GENETIC IMPROVEMENT OF SUGARCANE BY CONVENTIONAL AND MOLECULAR APPROACHES
Location: Sugarcane Research Unit
Title: Quick detection of Leifsonia xyli subsp. xyli by PCR and necleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates
Submitted to: Sugar Tech
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 23, 2008
Publication Date: December 4, 2008
Citation: Gao, S.-J., Pan, Y.-B., Chen, R.-K., Chen, P.-H., Zhang, H., Xu, L.-P. 2008. Quick detection of Leifsonia xyli subsp. xyli by PCR and necleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates. Sugar Tech. 10(4):334-340.
Interpretive Summary: The efficiency of detecting sugarcane ratoon stunting disease (RSD) by the nucleic acid-based polymerase chain reaction (PCR) assay is affected by the presence of PCR-inhibitory substances in xylem sap samples. Diagnosticians have to extract the DNA samples from xylem sap or plant tissue prior to PCR assays. The DNA extraction procedure takes more than 4 hours and involves two organic solvents, which limits its usage when conducting a large-scale field disease survey or RSD-resistance screening during varietal selection. We have developed a quick procedure that does not use any organic solvent. The RSD bacteria are precipitated from the xylem sap by centrifugation and then lyzed in a hot alkaline solution. The lysate is then neutralized before subjecting to PCR. The procedure takes less than an hour and produces the same level of detection. We used this procedure to detect RSD-infection status in 31 nursery-grown sugarcane varieties and found that 19 varieties (61.3%) tested positive, these included all of the FN-, GT-, and YT-series varieties developed in China. The same varieties also tested positive by PCR assays using extracted DNA templates and by two enzyme-immunoassays. All amplified PCR products had the same nucleotide sequences and the consensus sequence was deposited into GenBank as Accession EU723209. The results from this study indicated that the vast majority of the Chinese mainland varieties (16 out of 22) were RSD-infected, indicating the urgent need for a healthy seedcane program. In addition, all breeding programs in China need to include RSD-resistance as one of the major objectives and surveys are conducted to determine the extent of RSD infection in commercial sugarcane fields in China.
A quick polymerase chain reaction (PCR) assay was developed for the detection of Leifsonia xyli subsp. xyli (Lxx), the bacterial causal agent of ratoon stunting disease (RSD) of sugarcane, in crude juice samples from stalks. After removal of abiotic impurities and large molecular weight microorganisms by a 3000 rpm, 5 min centrifugation, the Lxx bacteria were precipitated from the xylem sap by a 12,000 rpm, 10 min centrifugation. The Lxx cells were re-suspended in 50 'l freshly prepared Buffer A (0.1M NaOH + 2% Tween 20), lysed by heating at 95oC for 10 min, cooled down on ice for 3 min, and neutralized by mixing with 50 'l of freshly prepared Buffer B (0.1M Tris-HCl, pH 8.0 + 2 mM EDTA). The resulting lysates were directly subjected to conventional PCR with Lxx-specific primers. Of the 31 varieties tested, 19 were positive for Lxx infection, including all FN, GT, and YT varieties. These varieties also tested positive by PCR on DNA templates extracted from the crude juice samples using a CTAB procedure and by two enzyme-immunoassays, dot-blot (DB-EIA) and direct antigen coating (DAC-ELISA). DB-EIA and DAC-ELISA detected Lxx in 90.3 and 93.5%, respectively, of the varieties while the detection rate with PCR was 61.3%. The modified PCR assay was quick and more economical. It did not require organic solvent usage or ethanol precipitation, but produced the same level of detection as that of the PCR using DNA samples prepared by the CTAB procedure. All the PCR amplicons were 439 bp in size sharing the same nucleotide sequence. This consensus sequence, GenBank Accession EU723209, aligned perfectly with three other Lxx nucleotide sequences available in the GenBank, AE016822, AF034641, and DQ232616. However, two mis-matches, a (G'A) transversion and a deletion, were found in another nucleotide sequence AF056003.