Search

Ratoon stunt, caused by the bacterium Leifsonia xyli subsp. xyli, is one of the major sugarcane diseases worldwide. The objectives of this study were to determine the variation in morphology and DNA sequence of L. xyli subsp. xyli strains isolated in China, to compare the changes that occurred in vascular ultrastructure and levels of endogenous hormone abscisic acid (ABA), auxins (indoleacetic acid [IAA]), and gibberellic acids (GA3) in sugarcane stalks. Experiments were also conducted with two sugarcane varieties, ‘ROC22’ and ‘Badila’, in the greenhouse to understand the cytological and physiological mechanisms of L. xyli subsp. xyli–induced growth stunting. There were three treatments in the experiments: (i) healthy plants (L. xyli subsp. xyli–free plants), (ii) infected plants (L. xyli subsp. xyli–infected seedcanes treated with hot water, and (iii) infected plants (healthy seedcanes dipped in L. xyli subsp. xyli cell culture). The results showed that sequence coverage of a locally isolated strain, LxxGXBZ01, was 99.99%, and the average nucleotide identity between LxxGXBZ01 and the other well-characterized Brazilian isolate LxxCTCB07 was 93.61%. LxxGXBZ01 occurred in different sizes and shapes in xylem vessels of infected plants. In comparison with healthy stalks, the secondary walls of the vessel element in L. xyli subsp. xyli–infected stalks were degraded with uneven wall thickness, deformities, sticky substances, and electron-dense substances accumulated inside the cells. Compared with the healthy and hot-water treatments, the contents of IAA and GA3 were significantly lower, while that of ABA was significantly higher in the L. xyli subsp. xyli–infected stalks. The information obtained in this study will expand our understanding of ratoon stunt etiology and cytological and physiological bases of the disease manifestation.

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTet(r) plasmids were constructed by cloning the promoter region of the Tet(r) gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTet(r) plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. ¹⁵N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N₂ associated with the host plants.