Greener Journal of Agricultural Sciences

ISSN: 2276-7770; ICV: 6.15

Vol. 6 (3), pp. 087-092, March 2016

Copyright ©2017, the copyright of this article is retained by the author(s)




Research Article (DOI:


Inoculation of Bradyrhizobium japonicum (Kirchner, 1896) Jordan (1982) (Bradyrhizobiaceae) on Inga edulis Mart. (Leguminosae, Mimosoideae)


Freitas J*1, Mônico AZ1, Lírio EJ2, Sarnaglia-Jnr VB2, Bonadeu F2, Silva JBNF3 & Hebling SA4


1Centro Universitário Norte do Espírito Santo – CEUNES/UFES. Rodovia BR 101 Norte, km. 60, Bairro Litorâneo, São Mateus – ES, Brazil, 29932-540.

2Escola Nacional de Botânica Tropical, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro. Rua Pacheco Leão, 915, sala 107, Jardim Botânico, Rio de Janeiro – RJ, Brazil, 22460-038.

3Centro de Ciências e Saúde (CCS), Universidade Federal do Rio de Janeiro. Av. Carlos Chagas Filho 373, Bloco J sala 25, Ilha do Fundão, Rio de Janeiro, RJ, Brazil, 22541-900.

4Faculdade Católica Salesiana do Espírito Santo. Av. Vitória, a950, Forte São João, Vitória – ES, Brazil, 29017-950.







Article No.: 063014287

DOI: 10.15580/GJAS.2016.3.063014287


Legumes that have the ability to fix atmospheric nitrogen through symbiosis with bacteria known as rhizobia, have great importance from an economic and ecological perspective, because in agricultural crops and reforestation, may waive the use of nitrogenous fertilizers and provide more resistant seedlings, minimizing impacts in nature and low cost. Bradyrhizobium japonicum (Kirchner, 1896) Jordan (1982) (Bradyrhizobiaceae) is a rhizobium which has association with several species of the subfamily Papilionoideae. This study aimed to analyze the initial growth of Inga edulis Mart (Leguminosae, Mimosoideae) inoculated with B. japonicum to test interaction in the subfamily Mimosoideae. We used the parameters: height, stem diameter and number of expanded leaves; data analysis consisted of the Shapiro-Wilk normality test. The inoculated seedlings showed a significant difference for height and number of leaves, but no  significant difference for stem diameter. From the results, it is inferred that I. edulis presents positive interaction with B. japonicum, i.e., the interaction of rhizobia extends to Mimosoideae, which allows to infer that the interaction of rhizobia can be shown as homologous synapomorphy of the clade Mimosoideae-Papilonoideae, which is supported by molecular phylogenies. The results are compared with other studies and the implications in the evolution of the clades of the Leguminosae are discussed in phylogenetic studies. To corroborate the hypothesis, further studies of species of different subfamilies of these clades should be tested in order to verify that indeed this interaction is a homologous synapomorphy of the clade or an apomorphy for Mimosoideae, Leguminosae and interaction with B. japonicum has arisen more than once in the evolution scale of the family.



Submitted: 30/06/2014

Accepted:  28/07/2014

Published: 28/03/2016


*Corresponding Author

Freitas J

E-mail: joelciofr@ gmail. com & naldinhozm@ gmail. com




Fabaceae, rhizobia, legume, agricultural crop









Leguminosas tem a capacidade de fixar nitrogênio atmosférico através da simbiose com bactérias, conhecidas como rizóbios, tem grande importância sob ponto de vista econômico e ecológico, pois em cultivos agrícolas e reflorestamentos, podem dispensar o uso de fertilizantes nitrogenados e propiciar mudas mais resistentes, o que minimiza os impactos desses insumos na natureza e diminui os custos. Bradyrhizobium japonicum (Kirchner, 1896) Jordan (1982) (Bradyrhizobiaceae) é um rizóbio com associação com diversas leguminosas de  Papilionoideae. O presente trabalho teve por objetivo analisar o crescimento inicial de indivíduos de Inga edulis (Leguminosae, Mimosoideae) submetidas à inoculação do rizóbio a fim de testar a interação deste em Mimosoideae, filogeticamente, grupo irmão de Papilionoideae. Foram utilizados os parâmetros altura, diâmetro do caule e número de folhas; a análise dos dados consistiu no teste de normalidade de Shapiro-Wilk. A partir dos resultados, é inferido que I. edulis apresenta interação positiva com B. japonicum. Os resultados são confrontados com outros trabalhos e as implicações na evolução dos clados de Leguminosae são discutidos com estudos filogenéticos.


Palavras-chave: Fabaceae, rizóbios, leguminosa, cultivo agrícola.





Nitrogen-fixing bacteria, known as rhizobia (e.g. Bradyrhizobium e Rhizobium) are species that live in a symbiotic relationship with plants, mainly legumes. The legume-rhizobium symbiosis is the source of fixed nitrogen that supply this element to the plant and in return receive carbon from the host plant (Taiz and Zeiger, 2004; Souchie et al., 2005). The rhizobia species can improve the development of seedlings in nurseries and tillage (Barnett and Baker, 1999; Moreira, 1994), increases disease tolerance (Green et al., 1999), maximizing the ability to establish these seedlings in the field, which assists in reforestation (Souchie et al., 2005).

The leguminosae are flowering plants with significant importance on component of forests in terms of richness and density, especially in biomes such as the Amazon, Cerrado and Atlantic Rainforest (BFG, 2015). The family has interaction with various rhizobial species (Jesus et al., 2005; Perret et al., 2000), most often the subfamilies Papilionoideae and Mimosoideae, uncommon in Caesalpinoideae (Souza et al., 1994; Moreira, 1994; Barberi et al., 1998). Molecular data support that the family is monophyletic, as well as the Mimosoideae and Papilionoideae subfamilies, but Caesalpinioideae is paraphyletic (Wojciechowski, 2003; Wojciechowski et al., 2004). In the present work, we use Caesalpinioideae term to refer this basal grade. For legumes there are records of interaction with several bacteria families: Xanthobacteraceae, Hyphomicrobiaceae, Phyllobacteriaceae, Methylobacteriaceae, Brucellaceae, Burkolderiaceae, Rhizobiaceae and Bradyrhizobiaceae (Moreira, 2008; Gyaneshwar et al., 2011).

Bradyrhizobium japonicum (Kirchner, 1896) Jordan (1982) (Bradyrhizobiaceae) is a important rhizobium species which has symbiotic interaction with many Papilionoideae (Leguminosae) species (Lírio et al., 2012) and is widely used to improve the production of Glycine max (L.) Merr, popularly known as “soy” in agricultural systems (Ruiz-Díez et al., 2012). However, no tests have been carried out with species of other legume subfamilies.

A typical legume species of South America is Inga edulis Mart. (subfamily Mimosoideae), found along the Atlantic Rainforest, Caatinga and Amazon Rainforest (Pennington, 1997; BFG, 2015) very much utilized by communities as food source, timber and medicine, especially by Andean countries (Pennington, 1997). Furthermore, is used in the Amazon in agroforestry systems to improve soil fertility and to shade in perennial crops as coffee and cacao (Lojka et al., 2010).

Once B. japonicum is capable of interacting with Papilionoideae species, and nodulation is considered by some authors an important taxonomic character (Faria and Sprent, 1994) the aim of this article was to evaluate whether the interaction of B. japonicum  extends to Mimosoideae, the Papilionoideae sister group. In this context, we evaluated the initial development of I. edulis inoculated with B. japonicum.





Study Area


The work was performed in the experimental field of the São Francisco de Assis School, in the municipality of Santa Teresa, Espírito Santo state, Brazil (19°56’10" S; 40°36’06" W). The elevation of the region is 650 m (Mendes and Padovan, 2000). The I. edulis seeds were collected in a private property localized in the municipality of Afonso Cláudio, Espírito Santo state, Brazil (20° 04' 26" S; 41°07' 32 W).


Experimental Design


A total of 200 seeds was used, 50% were inoculated and the rest used as control that were placed in tillage on soil, and after sowing, kept in full sun. We obtained B. japonicum (strain 5080) on peat substrate (Nitrosuper F45). To inoculation we diluted the bacteria in distilled water as carried out in Lírio et al. (2012). The dilution consisted of 1 mL of solution bacteria and 3 mL of distilled water for 5 g of seeds and they were protected from sunlight at the time of sowing. Height, stem diameter and number of expanded leaves parameters were measured two months after seeding.

Statistical Analysis


Statistical analysis was performed using the PAST software (Hammer et al., 2001). We used the Shapiro-Wilk (Shapiro and Wilk, 1965) test for normality. The t-test was used for the comparison of data obtained for the inoculated and control samples, with a significance level of 0.05%.





The data are normally distributed in the comparison of all parameters, which justified the use of the t-Student test (tab. 1). The treatment of I. edulis presents significant difference in the parameters height and number of sheets and did not show significant difference to stem diameter (fig. 1). The results suggest that the interaction of B. japonicum occur in I. edulis, included in the subfamily Mimosoideae.




Figure 1: Barchat showing the mean and standard error for height (n=20; t=2.338; p=0.03046), leaves number (n=20; t= 2.833; p= 0.01063) and stem diameter (n=20; t=0.3451; p= 0.7338), respectively. “Inc” = inoculated sample and “Ctrl” = control sample



Leblanc et al. (2005) isolated four Bradyrhizobium strains of I. edulis, and through BLAST analysis came to the conclusion that these strains are closely related (with molecular data) to B. japonicum (96% similar), however they consider that there was insufficient information of B. japonicum affiliation for strong conclusions.

The phylogenetic relationship of Leguminosae subfamilies is supported by molecular studies, which Papilionoideae and Mimosoideae are sister groups (Wojciechowski, 2003; Wojciechowski et al., 2004). Analyzing the interaction of legumes with rhizobia, other studies corroborate this pattern ,  where  it  is  proposed  that  there  are a gradual trend of interaction: Papilionoideae with the highest number of species with interaction, followed by Mimosoideae and Caesalpinioideae, with the lowest number of species (Souza et al., 1994; Barbieri et al., 1998). Possibly the smallest interaction with Caesalpinioideae is due the fact that the group is more basal in the evolution scale of Leguminosae, and that the interaction of the family with rhizobia arose in the period that Caesalpinioideae has been established (Faria et al., 1984; Barbieri et al., 1998). It is possible that the ability to interact with rhizobia has arisen more than once in the scale of evolution of the family, as has been proposed (Sprent, 2001; Doyle and Luckow, 2003), however, the specificity by rhizobia with different clades or species (Perret et al., 2000; Miller et al., 2007; Wang et al., 2012), permits the inference that the interaction of several species of rhizobia can be seen to be homologous synapomorphies of Leguminosae clades as inferred for B. japonicum in Mimosoideae + Papiloinoideae. In Papilionoideae 11 species nodulate with B. japonicum, which belong to the tribes Phaseoleae, Genisteae and Loteae (Lírio et al., 2012). Furthermore, Perret et al. (2000) cite three tribes as non-host of B. japonicum: Vicieae, Cicereae and Trifolieae. These absences of interaction can be explained as a single reversal, since phylogenetic analysis indicated that these tribes form a monophyletic clade called by Steele and Wojciechowski (2003) as "vicioid clade". Based on parsimony, is possible to infer that the nodulation of B. japonicum with Leguminosae may have arisen in the common ancestor of Papilionoideae and Mimosoideae.





According to the results, B. japonicum has positive interactions with I. edulis. This interaction may be a synapomorphy of the clade Papilionoideae + Mimosoideae. To corroborate our hypothesis that B. japonicum interacts with Papilionoideae and Momosoideae, but not with Caesalpinioideae, other studies with species of different subfamilies clades should be evaluated. Particularly between Mimosoideae and Caesalpinioideae to verify if this interaction is a homologous synapomorphy of Papilionoideae + Mimosoideae clade, or should be an apomorphy for Mimosoideae, and the interaction of B. japonicum group has arisen more than once in the scale of Leguminosae evolution.

Also we propose studies with native rhizobia species which can be isolated from other legumes, not only by the potential to elucidate evolutionary interactions between rhizobia and Leguminosae, but also by the economic and ecological importance that can make in agricultural crops and reforestation. Another aim to be evaluated is the effects caused by introduction of exotic rhizobia to local biodiversity. The studies should be performed by isolation of rhizobia in native legume species from adjacent crops areas, mainly of soy, once B. japonicum strains that have been inoculated in large scale (Ruiz-Díez et al. 2012). Furthermore, studies on competition with native rhizobia or negative interactions with local biodiversity are needed.





The authors thank the Escola Superior São Francisco de Assis, the Atlantic Forest Resaerch Institute (Museu de Biologia Prof. Mello Leitão), CAPES, and the biologists Adriano Totola, Braz Pereira Lopes, Eleisson Loss, Mariana Soares Colombi and Wesley Croce for their  contributions to this work.





The authors declare that they have no competing interest.





JF and AMZ conducted the experiments. VBSJ performed the statistical tests. JF, AMZ, EJL, BN and FB organized and discussed the results. SAH performed the study design and supervised the work. All authors read and approved the final manuscript.





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Cite this Article: Freitas J, Mônico AZ, Lírio EJ, Sarnaglia-Jnr VB, Bonadeu F, Silva JBNF and Hebling SA (2016). Inoculation of Bradyrhizobium japonicum (Kirchner, 1896) Jordan (1982) (Bradyrhizobiaceae) on Inga edulis Mart. (Leguminosae, Mimosoideae). Greener Journal of Agricultural Sciences, 6(3): 087-092,