@misc{oai:miyazaki-u.repo.nii.ac.jp:00004048,
 author = {Njoloma, Joyce Prisca},
 month = {2020-06-21},
 note = {Sugarcane (Saccharum spp. L.) is one of the most important agricultural crops whose
products are both for export and domestic consumption in most of the tropical and
subtropical countries of the world. Several recent studies have confirmed that biological
nitrogen fixation by endophytic bacteria contributes significantly to the nitrogen nutrition
of sugarcane. Endophytic diazotrophic bacteria, which have been isolated in association
with sugarcane plants, includes Acetobacter diazotrophicus and Herbaspirillum spp. In
this research work, the main objective was to study the mechanisms involved in the
establishment of nitrogen fixing system in sugarcane plant through the investigation of
the mode of infection and mechanism by which the endophytic diazotrophic bacteria
colonizes sugarcane after inoculation.
Herbaspirillum spp. B501gfp1 (B501gfp1), an isolate from wild rice was used as source
of inoculum to investigate the interaction between nitrogen-fixing endophytic bacterium
and micropropagated sugarcane plants under aseptic and non-sterile conditions. In the
first place an investigation was carried out to detect if the green fluorescence protein's
expression from B501gfp1 could be distinguished from the sugarcane tissue's
background fluorescence. B501gfp1 bacterial cells were spot inoculated on to sugarcane
tissues from 5 month-old sterile micro-propagated plants. Stem tissues and leaf sections
mounted on glass slides were directly inoculated with a single touch using the tip of a
syringe previously dipped into the inoculum containing 10(8) bacterial cells ml(-1). It was
observed that GFP fluorescence could be easily distinguished in the stem than in the leaf
tissues. However, the brightness level of the fluorescence varied with time as a result of
fluctuations in the bacterial cell density. The presence of chloroplasts in the leaf tissues of
sugarcane requires the use of bright GFP variants when monitoring bacteria-plant
interactions using gfp labeled bacteria.
In a study under sterile condition, two Japanese sugarcane plant cultivars (cvs) NiF8 and
Ni15 were inoculated using B501gfp1 in 2 inoculum doses of 10(8) and 10(2) bacterial cells
ml(-1) suspension. The results obtained showed that bacterial cells colonized both the root
and stem tissues, and colonization was apparent in the intercellular spaces. Higher
bacterial numbers detected in plant tissues corresponded to the higher inoculum
concentration treatment. Bacterial numbers also varied between the 2 cultivars with the
higher values determined in cv. Ni15. Colonization ability of B501gfp1 in the presence
of indigenous endophytes in sugarcane under non sterile condition was also carried out
using the two inoculum concentrations (10(2) and 10(8) bacterial cells ml(-1)) and in cv NiF8
only. Internal tissue colonization was observed in plants inoculated with both the 10(2) and
10(8) B501gfp1 bacterial cells ml(-1) inoculum concentrations. However, extensive
colonization and higher bacterial numbers were determined in the basal stem tissues of
plants inoculated with the 10(8) bacterial cells ml(-1). Infection and establishment of the
bacteria in the plants varied with respect to the inoculation technique. In the sterile plants
inoculated with intact roots and basal stem, more bacteria were detected in the root
tissues with the lateral root junctions as the main entry points. However, in some
instances internal tissue colonization was observed to have occurred through intact root
surfaces. On the other hand, plants which were inoculated with their stem bases bearing
cut openings under non-sterile condition had more bacteria colonizing the stem tissues
with dense establishments in the vascular bundle tissues. It can therefore be concluded
that the mode of infection and subsequent establishment mechanism in the internal tissue
is facilitated by the inoculation technique and availability of entry points. This study also
provides evidence for non-specificity of host plant among endophytes since
Herbaspirillum spp. B501gfp1, a rice isolate could colonize sugarcane plant tissues.},
 title = {Establishment Mechanism of Nitrogen Fixing System by Endophytic Bacteria in the Japanese Sugarcane Plant},
 year = {}
}