Transmission of biochemical characterization of Leifsonia xyli subsp xyli (Davis et. al) Evtushenko et.al 2000 causing ratoon stunting disease of sugarcane (Saccharum officinarum L.)

After numerous attempts tha causal agent of the ratoon stunting disease (RSD) of sugarcane was successfully isolated 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. Isolations were successful from sap extracts that were positively assayed for RSD using dot-blot immunoassay, phase contrast microscopy and polymerase chain reaction (PCR). The three isolates (8013, 6553 and 8583) were virulent to the susceptible sugarcane cultivars Phil 8013. Early symptoms of internal salmon-pink discoloration in immature nodes below the meristematic area were observed in young shoots (7 wk-old) of inoculated sugarcane plants. Sap extraction was made easier and simpler with the use of positive sap expression by pliers. Passing the sap through 0.45 um millipore filter (nitrocellulose membrane) significantly increased the number of positive isolations. Bacterial sap contaminants were larger than L. xyli subsp. xyli. The isolates were non-motile, did not produce spores, non-acid fast and Gram positive becoming Gram variable on old cells. There were aerobic and irregular shaped rods with club-shaped and y-cell formulations typical of the coryneform group. Cells measured 0.96 to 84 um by 0.19 to 0.29 um. Growth of the isolates in modified sugarcane (M-SC) medium was very slow. Colonies were evident only after 7 to 10 days 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. All biochemical characteristics of the three sugarcane isolates were comparable to previous reports of the corresponding pathogen. In plant inoculations, the bacterium was recovered in napier, corn, and sorghum 6 wk 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 RSD of sugarcane was identified as Leifsonia xyli subsp. xyli (Davis et al.) Evtushenko et al 2000