Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2006
Publication Date: 4/1/2007
Citation: Grisham, M.P., Pan, Y., Richard Jr, E.P. 2007. Early detection of Leifsonia xyli subsp. xyli in sugarcane leaves by real-time polymerase chain reaction. Plant Disease. 91:430-434. Interpretive Summary: Since a new crop of sugarcane is established by planting stalks of an established crop, it is important that growers use stalks as seed cane that are not infected with the bacterium that causes ratoon stunting disease (RSD), a major disease of sugarcane in Louisiana. Current tests used to determine if a plant is infected with the RSD require tissue from mature stalks for analysis, and by the time a sugarcane plant has formed stalks, it is too late to have them tested before they are cut for planting. Beginning in the spring when sugarcane plants in the field were approximately one-month old, we collected leaves from three different varieties every two weeks and tested them for the presence of the RSD bacterium with a technique based on a new technology called real-time polymerase chain reaction (PCR). We were able to detect the RSD bacterium with the real-time PCR technique at every stage of growth including the earliest sampling date, 14 weeks before stalk tissue was formed in the test plants. The technique has two major advantages, it is nondestructive since it only uses a small piece of leaf tissue, and it can be used to test sugarcane plants early enough in the growing season to select healthy stalks for planting, a definite advantage over currently used diagnostic tests which require mature stalks. Because of the early season detection capabilities, this diagnostic technique will be useful in disease management programs to limit the spread of RSD in the Louisiana sugarcane industry by helping growers identify the best source of stalks for planting, and thereby reducing the effects of this major disease.
Technical Abstract: A real-time, polymerase chain reaction (PCR) assay using SYBR green as the fluorescent, DNA-binding dye was developed to detect Leifsonia xyli subsp. xyli (Lxx), the causative agent of ratoon stunting disease (RSD) in sugarcane. Field samples were collected from second-ratoon crop plots that were established from either Lxx-inoculated cuttings or hot-water-treated cuttings to reduce the incidence of RSD. The youngest, fully expanded leaf with a visible dewlap (the collar between the leaf blade and sheath) was collected from Lxx-inoculated and hot-water-treated plots of three cultivars at two-week intervals beginning approximately one month after active growth began in the spring (11 April 2005). Plants infected with Lxx were detected on each sampling date; however, a decline in the number of positive assays occurred across all samples from an average of 42% of the samples positive over the first five sampling dates to 31% and 19% on the sixth (28 June) and seventh (19 July) sampling dates, respectively. To determine the effect of collecting samples from leaves at different positions on the stalk, the youngest, fully expanded leaf and the five leaves below it were sampled on 28 July and 21 October. The lowest number of samples testing positive for Lxx infection was among the samples from the youngest leaf, and the highest number of positive assays was found among the samples collected from the fourth leaf below the youngest leaf. Results suggest that the selection of tissue for analysis as a function of selection timing may affect the accuracy of the real-time PCR assays, especially for the latter samplings. The most common technique used for detecting Lxx infection of sugarcane in Louisiana is tissue-blot enzyme immunoassay (TB-EIA) in which tissue from the internode closest to the soil line is analyzed. In this study, the cultivars sampled did not produce sufficient stalk tissue for the TB-EIA until 14 weeks after the initial sampling date. By this date in the growing season, it becomes impractical to conduct large-scale testing of potential seed cane sources for Lxx infection. The real-time PCR assay is capable of detecting Lxx at early stages of plant growth, allowing sufficient time for growers to identify their best sources of seed cane for planting. The real-time PCR sampling protocol would also be useful for the RSD diagnosis of critical foundation plants used in micropropagation operations or when indexing plant material in quarantine programs.