Improvement of Quarantine Procedures for the Detection of Sugarcane Phytoplasmas

The importation of sugarcane varieties from other countries is necessary for the breeding of new varieties suited to the diverse environmental conditions in South Africa. To prevent the introduction and spread of new diseases, it is crucial that any imported pathogens are detected before the cane is released from quarantine. Although not present in South Africa, Grassy Shoot (GS) and Sugarcane White Leaf (SCWL) are economically significant phytoplasmal diseases that cause yield loss in sugarcane in a number of countries. Despite phytoplasmas being important pathogens that have been implicated in more than 300 plant diseases worldwide, these organisms have as yet not been cultured in vitro. The most reliable and rapid method of detection has been the use of nested polymerase chain reaction directed to conserved sequences in the 16S rRNA gene, where DNA isolation follows a phytoplasmal enrichment procedure. The process is both expensive and time consuming. This study investigated the use of FTA paper (Whatman BioScience) for DNA extraction, and the use of a radioactive probe in conjunction with a tissue blot procedure to detect phytoplasmas in sugarcane leaf samples exhibiting symptoms of GS and SCWL, and in Bermuda grass leaf samples showing symptoms of Bermuda Grass White Leaf disease. The FTA paper method was effective in extracting the DNA from which specific identifications could be made. As well as being more rapid, this method effected a two-thirds reduction in cost compared with the enrichment method. The radioactive probe was less useful because both mitochondrial and phytoplasmal DNA were detected.