Infection of sugar cane by the nitrogen-fixing bacterium Acetobacter diazotrophicus

Significant nitrogen fixation has recently been demonstratedin Brazilian sugar cane (Saccharum officinarum) cultivars knownto form associations with a number of diazotrophs, includingAcetobacter diazotrophicus, an acid-tolerant endophytic bacteriumwhich grows best on a sucrose-rich medium. In a series of experiments,aseptically-grown sugar cane plantlets were rooted in a liquidmedium and inoculated with A. diazotrophicus originally isolatedfrom field-grown sugar cane. After 4, 7, 9, and 15 d, plantswere examined under light, scanning and transmission electronmicroscopes and the presence of A. diazotrophicus on and withinplant tissues was confirmed by immunogold labelling. By 15 d,external bacterial colonization was seen on roots and lowerstems, particularly at cavities in lateral root junctions. Theloose cells of the root cap at root tips were a site of entryof the bacteria into root tissues. Both at lateral root junctionsand root tips, bacteria were also seen in enlarged, apparentlyintact, epidermal cells. After 15 d, bacteria were present inxylem vessels at the base of the stem, many connected via mucusto spiral secondary thickening. There was no obvious pathogenicreaction to the bacteria within the xylem. From these observations,it is proposed that, under experimental conditions, A. diazotrophicusfirstly colonized the root and lower stem epidermal surfacesand then used root tips and lateral root junctions to enterthe sugar cane plant where it was distributed around the plantin the transpiration stream. It is further suggested that thexylem vessels in the dense shoots of mature plants are alsoa possible site of N₂-fixation by diazotrophs as they providethe low pO₂ and energy as sucrose necessary for nitrogenaseactivity. Key words: Acetobacter diazotrophicus, endophyte, infection, nitrogen fixation, sugar cane.     

Herbaspirillum, an endophytic diazotroph colonizing vascular tissue 3Sorghum bicolor L. Moench

Leaves of Sorghum bicolor were examined at 5 d and 14 d afterinoculation with the N₂-fixing endophytic bacteria Herbaspirillumseropedicae and Herbaspirillum rubrisubalbicans. Plants inoculatedwith H. rubrisubalbicans expressed symptoms of ‘red stripedisease’ i.e. red stripes along the secondary veins ofthe leaf blade close to the inoculation point and spreadingup the leaves. Infected leaves showed dense colonization byH. rubrlsubalbicans in regions showing red stripe symptoms at5 d after inoculation. The infection was confined within thevascular system, in particular, the protoxylem and associatedlacunae, which were often completely filled with bacteria, withsome of the latter expressing nitrogenase. The bacteria wererecognized using H. rubrisubalbicans-speciflc antibodies andimmunogold labelling, which also showed that the antibody reactedwith material on the surface of the bacteria, and that thismucus was released into the lumen of the xylem. At 14 d afterinoculation, disease symptoms were slightly more severe, withboth meta- and protoxylem being even more heavily colonizedin parts of the leaf showing red stripes. However, a stronghost defence response was also apparent at this stage, withgums lining the walls of the vessels and enclosing the bacteria,although the latter were still actively dividing. At the edgesof visible disease symptoms, plant gums filled the xylem; bacteriahad formed distinct colonies within these gums, with some ofthe colonies associated with the xylem walls. Plants inoculatedwith H. seropedicae either did not express the disease or showedvery mild symptoms close to the inoculation point. In the lattercase, H. seropedicae were localized within protoxylem vesselsand the metaxylem was partly occluded with plant-derived gums.By contrast with H. rubrisubalbicans, H. seropedicae was alsolocalized in leaves at 14 d without disease symptoms and didnot always appear to elicit a host response, i.e. they colonizedthe walls of metaxylem, with the xylem vessels themselves remainingunoccluded and largely free of gums. The fine line separatingplant pathogens, endophytes and symbioses is discussed in lightof these results. Key words: Herbaspirillum, Sorghum bicolor, nitrogen fixation, endophyte, xylem