Effective methods of preserving SCWL-diseased sugarcane leaves for genomic DNA extraction and molecular detection of phytoplasma
Simple and economical methods to preserve sugarcane leaf tissues infected with sugarcane white leaf (SCWL) phytoplasma for later detection of SCWL using nested- PCR analysis were described. Diseased leaf tissues were preserved by air-drying and stored at room temperature or stored fresh at 4 or -20°C in two types of bags: brown paper bags and plastic bags with punctured holes. Leaf materials were stored for up to 32 days prior to DNA extraction. The best storage condition was placing fresh leaves at -20°C and air-drying leaves in paper bags at room temperature. High molecular weight plant genomic DNA bands were detected in all stored infected tissues. Genomic DNAs were used as templates for nested- PCR which showed a single band of 210 bp in the 16S-23S intergenic spacer region specific to SCWL phytoplasma. Nested-PCR was also performed on genomic DNA extracted from diseased leaves heattreated in a microwave oven and the SCWL-specific bands were effectively detected. Therefore, treating diseased sugarcane leaves by air-drying, freezing, refrigeration or microwave heat treatment were all effective for molecular detection of SCWL phytoplasma. However, air-drying and storage in paper bags was the most economical and practical, especially when large numbers of samples are to be stored or transported from the fields to a distant laboratory or exchanged among laboratories.
Key words: Sugarcane white leaf phytoplasma, nested-polymerase chain reaction (PCR), preservation, 16S- 23S intergenic spacer region.