A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.

Six closely related N 2 -fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15 N 2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.

Cereals such as maize, rice, wheat and sorghum are the most important crops for human nutrition. Like other plants, cereals associate with diverse bacteria (including nitrogen-fixing bacteria called diazotrophs) and fungi. As large amounts of chemical fertilizers are used in cereals, it has always been desirable to promote biological nitrogen fixation in such crops. The quest for nitrogen fixation in cereals started long ago with the isolation of nitrogen-fixing bacteria from different plants. The sources of diazotrophs in cereals may be seeds, soils, and even irrigation water and diazotrophs have been found on roots or as endophytes. Recently, culture-independent molecular approaches have revealed that some rhizobia are found in cereal plants and that bacterial nitrogenase genes are expressed in plants. Since the levels of nitrogen-fixation attained with nitrogen-fixing bacteria in cereals are not high enough to support the plant’s needs and never as good as those obtained with chemical fertilizers or with rhizobium in symbiosis with legumes, it has been the aim of different studies to increase nitrogen-fixation in cereals. In many cases, these efforts have not been successful. However, new diazotroph mutants with enhanced capabilities to excrete ammonium are being successfully used to promote plant growth as commensal bacteria. In addition, there are ambitious projects supported by different funding agencies that are trying to genetically modify maize and other cereals to enhance diazotroph colonization or to fix nitrogen or to form nodules with nitrogen-fixing symbiotic rhizobia.

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SummaryThe early nodulin ENOD40 has been proposed as playing a pivotal role in the organogenesis of legume root nodules. We have isolated the ENOD40 gene homologues ObENOD40 and OsENOD40 from the wild and cultivated rice genotypes Oryza brachyantha and Oryza sativa, respectively. Rice ENOD40s contain a sequence at the 5Ј end (region I) for encoding an oligopeptide that is highly conserved in all legume ENOD40s. Furthermore, at the 3Ј end (region II), the nucleotide sequence of rice ENOD40s exhibited a considerable homology to the corresponding region in legume ENOD40s. Among various organs of the rice plant, expression of OsENOD40 was detected only in stems. In situ hybridization studies revealed that, within the stem, transcription of OsENOD40 is confined to parenchyma cells surrounding the protoxylem during the early stages of development of lateral vascular bundles that conjoin an emerging leaf. Expression pattern of OsENOD40 promoter-GUS fusion in nodules developed on transgenic hairy roots of soybean was also found to be restricted to peripheral cells of nodule vascular bundles, thus evidencing that rice ENOD40 promoter activity is essentially the same as that of soybean ENOD40. Taken together, these results strongly suggest that OsENOD40 and legume ENOD40s share common, if not identical, functions in differentiation and/or function of vascular bundles.

Summary• Varieties of rice ( Oryza sativa ) differing in tolerance to aluminium (Al) were evaluated for their N-fixation ability after inoculation with a gusA -marked strain of Herbaspirillum seropedicae Z67.• Under axenic conditions, by 30 d, inoculation resulted in enhanced nitrogenase activity, d. wt, total N and total C content only in the Al-tolerant varieties, and one (cv. Moroberekan) showed significantly more 15 N 2 incorporation than an Al-sensitive variety ('IR45'). There were no differences in the number of the bacteria colonizing the different varieties, but the Al-tolerant ones secreted larger amounts of C in their root exudates, and bacteria colonizing the roots of cv. Moroberekan strongly expressed gusA and NifH proteins.• Under glasshouse conditions, by 30 d, inoculation resulted in increased growth of both cvs IR45 and Moroberekan, but the latter showed significantly greater nitrogenase activity and 15 N dilution. In a long-term experiment, by 120 d, cv. Moroberekan showed a significant increase in N content after inoculation.• Herbaspirilla were localized on and within roots and aerial parts of cvs Moroberekan and IR45 under both growth conditions. The role of N fixation in growth promotion of rice by H. seropedicae is discussed in terms of availability of C.

SummaryCajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F 1 hybrids. The F 1 hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F 2 , F 3 and F 4 progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F 0 parental cross.As a route of introgression, diploid F 1 hybrids were backcrossed with cultivated pigeonpea and BC 1 progeny obtained by in vitro culture of aborting embryos. BC 2 plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F 1 and BC 1 plants, it was possible to produce BC 2 and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.