Axenic culture, transmission and biochemical characterization of Leifsonia xyli subsp. xyli (Davis et.al) Evtushenko et.al. 2000, the cause of ratoon stunting disease of sugarcane (Saccharum officinarum L.)

The causal agent of the ratoon stunting disease (RSD) of sugarcane was successfully isolated after numerous attempts from three Philippine sugarcane cultivars, namely: Phil 8013, Phil 6553 and Phil 8583. The RSD bacterium was isolated using the sugarcane medium developed by Davis in 1980 with some modifications. Isolation were successful from sap extracts that were positively assayed for RSD using dot-blot immunoassay and phase-contrast microscopy. The three isolates (8013, 6553, and 8583) were virulent to the susceptible sugarcane cultivar Phil 8013. Early symptom of internal salmon-pink discoloration in immature nodes below the meristematic area was observed in young shoots (7 weeks old) of the inoculated sugarcane plants. Sap extraction was made easier and simpler by the use of pliers to express the sap. Passing the sap through 0.45 um millipore filter (nitrocellulose membrane) significantly increased the number of positive isolations due to the great reduction of contaminants. These significant results led to the development of the modified isolation scheme for the ratoon stunting disease bacterium. The modified isolation scheme eliminated much of the tedious process of aseptic preparations and significantly increased the chances of isolating L. xyli subsp xyli from sugarcane sap extracts. The isolates were non-motile, did not produce spores, non-acid fast and gram positive becoming gram variable in old cells. They were aerobic and irregularly shaped rods with club-shaped and V-cell formations typical of the Coryneform group. Cells measured 0.96 to 3.84 um by 0.19 to 0.29 um. Growth of the isolates in modified sugarcane (MSC) medium was very slow. Colonies were evident only after 7 to 9 days of incubation at 28 deg C. Full colony development required 2 to 3 weeks and reached diameters of 0.2 to 0.3 mm. Colonies were white which turned off-white with age. No growth was observed at 32 deg C to 37 deg C. Fastest growth occurred at 28 deg C-30 deg C. All of the biochemical characteristics of the three sugarcane isolates were comparable to previous reports for the corresponding pathogen. The isolates 8013, 6553, and 8553 were positively detected by the polymerase chain reaction analysis. The specific primers LxxITS 5 and LxxITSr 5 gave an amplified DNA product of a single band which was comparable to the single band obtained with the DNA of L. xyli subsp. xyli having a size of 305 bp. In plant inoculations, the bacterium was detected in napier, corn and sorghum six weeks after inoculation by means of dot-blot immunoassay and phase-contrast microscopy. However, no visible RSD symptoms were observed from these experimentally infected plants. On the basis of morphological, cultural, biochemical and pathological properties of the three isolates, the pathogen of ratoon stunting disease of sugarcane was identified as Leifsonia xyli subsp. xyli (Davis et al.) Evtushenko et al. 2000