DETECTING SUGARCANE DISEASE PATHOGENS BY DNA BASED TECHNOLOGY

Authors: Pan, Yong-Bao; Grisham, Michael; Burner, David; Legendre, Benjamin

Submitted to: Inter-American Sugar Cane Seminars Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 9/9/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: The majority of sugarcane cultivars grown in Louisiana commercial fields are susceptible to two major bacterial diseases: Leaf scald and ratoon stunting disease (RSD). Both disease cannot be readily identified visually and there is a potential risk of disease outbreak in the sugarcane industry. Reliable and inexpensive procedures are needed to identify the presence of these diseases in sugarcane fields. In response to this need, we have developed DNA- based diagnostic protocols to detect these pathogens in tissue: a polymerase chain reaction (PCR) protocol for the leaf scald and a DNA-hybridization protocol for RSD. With primers Ala 4 and L1, the PCR protocol amplified a unique product from the DNA of leaf scald bacterium, or without DNA extraction, directly from live or dead cells, infected leaf diffusate and sap. Similarly, another unique DNA product was amplified from the RSD bacterium with primers L1 and G1. Preliminary data indicated that this RSD-derived DNA product can be used as a probe to detect RSD infection by tissue- and dot-blot hybridizations. These DNA-based diagnostic protocols should be especially useful in quarantine and clean-seed programs.

Technical Abstract: Leaf scald and ratoon stunting disease (RSD) are two major sugarcane bacterial diseases in Louisiana. The majority of the commercial cultivars grown in commercial fields in Louisiana are susceptible to both diseases. There is a potential risk of disease outbreak that can cause severe yield and quality loss in the sugarcane industry. Reliable and inexpensive procedures are needed to identify the presence of these diseases in sugarcane fields. In response to this need, we have developed DNA-based diagnostic protocols to detect these pathogens in tissue: a polymerase chain reaction (PCR) protocol for the leaf scald and a DNA-hybridization protocol for RSD. With primers Ala 4 and L1, the PCR protocol amplified a unique 360 base pair (bp) ribosomal DNA product from the leaf scald bacterium in less than 2 hours. This 360 bp DNA product was amplified from the genomic DNA of 71 strains of leaf scald bacterium collected worldwide, including representatives of the 3 known serovars (I, II, and III), or without DNA extraction, directly from cultures of live Xa cells, Xa-infected sugarcane leaf diffusate and sap, and sacrificed Xa cells. Similarly, a 560 bp ribosomal DNA product was PCR amplified from the genomic DNA of RSD bacterium with primers L1 and G1. Preliminary data indicated that this 560 bp DNA product did not cross hybridize to PCR products from Xa and several other sugarcane saprophytes, indicating its potential use in detecting RSD infection by tissue- and dot-blot hybridizations. These DNA-based diagnostic protocols should be especially useful in quarantine and clean-seed programs.