Bukholderia strains promote Mimosa spp. growth but not Macroptilium atropurpureum1 1 Dados extraídos parcialmente da Dissertação de Mestrado da primeira autora

Estirpes de Burkholderia promovem o crescimento de Mimosa spp. mas não o de Macroptilium atropurpureum

Kaliane Sírio Araújo Fernanda de Carvalho Fatima Maria de Souza Moreira About the authors

ABSTRACT

The aim of this study was to evaluate the relationship and symbiotic efficiency of 14 strains of Burkholderia isolated from rupestrian grasslands, using M. atropurpureum and Mimosa tenuiflora as trap plants, with the species M. atropurpureum, Mimosa bimucronata and M. foliolosa. For the nodulation and symbiotic efficiency test in M. atropurpureum, long-neck bottles containing nutrient solution were used. The experiments with Mimosa spp. were carried out in tubes containing vermiculite (160 cm3) and sand (80 cm3) (2:1). The parameters under evaluation were number of nodules, nodules dry matter production, shoots dry matter, roots dry matter, and total dry matter production for all the species analyzed; and plant height, diameter, and the Dickson quality index for Mimosa species. Of the 14 tested strains, two nodulated M. atropurpureum; however, they were ineffective in promoting plant growth. All the tested strains established symbiosis with M. bimucronata, and 12 strains nodulated M. foliolosa. Of these, six promoted growth in M. bimucronata, and seven presented symbiotic efficiency in M. foliolosa. The strains UFLA 01-739, UFLA 01-748 and UFLA 01-751, isolated from M. tenuiflora, and UFLA 04-260 and UFLA 04-405, isolated from M. atropurpureum, stood out as potential inoculants for the Mimosa species evaluated in this study.

Key words:
Rupestrian grasslands; Nitrogen-fixing bacteria; Symbiosis; Mimosa bimucronata; Mimosa foliolosa

RESUMO

O objetivo deste trabalho foi avaliar a relação e a eficiência simbiótica de 14 estirpes de Burkholderia isoladas de campos rupestres, utilizando M. atropurpureum e Mimosa tenuiflora como plantas iscas, com as espécies: M. atropurpureum, Mimosa bimucronata e M. foliolosa. Para o teste de nodulação e eficiência simbiótica em M. atropurpureum foram utilizadas garrafas do tipo long neck, contendo solução nutritiva. Os experimentos com as Mimosa spp. foram realizados em tubetes contendo vermiculita (160 cm3) e areia (80 cm3) (2:1). Os parâmetros avaliados foram número de nódulos, produções de massa de matéria seca de nódulos, da parte aérea, da raiz e total para todas as espécies vegetais analisadas, e altura da planta, diâmetro, e o índice de qualidade de Dickson para as espécies de Mimosa. Das 14 estirpes avaliadas, duas foram capazes de nodular M. atropurpureum. Entretanto, estas foram ineficientes na promoção de crescimento vegetal. Todas as estirpes testadas estabeleceram simbiose com M. bimucronata e 12 estirpes nodularam M. foliolosa. Destas, seis promoveram o crescimento de M. bimucronata e sete apresentaram eficiência simbiótica em M. foliolosa. Destacam-se as estirpes UFLA 01-739, UFLA 01-748, UFLA 01-751 isoladas de M. tenuiflora e UFLA 04-260 e UFLA 04-405 isoladas de M. atropurpureum como potenciais inoculantes para as espécies de Mimosa avaliadas.

Palavras-chave:
Campos rupestres; Bactérias fixadoras de nitrogênio; Simbioses; Mimosa bimucronata; Mimosa foliolosa

INTRODUCTION

Bacteria of the Burkholderia genus occupy a variety of ecological niches, and are exploited for several purposes, such as biological control, bioremediation, and plant growth promotion (COENYE; VANDAMME, 2003COENYE, T.; VANDAMME, P. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environmental Microbiology, v. 5, n. 9, p. 719-729, 2003.). The first Burkholderia strains with nodulation capacity were proven in Macroptilium atropurpureum (DC), which were isolated from nodules of Aspalathus carnosa Bergius and Machaerium lunatum (L. f.) Ducke (MOULIN et al., 2001MOULIN, L. et al. Nodulation of legumes by members of the b-subclass of Proteobacteria. Nature, v. 411, n. 6840, p. 948-950, 2001.).

Since then, it has been proven the ability of Burkholderia species to nodulate M. atropurpureum (ANGUS et al., 2013ANGUS, A. A. et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant and Soil, v. 369, n. 1, p. 543-562, 2013.; ELLIOT et al., 2007ELLIOTT, G. N. et al. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum Annals of Botany, v. 100, n. 7, p. 1403-1411, 2007.; LIMA et al., 2009LIMA, A. S. et al. Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant and Soil, v. 319, n. 1, p. 127-145, 2009.), Cyclopia spp. (ELLIOT et al., 2007), Vigna unguiculata (L.) Walp (SOARES et al., 2014SOARES, B. L. et al. Cowpea symbiotic efficiency, pH and aluminum tolereance in nitrogen-fixing bactéria. Scientia Agricola, v. 71, n. 3, p. 171-180, 2014.), Phaseolus vulgaris L. (FERREIRA et al., 2012FERREIRA, P. A. A. et al. Efficient nitrogen-fixing Rhizobium strains isolated from amazonian soils are highly tolerant to acidity and aluminium. World Journal of Microbiology and Biotechnology, v. 28, n. 5, p. 1947-1959, 2012.; TALBI et al., 2010TALBI, C. et al. Burkholderia phymatum strains capable of nodulating Phaseolus vulgaris are present in Moroccan soils. Applied and Environmental Microbiology, v. 76, n. 13, p. 4587-4591, 2010.), and Mimosa spp. (BARRET; PARKER, 2005BARRET, C. F.; PARKER, M. A. Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama. Systematic and Applied Microbiology, v. 28, n. 1, p. 57-65, 2005.; BONTEMPS et al., 2010BONTEMPS, C. et al. Burkholderia species are ancient symbionts of legumes. Molecular Ecology, v. 19, n. 1, p. 44-52, 2010.; CHEN et al., 2003CHEN, W. M. et al. Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature. Journal of Bacteriology, v. 185, n. 24, p. 7266-7272, 2003., 2005CHEN, W. M. et al. Proof that Burkholderia forms effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America. Applied and Environmental Microbiology, v. 71, n. 11, p. 7461-7471, 2005., 2006CHEN, W.-M. et al. Burkholderia mimosarum sp. nov., isolated from root nodules of Mimosa spp. From Taiwan and South America. International Journal of Systematic and Evolutionary Microbiology, v. 56, n. 8, p. 1847-1851, 2006.; REIS JÚNIOR et al., 2010REIS JÚNIOR, F. B. dos et al. Nodulation and nitrogen fixation by Mimosa spp in the Cerrado and Caatinga biomes of Brazil. New Phytologist, v. 186, n. 4, p. 934-946, 2010.).

Although it has been proven the nodulation of M. atropurpureum and Mimosa spp. by Burkholderia strains, the symbiotic effectiveness of these relationships is still unknown. The efficiency of these strains has been mainly reported by means of observations by the nodules internal color, and it was verified the presence or absence of leghemoglobin. Moulin et al. (2001)MOULIN, L. et al. Nodulation of legumes by members of the b-subclass of Proteobacteria. Nature, v. 411, n. 6840, p. 948-950, 2001. and Barrett and Parker (2005)BARRET, C. F.; PARKER, M. A. Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama. Systematic and Applied Microbiology, v. 28, n. 1, p. 57-65, 2005. verified that nodules formed in M. atropurpureum were ineffective due to the whitish color, and did not observe the presence of leghemoglobin. Chen et al. (2005)CHEN, W. M. et al. Proof that Burkholderia forms effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America. Applied and Environmental Microbiology, v. 71, n. 11, p. 7461-7471, 2005. suggested that the strains of Burkholderia Br3407, Br 3454, Br 3461, Br3469 and MAP3-5 were efficient in Mimosa pudica L., M. diplotricha C. Wright ex Sauvalle, M. pigra L. and M. acutispula Benth. due to the internal reddish color of the nodule. In addition to nodules internal color, some authors report the efficiency and/or inefficiency of Burkholderia strains by means of the evaluation of nitrogenase activity in Mimosa spp. nodules. (CHEN et al., 2005CHEN, W. M. et al. Proof that Burkholderia forms effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America. Applied and Environmental Microbiology, v. 71, n. 11, p. 7461-7471, 2005.; ELLIOTT et al., 2007ELLIOTT, G. N. et al. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum Annals of Botany, v. 100, n. 7, p. 1403-1411, 2007.; REIS JÚNIOR et al., 2010REIS JÚNIOR, F. B. dos et al. Nodulation and nitrogen fixation by Mimosa spp in the Cerrado and Caatinga biomes of Brazil. New Phytologist, v. 186, n. 4, p. 934-946, 2010.), and few studies have evaluated the development of the plants inoculated with Burkholderia strains. It was observed the efficiency of Burkholderia phymatum (STM815T) to promote the growth of Mimosa pudica (ELLIOTT et al., 2007), but the same was not observed when this species was inoculated with Burkholderia Hpud10.4(C) (BARRETT; PARKER, 2006BARRET, C. F.; PARKER, M. A. Coexixtence of Burkholderia, Cupriavidus and Rhizobium sp. nodule bacteria on two Minosa spp. in Costa Rica. Applied and Environmental Microbiology, v. 72, n. 2, p. 1198-12016, 2006.). On the other hand, in M. atropurpureum, it was verified greater dry matter production and shoot length of plant when it was inoculated with Burkholderia tuberum (STM678T). However, this increase was attributed to other processes rather than biological nitrogen fixation, since the observed nodules were ineffective (ANGUS et al., 2013ANGUS, A. A. et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant and Soil, v. 369, n. 1, p. 543-562, 2013.).

Considering the economic and ecological importance of M. atropurpureum and Mimosa spp., as well as the biotechnological potential of Burkholderia strains, this study aimed to verify the relationship and symbiotic efficiency of Burkholderia strains isolated from nodules of M. atropurpureum and Mimosa tenuiflora (Willd.) Poir. cultivated in rupestrian grasslands soils in M. atropurpureum, Mimosa bimucronata (DC.) Kuntze and Mimosa foliolosa Benth. subsp. pachycarpa (Benth.) Barneby var. pachycarpa.

MATERIAL AND METHODS

The 14 Burkholderia sp. strains evaluated regarding their relationship and symbiotic efficiency in Macropitilium atropurpureum, Mimosa bimucronata and M. foliolosa were isolated from nodules of M. atropurpureum and M. tenuiflora grown in distinct soils of rupestrian grasslands, located in Serra do Cipó, Minas Gerais, Brazil (CARVALHO, 2010CARVALHO, F. Abundância de espécies de plantas e diversidade de simbiontes radiculares em campos rupestres da serra do cipó, MG. Tese (Doutorado em Ecologia, Conservação e Manejo da Vida Silvestre) - Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 2010.). Table 1 shows the cultural characteristics and origin of these strains. The 14 strains were deposited in the GenBank under the accession number KT957898 to KT957912.

Table 1
Identification and origin of the 14 Burkholderia strains used in authentication trials, and symbiotic efficiency in M. atropurpureum,M. bimucronata and M. foliolosa, (CARVALHO, 2010CARVALHO, F. Abundância de espécies de plantas e diversidade de simbiontes radiculares em campos rupestres da serra do cipó, MG. Tese (Doutorado em Ecologia, Conservação e Manejo da Vida Silvestre) - Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 2010.)

Three experiments were carried out in a greenhouse. Each experiment consisted of 17 treatments: 14 treatments corresponded to the inoculation of each Burkholderia strain in nutrient solution (HOAGLAND; ARNON, 1950HOAGLAND, D. A.; ARNON, D. D. The water-culture method for growing plants without soil. Berkeley: California Agricultural of Experiment Station, 1950. 32 p. (Circular 347).), containing low mineral N concentration (5.25 mg L-1); two uninoculated negative controls, one with low mineral N concentration (5.25 mg L-1), and another with high mineral N concentration (52.5 mg L-1); and one positive control in nutrient solution with low mineral N concentration (5.25 mg L-1), inoculated with a nodulating strain, according to the host plant species UFLA 04-212 - Bradyrhizobium sp, for M. atropurpureum (FLORENTINO et al., 2009FLORENTINO, L. A. et al. Sesbania virgata stimulates the occurrence of its microsymbiont in soils but does not innibit microsymbiont of other species. Scentia Agricola, v. 66, n. 5, p. 667-676, 2009.), BR3460 - Burkholderia gladioli for M. bimucronata (FARIA et al., 1997FARIA, S. M. et al. Obtenção de estirpes de rizóbio eficientes na fixação biológica de nitrogênio para espécies florestais. Seropédica: Embrapa Agrobiologia, 1997. p. 1-4. (Recomendação Técnica, 3).) and UFLA 01-750 - Burkholderia sp. for M. foliolosa. The mineral N sources used were Ca(NO3)2.4H2O; KNO3 and NH4H2PO4.

Each treatment in the experiment with M. atropurpureum consisted of three replications. For the experiment with M. bimucronata and M. foliolosa, each treatment consisted of eight replications.

The experiment with M. atropurpureum was carried out from September 17th 2013 to October 28th 2013, and temperature ranged between 16 to 27 °C. Long neck bottles containing nutrient solution (HOAGLAND; ARNON, 1950HOAGLAND, D. A.; ARNON, D. D. The water-culture method for growing plants without soil. Berkeley: California Agricultural of Experiment Station, 1950. 32 p. (Circular 347).) were used in the experiment. The bottles were autoclaved for one hour, at 1.5 kg cm-2 pressure and 127 °C. M. atropurpureum seeds were surface sterilized using 98% ethyl alcohol (30 seconds), 2% sodium hypochlorite (2 minutes), and then scarified with pure sulfuric acid (20 minutes). After this period, seeds were subjected to successive washes in sterile distilled water. Afterwards, seeds were placed in petri dishes containing sterilized filter paper and moistened cotton, where they remained for 48 hours in a growth chamber, at 28 °C, for the pre-sprouting seed. One plantlet per bottle was used. The 17 strains were grown in liquid culture medium "79" (FRED; WAKSMAN, 1928FRED, E. B.; WAKSMAN, S. A. Laboratory manual of general microbiology. New York: McGraw-Hill Book, 1928. 143 p.) for 3 days for Burkholderia strains, and for 5 days for UFLA 04-212 (Bradyrhizobium sp.), due to differences in the growth time of the strains under shaking (110 rpm), at 28 °C. In each inoculated treatment, plantlets were microbiolyzed with 2 ml inoculum in minimum concentration of 1x108 bacterial cells mL-1. At 40 days, plants were collected to determine number of nodules (NN), shoot dry matter production (SDM), root dry matter (RDM), and total dry matter (TDM), and the result was obtained by the sum of SDM and RDM. Data were subjected to analysis of variance, by using the statistical analysis program SISVAR, version 5.3 (FERREIRA, 2011FERREIRA, D. F. Sisvar: a computer statistical analysis system. Ciência e agrotecnologia, v. 35, n. 6, p. 1039-1042, 2011.). The effects of the treatments were compared by the Scott-Knott test at 5% probability.

Experiments in M. bimucronata and M. foliolosa were carried out, respectively, from July 22nd to October 2nd, 2013, and from September 16th to November 25th, 2013. The temperatures ranged from 13 to 26 °C and from 16 to 27 °C, respectively. Plants were cultivated in 240 cm3 polypropylene tubes, containing vermiculite (160 cm3) and sand (80 cm3) (2:1), and irrigated with nutrient solution (HOAGLAND; ARNON, 1950HOAGLAND, D. A.; ARNON, D. D. The water-culture method for growing plants without soil. Berkeley: California Agricultural of Experiment Station, 1950. 32 p. (Circular 347).), according to the plants need. M. bimucronata and M. foliolosa seeds were disinfected using the same procedure applied in M. atropurpureum seeds, and went through breaking dormancy process for 10 minutes in pure sulfuric acid. Afterwards, they were subjected to successive washings in sterile distilled water. After pre-sprouting seed in growth chamber (28 °C for 48 hours), one plantlet was transferred to each tube. Plants were harvested after 70 days to determine plant height and diameter, number of nodules (NN), nodules dry matter production (NDM), shoot dry matter (SDM), root dry matter (RDM), and total dry matter (TDM), which was obtained by the sum of SDM and RDM. Dickson quality index (DQI) was obtained by the following equation (1):

(1)

In which: SRR is the ratio between shoot dry matter and root dry matter, and HDR is the ratio between shoot height and stem diameter (DICKSON; LEAF; HOSNER, 1960DICKSON, A.; LEAF, A. L.; HOSNER, J. F. Quality appraisal of white spruce and white pine seedling stock in nurseries. Forest Chronicle, v. 36, n. 1, p. 10-13, 1960.).

Data were subjected to analysis of variance by using the statistical analysis program SISVAR version 5.3 (FERREIRA, 2011FERREIRA, D. F. Sisvar: a computer statistical analysis system. Ciência e agrotecnologia, v. 35, n. 6, p. 1039-1042, 2011.). The effects of the treatments were compared by the Scott-Knott test at 5% probability. NN data and NDM were transformed into square root of Y + 0.5. Graphs were obtained by the R software.

RESULTS AND DISCUSSION

UFLA 04-212 (Bradyrhizobium sp.) was able to nodulate and promote growth of M. atropurpureum (Figura 1), which indicates that the experimental conditions were suitable for nodulation. However, of the 14 Burkholderia strains (Table 1), only UFLA 01-726 and UFLA 04-248 (14%) nodulated M. atropurpureum, but inefficiently, i.e., growth was similar to that of the control without inoculation. The species M. atropurpureum has been used as trap plant in studies on diversity since they capture bacteria of the alphaproteobacteria and betaproteobacteria classes (LIMA et al., 2009LIMA, A. S. et al. Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant and Soil, v. 319, n. 1, p. 127-145, 2009.). Although the interaction between Burkholderia and M. atropurpureum enable the formation of nodules (ANGUS et al., 2013ANGUS, A. A. et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant and Soil, v. 369, n. 1, p. 543-562, 2013.; ELLIOT et al., 2007ELLIOTT, G. N. et al. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum Annals of Botany, v. 100, n. 7, p. 1403-1411, 2007.; LIMA et al., 2009LIMA, A. S. et al. Nitrogen-fixing bacteria communities occurring in soils under different uses in the Western Amazon Region as indicated by nodulation of siratro (Macroptilium atropurpureum). Plant and Soil, v. 319, n. 1, p. 127-145, 2009.; MISHRA et al., 2012MISHRA, R. P. N. et al. Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia. Microbiology Ecology, v. 79, n. 2, p. 487-503, 2012.), this interaction is not always considered effective (ANGUS et al., 2013ANGUS, A. A. et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant and Soil, v. 369, n. 1, p. 543-562, 2013.; BARRET; PARKER, 2005BARRET, C. F.; PARKER, M. A. Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama. Systematic and Applied Microbiology, v. 28, n. 1, p. 57-65, 2005.; MOULIN et al., 2001MOULIN, L. et al. Nodulation of legumes by members of the b-subclass of Proteobacteria. Nature, v. 411, n. 6840, p. 948-950, 2001.). Angus et al. (2013)ANGUS, A. A. et al. Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors. Plant and Soil, v. 369, n. 1, p. 543-562, 2013. reported that B. tuberum (STM 678T) favored the growth of M. atropurpureum. However, this effect was attributed to other processes that promote plant growth, since nodules were ineffective. Furthermore, there was no comparison with a positive control for nodulation and efficiency, but with an uninoculated control and with CIAT 899 (Rhizobium tropici) strain, which was inefficient.

Figure 1
Shoot dry matter, root dry matter and total dry matter (g), with coefficient of variation (CV) of 45.3, 23.3 and 31.4%, respectively, of Macroptilium atropurpureum, with the different treatments: treatments with individual inoculation with strains (Table 1 indicates the identification of strains); and two treatments without inoculation: one containing high mineral N concentration - 52.5 mg L-1 (With N), and another containing low mineral N concentration- 5.25 mg L-1 (Minimum N) concentration. All inoculated treatments received low mineral N concentration- 5.25 mg L-1. Columns followed by the same letter do not statistically differ by the Scott-Knott test at 5% probability

Of the four strains isolated from M. atropurpureum nodules (Table 1), only one (UFLA 04-248) nodulated while re-inoculated in this legume. This behavior was also observed by other authors, when Burkholderia fungorum strains isolated from M. atropurpureum did not nodulate after re-inoculation in the same species (SILVA et al., 2012SILVA, K. et al. Diazotrophic Burkholdria species isolated from the Amazon region exhibit phenotypical, functional andd genetic diversity. Systematic and Apllied Microbiology, v. 35, n. 4, p. 253-262, 2012). However, Burkholderia was able to nodulate common bean (FERREIRA et al., 2012FERREIRA, P. A. A. et al. Efficient nitrogen-fixing Rhizobium strains isolated from amazonian soils are highly tolerant to acidity and aluminium. World Journal of Microbiology and Biotechnology, v. 28, n. 5, p. 1947-1959, 2012.).

Burkholderia strains presented higher capacity of establishing symbiosis with the plants of the genus Mimosa, since 100% of the tested strains nodulated M. bimucronata species, and 86% nodulated M. foliolosa species (Tables 2 and 3). The four strains isolated from M. atropurpureum nodulated M. bimucronata, and three nodulated M. foliolosa (Tables 2 and 3). The present study corroborates other researches, which show higher affinity of Burkholderia strains with Mimosa genus. This affinity was indicated by the high frequency of Burkholderia strains isolated from Mimosa spp. located in different ecosystems (BARRET; PARKER, 2005BARRET, C. F.; PARKER, M. A. Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama. Systematic and Applied Microbiology, v. 28, n. 1, p. 57-65, 2005.; BONTEMPS et al., 2010BONTEMPS, C. et al. Burkholderia species are ancient symbionts of legumes. Molecular Ecology, v. 19, n. 1, p. 44-52, 2010.; CHEN et al., 2003CHEN, W. M. et al. Legume symbiotic nitrogen fixation by beta-proteobacteria is widespread in nature. Journal of Bacteriology, v. 185, n. 24, p. 7266-7272, 2003., 2005CHEN, W. M. et al. Proof that Burkholderia forms effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America. Applied and Environmental Microbiology, v. 71, n. 11, p. 7461-7471, 2005., 2006CHEN, W.-M. et al. Burkholderia mimosarum sp. nov., isolated from root nodules of Mimosa spp. From Taiwan and South America. International Journal of Systematic and Evolutionary Microbiology, v. 56, n. 8, p. 1847-1851, 2006., 2007CHEN, W. M. et al. Burkholderia nodosa sp. nov., isolated from root nodules of the woody Brazilian legumes Mimosa bimucronata and Mimosa scabrella International Journal of Systematic and Evolutionary Microbiology, v. 57, n. 5, p. 1055-1059, 2007., 2008CHEN, W. M. et al. Burkholderia sabiae sp. nov., isolated from root nodules of Mimosa caesalpiniifoliaInternational Journal of Systematic and Evolutionary Microbiology, v. 58, n. 9, p. 2174-2179, 2008.; MISHRA et al., 2012MISHRA, R. P. N. et al. Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta-rhizobia. Microbiology Ecology, v. 79, n. 2, p. 487-503, 2012.; REIS JÚNIOR et al., 2010REIS JÚNIOR, F. B. dos et al. Nodulation and nitrogen fixation by Mimosa spp in the Cerrado and Caatinga biomes of Brazil. New Phytologist, v. 186, n. 4, p. 934-946, 2010.). However, Elliot et al. (2007)ELLIOTT, G. N. et al. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum Annals of Botany, v. 100, n. 7, p. 1403-1411, 2007. found that the strain Burkholderia tuberum STM678T isolated from nodules of Aspalathus carnosa (MOULIN et al., 2001MOULIN, L. et al. Nodulation of legumes by members of the b-subclass of Proteobacteria. Nature, v. 411, n. 6840, p. 948-950, 2001.) failed to nodulate three species of Mimosa (Mimosa pigra, M. diplotricha and M. pudica), probably for presenting nodulation gene sequences different from those of nodulating Burkholderia species described, and which establish symbiosis with Mimosa spp. (GYANESHWAR et al., 2011GYANESHWAR, P. et al. Legume-nodulating betaproteobacteria: diversity, host range, and future prospects. The American Phytopathological Society, v. 24, n. 11, p. 1276-1288, 2011.).

Table 2
Number of nodules (NN), nodules dry matter (NDM), shoot dry matter (SDM), root dry matter (RDM), and total dry matter (TDM), plant height and diameter in Mimosa bimucronata and Dickson quality index (DQI) in different treatments
Table 3
Number of nodules (NN), nodules dry matter (NDM), shoot dry matter (SDM), root dry matter (RDM), and total dry matter(TDM), height and diameter of Mimosa foliolosa and Dickson quality index (DQI), in different treatments

In the experiment carried out with the species M. bimucronata, there was significant effects of treatments for most variables NN, NDM, SDM, plant height, diameter, and DQI (Table 2). The strains UFLA 01-732, UFLA 01-733, UFLA 01-739, UFLA 01-748, UFLA 01- 751, UFLA 01-756, UFLA 04-248 and UFLA 04-405 presented higher number of nodules, and were grouped with the inoculant strain for this culture (BR 3460) (Table 2).

These strains, except for UFLA 01-756, also had higher nodules dry matter production. The strains UFLA 01-744, UFLA 01-753, UFLA 04-260, UFLA 04-269, despite not presenting number of nodules similar to that of the inoculant strain (BR 3460), presented equivalent nodules dry matter production (Table 2). In relation to shoot dry matter production, the strains UFLA 01-731, UFLA 01-739, UFLA 01-748, UFLA 04-260, UFLA 04-405 were superior to the inoculant strain, and equivalent to the control with high mineral N concentration (Table 2). There were no differences between treatments in relation to root dry matter and total dry matter (Table 2).

In relation to the height of M. bimucronata, the strains UFLA 01-731, UFLA 01-732, UFLA 01-739, UFLA 01-748, UFLA 01-756, UFLA 04-260, UFLA 04-40 were similar to the strain BR 3460, which were superior to the control with high nitrogen concentration (Table 2). All these strains plus UFLA 01-744, UFLA 01-753, and UFLA 04-248 also promoted higher diameter growth, and were equivalent to the control with high mineral N concentration.

For the Dickson quality index (DQI), the strains UFLA 01-739, UFLA 01-744, UFLA 01-748, UFLA 01-753, UFLA 01-756, UFLA 04-248, UFLA 04-260, UFLA 04-269 and UFLA 04-405 were equivalent to the inoculant strain (BR 3460) and to the control with high N concentration (Table 2). However, the strains UFLA 01-739, UFLA 01-748 and UFLA 04-405 should be highlighted. Of the 14 tested strains, in the experiment carried out with M. foliolosa, only two did not nodulate this species, UFLA 01-731 and UFLA 04-269, which were isolated from Mimosa tenuiflora and Macroptilium atropurpureum, respectively (Table 3). M. folilosa is an endemic species of rupestrian grassland of Serra do Cipo, MG, and this is the first study that evaluates the nodulation and efficiency of nitrogen-fixing strains in its development; therefore, no strains have been approved by MAPA as inoculant yet.

The strains that presented higher number of nodules production were UFLA 01-726, UFLA 01-733, UFLA 01-748 and UFLA 04-248. With the exception of UFLA 01-733, these strains also promoted greater nodules dry matter production (Table 3).

All parameters showed significant effects; however, no strain was able to surpass, nor was equivalent to the results obtained with the control with high nitrogen concentration for shoot dry matter, total dry matter, height and diameter (Table 3). Root dry matter production by the strains UFLA 01-731, UFLA 01-733, UFLA 01-751, UFLA 04-260 and UFLA 04-269 was equivalent to that of the control with high mineral N concentration.

The values obtained by the Dickson quality index by the strains UFLA 01-726, UFLA 01-739, UFLA 01-748, UFLA 01-750, UFLA 01-751, UFLA 04-248, UFLA 04-260, and UFLA 04-405 were equivalent to that of the control with high N concentration (Table 3).

The Mimosa species evaluated this study are promising to be used in the recovery of degraded areas. The use of native legumes species that establish symbosis with nitrogen-fixing bacteria is of great interest for degraded areas restoration, since they established themselves in the field more quickly, and promote greater biomass accumulation and contribute to the improvement of soil fertility, favoring other species that are not able to establish this type of symbiosis (CHAER et al., 2011CHAER, G. M. et al. Nitrogen-fixing legume tree species for the reclamation of severely degraded lands in Brasil. Tree Physiology, v. 31, p. 139-149, 2011.).

This study is the first to evaluate the potential of bacteria strains from nodules of legume species cultivated in rupestrian grasslands soils. The soils of this region are oligotrophic and acid (CARVALHO et al., 2012CARVALHO, F. et al. The mosaico f habitats in the high-altitude brazilian rupestrian fields is a hotspot for arbuscular mycorrhizal fungi. Applied Soil Ecology, v. 52, n. 1, p. 9-19, 2012.; 2014CARVALHO, F. et al. Relationship between physical and chemical soil attributes and plant species diversity in tropical mountain ecosystems from Brazil. Journal of Mountain Science, v. 11, n. 4, p. 875-883, 2014.; NEGREIROS; MORAIS; FERNANDES, 2008NEGREIROS, D.; MORAIS, M. L. B.; FERNANDES, G. W. Caracterização da fertilidade dos solos de quatro leguminosas de campos rupestres, serra do cipó, MG, Brasil. Revista de la Ciência del Suelo y Nutrición Vegetal, v. 8, n. 3, p. 30-39, 2008.), and the selection of strains adapted to these conditions may be used to obtain seedlings for the recovery of areas which have soils with these characteristics.

Although Mimosa foliolosa is an endemic species of the same region where the Burkholderia strains were obtained, they were more efficient in Mimosa bimucronata.

It can be considered that the strains UFLA 01-739, 01-748, 01-751, 04-260, 04-405 were in general the most effective in promoting the growth of Mimosa species. UFLA 01-739, 01-748, and 04-405 were considered potential inculants for M. bimucronata; and UFLA 01-751 and UFLA 04-260 were considered potential inoculants for M. foliolosa.

CONCLUSIONS

  1. Burkholderia strains establish symbiosis with the species Macroptilium atropurpureum, Mimosa bimucronata and Mimosa foliolosa, and present greater specificity for species of the genus Mimosa;

  2. Macroptilium atropurpureum nodulation by Burkholderia strains occurred only with two of the 14 strains, and was inefficient;

  3. Burkholderia strains isolated from Macroptilium atropurpureum and Mimosa tenuiflora cultivated in rupestrian grasslands soils promote plant growth of the species Mimosa bimucronata and Mimosa foliolosa, with potential to be used as inoculants for these species.

  4. Burkholderia strains promote Mimosa spp. growth, but not Macroptilium atropurpureum.

  • 1
    Dados extraídos parcialmente da Dissertação de Mestrado da primeira autora

ACKNOWLEDGEMENTS

To the National Council for Scientific and Technological Development (CNPq) and to the Coordination for the Improvement of Higher Education Personnel (CAPES) for the finantial support to this research project, and for the study and research scholarships.

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Publication Dates

  • Publication in this collection
    Jan-Mar 2017

History

  • Received
    25 Nov 2015
  • Accepted
    20 May 2016
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