysis. Sequences with high similarity retrieved from GenBank were included in this analysis as references and one sequence from a more distantly related taxon was included as an out group. The phylogenetic tree was constructed using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993).