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.
We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N 2 -fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N 2 fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N 2 fixation arises in association with nondiazotrophic endophytes.Microbes are not always culturable even though their biological activities may be detectable (1,15,20). This is true for some N 2 -fixing bacteria associated with plants, such as Azoarcus endophytes (16) and rhizobial bacteroids (22). Although the functional significance of microbial consortia in biofilms, for example (1, 3), has been emphasized, there are few concrete examples of their specific functions.The availability of fixed nitrogen limits primary productivity in plant ecosystems. During their evolution, legumes have acquired a symbiotic relationship with rhizobia that fix atmospheric nitrogen. Among nonleguminous plants, several diazotrophs have been isolated and characterized as nitrogenfixing endophytes, including Acetobacter (18), Azoarcus (11,16), and Herbaspirillum (6,8). Endophytes are microorganisms that spend most of their life cycles inside plant tissues without causing symptoms of plant damage (16). We still do not know whether these diazotrophic endophytes contribute substantially to the nitrogen economy of grasses (11,12). It is possible that we have overlooked the real contributors to nitrogen fixation in nonleguminous plants. Indeed, nitrogenase transcript analysis has indicated that endophytes, such as Azoarcus sp. and others in an apparently unculturable state, fix nitrogen in plants (11).Wild grasses can often grow in nitrogen-deficient soils, suggesting that functioning diazotrophic bacteria are associated with them. We therefore tried to isolate and characterize diazotrophic bacteria associated with wild rice species in situ and pioneer plants growing on a devastated lahar area with volcanic eruptions. For this work, we used mainly the aerial parts of plants as isolation materials to avoid bacterial contamination from soils. During efforts to isolate endophytic diazotrophs from these plants, we faced problems with unculturable diazotrophic bacteria and found an anaerobic nitrogen-fixing consortium (ANFICO) consisting of N 2 -fixing clostridia and diverse nondiazotrophic bacteria. The objective of this work was to clarify the members of ANFICOs and their interactions. MATERIALS AND METHODSIsolation of nitrogen-fixing bact...
Microbial isolate Z143-1 found to be associated with an unidentified tunicate was characterized due to its significant antimicrobial activity. Z143-1 is similar to Pseudovibrio ascidiaceicola and Pseudovibrio denitrificans in morphological, physiological and biochemical characteristics, except for its ability to ferment glucose and produce a characteristic red pigment. Fatty acid methyl ester analysis revealed a predominance of the fatty acid 18:1 omega7c at 80.55%, at levels slightly lower than the Pseudovibrio denitrificans type strain DN34(T) (87.7%). The mol% G+C of Z143-1 is 54.02, relatively higher than the Pseudovibrio denitrificans type strain DN34(T) and Pseudovibrio ascidiaceicola with mol% G+C of 51.7 and 51.4, respectively. However, phylogenetic analysis of the 16S rRNA gene sequence of Z143-1 showed 100% similarity with the Pseudovibrio denitrificans type strain DN34(T). In this study, the bacterium Z143-1 is reported as a new strain of Pseudovibrio denitrificans. While there is no report of a secondary metabolite for Pseudovibrio denitrificans, Z143-1 produces the red pigment heptylprodigiosin, also known as 16-methyl-15-heptyl-prodiginine, which shows anti-Staphylococcus aureus activity.
Acid- and gas-producing nitrogen-fixing bacteria associated with rice roots and leaf sheaths were isolated. These isolates along with reference enterobacteria strains were characterized biochemically and serologically. Using selected cultural and biochemical tests, all isolates from rice were identified as Enterobacter cloacae, except two strains which were found similar to Klebsiella pneumoniae. However, further biochemical tests showed that the Klebsiella isolates were K. planticola. Antisera and fluorescent antibodies (FA) were prepared against K. pneumoniae M5a1, K. planticola DWUL2, and E. cloacae EnSs. The results of FA cross-reactions of 28 strains isolated from rice plants and of other type cultures showed a separation into two different serogroups: E. cloacae and K. planticola. However, the cross-reactions in gel immunodiffusion were found to be extensive and widespread. All strains of Enterobacteriaceae tested produced at least one immunodiffusible precipitin band with any one or all antisera. The population of N2-fixing enterobacteria associated with the root and stem of rice fell within the range of 103 and 105 per gram dry weight. The percentage incidence of N2-fixing enterobacteria among the aerobic heterotrophic bacteria in the root and stem was found to be less than 1%.
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