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New method speeds detection of ratoon stunting disease

A faster method for detecting ratoon stunting disease--the most important disease affecting sugarcane production worldwide--has been developed by the Agricultural Research Service and cooperators.

RSD has been reported in almost every geographic area where sugarcane is grown. It causes an average 5 percent yield loss, but under drought conditions, yield reductions can be as high as 50 percent. RSD is hard to detect because it has no unique external symptoms, so growers have no way of knowing if their fields have been infected. The bacterium that causes RSD, Leifsonia xyli subsp. xyli, also called "Lxx," is extremely difficult to isolate and culture, making it hard to diagnose for further studies.

ARS plant molecular geneticist Yong-Bao Pan with the agency's Sugarcane Research Laboratory in Houma, La., and cooperators in China have developed a rapid and more reliable technique for detecting RSD using standard laboratory equipment.

Popular RSD detection methods require the use of either antibodies, which may not be available, or bacterial DNA isolation using toxic organic solvents such as chloroform and beta-mercaptoethanol, a process which can take up to four hours. Due to the low concentration of the bacterium in the host, serological methods--tests that use antibodies to detect infection--have limited use when conducting a large-scale field RSD disease survey or RSD-resistance screening during breeding selection.

Pan's method uses xylem sap to test for RSD. The plant's xylem--the tissue responsible for the transport of water and soluble mineral nutrients from the roots throughout the plant--contains optimum concentrations of Lxx, making it an ideal area for collecting DNA. This safe technique uses two inorganic buffers to isolate Lxx's DNA, taking less than an hour to complete. The new method also utilizes general equipment, such as a centrifuge and a polymerase chain reaction machine, which are more commonly found in labs worldwide.

The PCR method is more sensitive than serological methods and amplifies the DNA sequences of the bacterial genome in areas where it is hard to find, such as in xylem. It is thus a more practical method for labs throughout the world, especially those in developing countries, to detect RSD.


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