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Early responses of rice (Oryza sativa L.) seedlings to inoculation with beneficial diazotrophic bacteria are dependent on plant and bacterial genotypes

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Abstract

Background and aims

Rice cultivars, in combination with diazotrophic bacteria, can obtain variable contributions from Biological Nitrogen Fixation (BNF). Plant genetic controls and the genotype of the bacterial strains might regulate in particular ways the plant responses during these associations. Some of this regulation is likely to occur during the first stages of bacterial infection and plant colonization, and some of the early plant responses might involve ethylene signaling, as previously reported for sugarcane. The aim of this work was to investigate whether rice early responses to inoculation with beneficial diazotrophic bacteria and the expression of ethylene receptors (ERs) by the host are dependent on plant and bacterial genotypes.

Methods

Bacterial colonization, lateral root development and expression of ERs were measured in seedlings of two rice cultivars, IR42 and IAC4440, which, respectively, are known to have higher and lower BNF capabilities. Inoculation experiments were performed with two strains of bacteria: Azospirillum brasilense sp245 and Burkholderia kururiensis M130.

Results

Cultivar IR42, which has a relatively high BNF capability, was more colonized by the bacteria early after inoculation in comparison with cv. IAC 4440, which has a lower BNF capability. However, although both cultivars showed a significant increase in lateral root numbers 10 days after inoculation with A. brasilense sp245, plants inoculated with B. kururiensis M130 did not. In addition, a differential ER expression pattern was observed between IR42 and IAC4440 in response to inoculation with the two strains. The expression of ERs was higher in the more BNF-effective cultivar IR42, than in the less BNF-effective cv. IAC4440, especially when cv. IR42 was inoculated with A. brasilense sp245. Moreover, plants associated with B. kururiensis M130 showed a different ER expression profile compared to those inoculated with A. brasilense sp245, with the latter exhibiting greater increases of ER mRNA levels three days after bacterial inoculation.

Conclusions

We have shown that two rice genotypes contrasting in their BNF capability responded differentially to early inoculation with different strains of diazotrophic bacteria. The two rice genotypes were colonized with different bacterial numbers during their early association with two strains of diazotrophic bacteria, and the two strains had different effects on the promotion of lateral root development early after inoculation. They also triggered distinct ER expression patterns in the two rice cultivars. These data thus suggest that rice ethylene responses to beneficial diazotrophic bacteria are controlled by both plant and bacterial genotypes.

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Acknowledgments

The authors are grateful to Dr. Emanuel Maltempi de Souza and Dr. Euan James for critical reading of the manuscript and to Centro Nacional de Pesquisa em Agrobiologia (CNPAB)/Empresa Brasileira de Pesquisa Agropecuária (Embrapa) for providing rice genotypes and endophytic diazotrophic bacteria strains. L.V. is indebted to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and T.L.G.C. is indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for graduate fellowships. A.S.H. and P.C.G.F. receive support from a CNPq research grant. The research was supported by Instituto Nacional de Ciência de Tecnologia (INCT) in Biological Nitrogen Fixation, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and CNPq.

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Authors and Affiliations

  1. Laboratório de Biologia Molecular de Plantas, Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Brigadeiro Trompowsky s/no. Bl. B sala 33A, Cidade Universitária lha do Fundão, CEP: 21941-590, Rio de Janeiro, RJ, Brazil

    Lívia Vargas, Thais Louise Gurjão de Carvalho, Paulo Cavalcanti Gomes Ferreira & Adriana Silva Hemerly

  2. Centro Nacional de Pesquisa em Agrobiologia/Empresa Brasileira de Pesquisa em Agropecuária, BR465, Km47 23851-970, Seropédica, RJ, Brazil

    Vera Lúcia Divan Baldani & José Ivo Baldani

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  1. Lívia Vargas
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  2. Thais Louise Gurjão de Carvalho
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  3. Paulo Cavalcanti Gomes Ferreira
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  4. Vera Lúcia Divan Baldani
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  5. José Ivo Baldani
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  6. Adriana Silva Hemerly
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Correspondence to Adriana Silva Hemerly.

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Responsible Editor: Euan K. James.

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Figure S1

Relative levels of diazotrophic bacteria colonizing rice tissues. Colonization of rice seedlings by (a) A. brasilense sp245 or (b) B. kururiensis M130 was quantified by qRT-PCR at 3 and 10 days after inoculation (in 7 and 14 day-old plants, respectively). Day 0 represents plants before inoculation and it is a control for quantification. Bacterial 16 S rRNA levels were normalized with rice 28 S rRNA levels. The relative expression was calculated by minimizing the Ct from inoculated RNA samples and the Ct from uninoculated controls, and then the result (deltaCt) is submitted to the formula 2-deltaCt. Each sample was representative of 10 to 15 whole seedlings, with three technical replicates. The data from one representative experiment are shown. A biological repeat is shown in Fig. 1. Bars indicate mean ± standard error. (GIF 44 kb)

High resolution image (EPS 1776 kb)

Figure S2

Profile of ER expression in response to inoculation with A. brasilense sp245. Relative mRNA levels of OsERS1, OsERS2, OsETR2 and OsETR3 were analyzed by qRT-PCR and they were normalized with 28 S rRNA levels and with uninoculated control plants. Expression was analyzed in whole seedlings of cvs IR42 and IAC4440 3 days after inoculation with A. brasilense sp245. Data were compared with uninoculated control plants. Each sample of the biological repeats was a pool of 10 to 15 whole seedlings. The mean values of three biological repeats are shown in this graph. One representative experiment is presented in Fig. 4a. A statistical analysis was performed using the Kruskal-Wallis test with P < 0.05 (n = 3). Asterisks (*) indicate a significant difference in expression between control and inoculated plants. Bars indicate mean ± standard deviation. (GIF 32 kb)

High resolution image (EPS 4725 kb)

Figure S3

Profile of ER expression in response to inoculation with B. kururiensis M130. Relative mRNA levels of OsERS1, OsERS2, OsETR2 and OsETR3 were analyzed by qRT-PCR and they were normalized with 28 S rRNA levels and with uninoculated control plants. Expression was analyzed in whole plants of cvs IR42 and IAC4440 3 days after inoculation with B. kururiensis M130. Data were compared with control non inoculated plants. Each sample of the biological repeats was a pool of 10 to 15 whole plants. The mean values of three biological repeats are shown in this graph. One representative experiment is presented in Fig. 4b. A statistical analysis was performed by using the Kruskal-Wallis test with P <0.05 (n = 3). Asterisks (*) indicate a significant difference in expression between control and inoculated plants. Bars indicate mean ± standard deviation. (GIF 29 kb)

High resolution image (EPS 4686 kb)

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Vargas, L., de Carvalho, T.L.G., Ferreira, P.C.G. et al. Early responses of rice (Oryza sativa L.) seedlings to inoculation with beneficial diazotrophic bacteria are dependent on plant and bacterial genotypes. Plant Soil 356, 127–137 (2012). https://doi.org/10.1007/s11104-012-1274-8

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