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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.32 no.3 São Paulo Aug./Oct. 2001 



Olmar B. Weber1*; Leonardo M. Cruz2; José I. Baldani3; Johanna Döbereiner4

1Embrapa ¾ Centro Nacional de Pesquisa de Agroindústria Tropical, Fortaleza, CE, Brasil. 2Departamento de Bioquímica - Universidade Federal do Paraná, Curitiba, PR, Brasil. 3Embrapa ¾ Centro Nacional de Pesquisa de Agrobiologia, Seropédica, RJ, Brasil. 4In memoriam

Submitted: December 11, 2000; Returned to authors for corrections: March 12, 2001; Approved: September 03, 2001





Diazotrophic bacteria isolated from banana plants were characterized by morphological and physiological aspects. Three different groups of these plant-bacteria could be established. Two of them showed similarity to species of the Herbaspirillum genus. The third one was different because used only a few carbon substrates and produced water diffusible compounds that fluoresced under UV light. All three bacterial groups were thin rods with mono or bipolar flagella, presented negative reaction in Gram stain, showed catalase activity, were able to reduce nitrate and grew better in semi-solid JNFb medium at 31ºC. The nitrogenase activity was detected in semi-solid N-free JNFb medium and expressed higher values when pH ranged from 6.5 to 7.0 (groups I and II) and 6.0 to 6.5 (group III). The diazotrophs isolated from banana plants were distinct from species of Herbaspirillum previously identified in gramineous plants.

Key words: Herbaspirillum, Herbaspirillum-like, Musa spp., microbial ecology



Over the last decades there has been a great interest in nitrogen fixing-bacteria associated to non-legumes, principally gramineous plants. The bacterial genera Azospirillum (10,27), Herbaspirillum (2,3,13,23), Burkholderia (4,16) and Gluconoacetobacter (8) were identified in these plants. In addition, diazotrophs also occurred in sweet potato (17), cassava (5), coffee (19), pineapple (27,29) and banana plants (29).

The banana fruit crop is widely cultivated in tropical areas where high dosages of fertilizers are commonly applied. The biological nitrogen fixation could be an alternative for this crop system, once the plants are able to establish association with Herbaspirillum-like and Burkholderia related bacteria (29), which are not well known yet. This work aimed physiological and morphological characterization of the Herbaspirillum-like bacteria isolated from banana plants.

Among more than twenty-five strains of Herbaspirillum-like bacteria previously obtained (29), the representative diazotrophs from root samples of banana Caipira (BA12), stems of cultivars Butuhan (BA10) and Caipira (BA14, BA22), and leafs of cultivars Butuhan (BA11), Caipira (BA13), Prata Anã (BA22) and Maçã (BA87) were included in this study. All isolates were activated in semi-solid NFb medium (11) and grown on solid NFb and 79 + potato agar media (4) during three days at 30ºC. Controls (Bacillus sp. and Escherichia coli), grown on the last medium, and diazotrophs were evaluated for Gram stain, catalase and oxidase reactions (26). The nitrate reduction (7) with nitrite accumulation (21) of diazotrophs was evaluated after two days incubation in liquid DYGS medium (25) modified by adding 1 g/L of malate and pH adjusted to 6.5. All isolates and a strain of Pseudomonas fluorescens were also evaluated for fluorescence of siderophores on King B medium under UV light after two days incubation at 30ºC (20).

Nitrogenase activity of diazotrophic bacteria isolated from banana and type strains Herbaspirillum seropedicae Z67T and Herbaspirillum rubrisubalbicans M4T was evaluated in JNFb medium, at different pH and temperature of incubation (3). The inocula were prepared in liquid DYGS medium incubated for 24 h at 30ºC. Adjustment of pH (5.0, 5.5, 6.0, 6.5, 7.0 and 7.5) was made by adding sterile 5% H2SO4 or 10% KOH to sterile semi-solid JNFb medium. Three vials containing 5 mL of this medium were inoculated with 15 µL aliquots of bacterial suspensions and incubated at 30ºC during 52 h. In the temperature experiment, the pH of semi-solid JNFb medium was adjusted to 6.0 and incubated for 48 h at 28, 31, 34, 37 and 41ºC. Nitrogenase activity in both experiments was determined by the C2H2 reduction method, after 1 h of incubation at 30ºC.

The cell morphology of isolates BA10, BA12 and BA22 grown on 79 + potato agar medium during two days at 30ºC was observed under transmission electron microscopy (TEM) ZEISS M-900 operated at 80 kW. The cells were fixed on silver 200 Hex Mesh grids coated with Formvar and stained with 5% uranil acetate solution (22).

The capability of isolates BA10, BA11, BA12, BA13, BA22, BA23 and Z67T to survive in soil was also evaluated. All strains were grown in liquid DYGS medium for 24 h at 30ºC, centrifuged for 20min at 4.000 rpm and suspended in saline solution to 1.0 OD at 600 nm. Aliquots (1 mL) of bacterial suspensions were inoculated into 10 g samples of red-yellow podzolic soil (typic Hapludult). The soil samples were moistened to 70% field capacity and incubated at 30ºC. Three samples of each soil treatment were harvested at different times (1 h and 8, 16 and 24 days) and submitted to estimation of MPN diazotrophs, according to Döbereiner et al. (11).

On solid NFb medium, the banana plant Herbaspirillum-like bacteria formed colonies with blue centers. In semi-solid N-free JNFb medium, they formed veil-like membranes near the surface. All isolates were Gram negative, catalase and oxidase positive and were able to reduce nitrate in liquid DYGS medium similarly to H. seropedicae (3) and H. rubrisubalbicans (2,23).

The three bacterial groups were able to form good pellicles in semi-solid media containing D-mannitol, D-sorbitol, glycerol, citrate, a-ketoglutarate, succinate, fumarate and malate as sole carbon source. The isolates of group I were able to use N-acetyl-glucosamine, those of group II used meso-erythritol + NH4, and those of group III were unable to use these carbon sources in semi-solid media (Table 1). The characteristic observed in isolates of group I was similar to that described for H. seropedicae (3), while those of group II was similar to H. rubrisubalbicans (2). However, differently from these species of Herbaspirillum, the two first groups were able to use L-tartrate as sole carbon source. The groups I and II had the same pattern as H. rubrisubalbicans in ARDRA analysis and showed phylogenetic relationship among the Herbaspirillum cluster (9). Both bacterial groups failed to react with species-specific oligonucleotide probes (29) and may represent new Herbaspirillum species.

The isolates from group III were able to produce blue fluorescent compounds on King B medium under UV light (Table 1), which has never been observed with diazotrophs before. The pigment production has been detected in plant pathogenic Pseudomonas species (18), Burkholderia cepacia (14) and members of Comamonadaceae family (30) without nitrogenase activity. The phylogenetic relationship of ARDRA types from the bacterial group III with the Comamonadaceae was observed (9). The pigment production was a characteristic described for Hidrogenophaga, Xilophilus and Brachymonas genera (30).

The bacterial group III may represent a new genus of diazotrophic bacteria. The representative isolate (BA22) from the group was a straight rod (1.8 µm long and 0.6 to 0.8 µm wide) with only one polar flagellum (Table 1 and Fig. 1), distinguishing it from the known species of Herbaspirillum genus identified in gramineous plants. Short rods with one polar flagellum were observed in N2-fixing Pseudomonas sp. isolated from rhizosphere of Oryza sativa (6) and described for Brachymonas, Hidrogenophaga and Xilophilus genera (30). However, these bacteria fermented sucrose, except the Brachymonas genus, which occurred in activated sludge (30). Another dinitrogen-fixing Pseudomonas sp., isolated from Deschampsia caespitosa, was a slightly curved rod and presented tuff polar flagella (15).

The bacterial groups I and II presented curved rod-shaped cells (Table 1 and Fig. 1) and were mobile in water, with increased movement next to air bubbles. The isolate BA10 (representative from group I) measured 2.0 µm by 0.6 to 0.7 µm and presented one to three polar flagella or 1+1 and 1+2 flagella in both cell poles, similarly to H. seropedicae (3). The isolate BA12 (representative of group II) presented 1.9 µm by 0.7 to 0.8 µm, with flagella similar to isolate BA10. Bipolar flagella had never been observed in strains of H. rubrisubalbicans (23).

Isolates from groups I and II presented higher nitrogenase activity in N-free JNFb medium when the pH ranged from 6.5 to 7.0 (Fig. 2) and the temperature was 31ºC (Fig. 3). No nitrogenase activity was detected in semi-solid JNFb medium at 41ºC. The optimal pH value for H. seropedicae ranged from 5.0 to 8.0 in malate nitrogen-free semi-solid medium, at 34ºC (3). Concerning the bacterial group III, we detected higher nitrogenase activity when the pH ranged from 6,0 to 6.5 (Fig. 2) and temperature of 31ºC (Fig. 3). The dinitrogen-fixing Pseudomonas sp. from rice plants grew better on semi-solid NFb medium with 0.5% glucose and 0.01% yeast extract, with pH ranging from 6.5 to 7.0 and temperature ranging from 30 to 35ºC (28).




The three bacterial groups survived poorly in a red-yellow podzolic soil. After three weeks, bacteria from groups II and III were no more detectable in the soil while less than 101 bacteria/g was observed for bacteria from group I. The poor surviving capability was also observed for bacteria of Herbaspirillum genus (23) and for Burkholderia brasilensis (4) and could be an indication of endophytic diazotrophs, as related by Döbereiner (12) and Baldani et al. (1). Further, the populations (102 to 103 bacteria per gram) of native Azospirillum brasilense and Azospirillum lipoferum present in the control soil showed no decrease during the incubation time. This genus of diazotrophic bacteria has been originally isolated from rhizosphere of gramineous plant (12) and behaves as facultative endophytic bacterium (1).



The authors would like to express deep appreciation to Dr. Francisco Freire of Embrapa Tropical Agroindustry, for critically reviewing the manuscript.




Bactérias do tipo Herbaspirillum em bananeira

Bactérias diazotróficas do tipo Herbaspirillum isoladas de bananeiras foram avaliadas pelas características morfológicas e fisiológicas de crescimento. Três grupos de bactérias foram estabelecidos, sendo dois relacionados às espécies de Herbaspirillum e diferentemente o terceiro grupo apresentou habilidade em crescer com poucos substratos orgânicos e produziu substância fluorescente em meio B de King. As bactérias dos três grupos eram bastonetes com flagelos mono ou bipolares, apresentaram reação negativa na coloração de Gram, expressaram atividade de catalase e oxidade, foram capazes de reduzir o nitrato e cresceram melhor em meio JNFb semi-sólido incubado a 31ºC. A atividade da nitrogenase, medida através da atividade de redução de acetileno, foi máxima em meio JNFb semi-sólido, após o ajuste de pH na faixa de 6,0 a 7,0 (grupos I e II) e 5,5 a 6,5 (grupo III). As bactérias diazotróficas associadas às bananeiras são diferentes das espécies de Herbaspirillum anteriormente identificadas em gramíneas.

Palavras-chave: Herbaspirillum, bactérias tipo Herbaspirillum, Musa spp., ecologia microbiana




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* Corresponding author. Mailing address: Rua Dra. Sara Mesquita, 2270, Planalto Pici, 60511-110, Fortaleza, CE, Brasil, Fax: (+55 85) 299-1833, E-mail:

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