Correspondence to: Ariel D. Abstract A new role for the plant growth-promoting nitrogen-fixing endophytic bacteria Gluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions. Living G. Results point toward a form of induction of systemic resistance in sugarcane-G.
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Search Menu Abstract Sugar cane Saccharum spp. In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted. After 10 d there was extensive anatomical evidence of endophytic colonization by G. The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion.
No hypersensitive response HR was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment 7 d.
Close to the point of inoculation, G. In leaf samples taken up to 4 cm from the inoculation points, G. Sugar cane , Gluconacetobacter diazotrophicus , endophytic bacteria , nitrogen fixation , immunogold labelling.
Introduction Applications of mineral N fertilizer to Brazilian sugar cane interspecific hybrids of Saccharum sp. It has long been hypothesized that the apparent deficit in N inputs is made up by biological nitrogen fixation BNF Patriquin et al. Boddey et al. It has been suggested that as these bacteria are endophytes, and hence live within the plant tissues, they can fix N2 more efficiently than diazotrophs that remain in the rhizosphere or on the rhizoplane Patriquin et al. In addition, endophytic bacteria will not have to compete with other soil microbes for scarce resources Sprent and James, ; Hallmann et al.
Although endophytic diazotrophs, such as G. After 4 d the bacteria had colonized the surface of the roots, with the lateral root junctions and the root tips being the most preferred sites. After a further 11 d, G. The bacteria in the xylem were confirmed to be G. Since that study was published, further micrographs showing xylem vessels colonized by G.
However, results have been presented that contrast with these studies Dong et al. These authors suggested that the intercellular spaces in the sucrose storage parenchyma of the stems of mature sugar cane stalks were the most likely location of a symbiosis with G. In a later paper Dong et al. Furthermore, Dong et al. Dong et al. James et al. In order to resolve these differences, two sets of experiments are performed.
Reis et al. One of the aims of this infection study was to confirm the data presented previously James et al. This is a method commonly used by sugar cane pathologists to determine resistance or susceptibility to bacteria, such as Xanthomonas albilineans and X.
This inoculation technique has been used successfully to examine the infection of sugar cane and sorghum leaves by Herbaspirillum spp Olivares et al. Materials and methods Organisms, growth conditions, bacterial inoculation, and counts Experiment 1 Micropropagated sugar cane plantlets cv. The plantlets were then inoculated with 0. Plants were harvested at 10 d after inoculation, and pieces of roots, leaves and stems from five inoculated and five control plants were taken for microscopical analysis.
Dry weights were determined according to Reis et al. Acetylene reduction assays were performed according to the methodology of James et al. Five replicate plants were inoculated, and five control plants were inoculated with sterile water, with the exuded excess fluid in both cases being immediately mopped up with sterile cotton wool. At 7 d after inoculation, leaves with visible inoculation points were taken for microscopy and bacterial counts. The leaves were prepared for the counts according to the method of Olivares et al.
Olivares et al. Pieces of leaves from Experiment 2 were sampled around the inoculation point and up to 4 cm above the inoculation point, and these were fixed and embedded as above. Optical and transmission electron microscopy TEM were performed on the samples James et al. Two antibodies were used in the immunogold analysis: a polyclonal antibody raised in a rabbit against G.
Results Experiment 1: infection of micropropagated plants At 10 d after inoculation none of the plantlets from either treatment exhibited any macroscopically visible disease symptoms data not shown.
The dry weights of the G. There was a slight red colour on most of the G. The surfaces of the roots were colonized by the bacteria in a manner similar to that reported previously James et al. As in these studies, the bacteria accumulated at lateral root junctions and colonized damaged epidermal cells not shown , but in the present study there were no indications that they had actually penetrated beyond the root epidermis.
This infection generally took the form of one or two of the metaxylem vessels being almost completely filled with bacteria Fig. No bacteria were apparent within control plants Fig.
The bacteria in the infected xylem vessels Fig. Further into the wound sites, intercellular and intracellular bacteria Fig. The intracellular bacteria were observed only in cells that appeared to be dead or damaged, without intact cytoplasm or organelles Fig.
Some xylem vessels in this region Fig. Deeper into the stem cortex, and away from the wound sites, bacteria were seen within apparently unoccluded xylem vessels Fig. No intercellular bacteria were observed within the stem cortex away from the wound sites Fig. Although they were not usually seen within leaves, in some specimens bacteria were observed entering via stomata, and the latter appeared to be damaged by this invasion process not shown. Both the bacteria and the matrix reacted with the G.
Open in new tab Download slide Micrographs from Experiment 1 showing colonization of micropropagated sugar cane plantlets by Gluconacetobacter diazotrophicus at 10 d after inoculation. This section was incubated with an antibody raised against G.
The gold labelling was visualized for light microscopy using silver enhancement, and the background was lightly stained with a dilute solution of toluidine blue 0. This is taken from a serial section to that in a and was not immunogold labelled. This section was treated as a and again shows immunogold labelled G.
Note that much of the antibody reaction is with extracellular material arrows. There are no gold particles on the section. Large numbers of bacteria have accumulated at the surface of the wound, and many have colonized damaged cells small arrows.
The space contains a number of bacteria that are immunogold labelled with an antibody raised against G. The xylem vessels, which appear to be unoccluded cf. P, phloem. This section was immunogold labelled with an antibody raised against the Fe NifH — protein of nitrogenase. One of the bacteria is clearly senescent in appearance, with disintegrating cytoplasm S , and is not immunogold labelled.
W, xylem cell wall.
With the sequenced genome available to researchers, new strides have been made in understanding the many processes of G. Recently, with the aid of the sequenced Pal5 genome, a putative FeSII coding gene was identified which opened the possibility of G. However, oxygen is not the only inhibitor of nitrogenase, reactive oxygen species ROS , by-products of aerobic metabolism critical in the production of ATP for the high energy-demanding process of nitrogen fixation, have also proven to be inhibitors of nitrogenase [ 37 , 93 ]. While ROS levels were expected to increase during nitrogen fixation and elevated aerobic respiration, they in fact decreased within G. Asparagine, important to microbial growth promotion, is also a nitrogenase inhibitor and has been found in high amounts in many of G. Genome sequencing has shown that G.