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Endophytic Micromonospora from Medicago sativa are apparently not able to fix atmospheric nitrogen
Soil Biology & Biochemistry 74 (2014) 201e203
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Endophytic Micromonospora from Medicago sativa are apparently not able to fix atmospheric nitrogen Pilar Martínez-Hidalgo a, d, José Olivares c, Antonio Delgado b, Eulogio Bedmar c, Eustoquio Martínez-Molina a, d, * a
Department of Microbiology and Genetics, University of Salamanca, Doctores de la Reina s/n, 37007 Salamanca, Spain Instituto Andaluz de Ciencias de la Tierra, CSIC-Universidad de Granada, Avda. de las Palmeras. 4, 18100 Armilla, Granada, Spain Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, 18008 Granada, Spain d Unidad Asociada USAL-CSIC “Interacción Planta-Microorganismo”, Salamanca, Spain b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2013 Received in revised form 10 March 2014 Accepted 16 March 2014 Available online 31 March 2014
It has been reported that Micromonospora strains isolated from root nodules of Casuarina and Lupinus could be capable of fixing atmospheric nitrogen (N2) both inside root nodules and as free-living bacteria. Given the potential significance of such findings, we performed similar studies using isolates from Medicago root nodules in order to confirm if this phenomenon was a universal characteristic in nodulederived Micromonospora isolates. Our results showed that of the Micromonospora strains studied: (i) they did not grow in N-free culture media, (ii) nitrogenase activity or incorporation of N2 to the biomass was not detected, neither in trials with free-living bacteria, nor in symbiosis with plants and (iii) the presence of genes for nitrogen fixation in these strains was impossible to confirm. It can be concluded that, under the conditions tested and with the variety of techniques used, Micromonospora strains employed do not necessarily fix N2 as free-living diazotrophs nor in symbiosis with alfalfa plants. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Actinobacteria Micromonospora Biological N2 fixation Plantemicrobe interactions
Over the past decade, several new micro-organisms capable of fixing N2 in symbiosis with legumes have been found. Valdés et al. (2005) described an actinobacteria isolated from Casuarina nodules, that belong to the genus Micromonospora that could fix N2. In subsequent works (Trujillo et al., 2010) isolated Micromonospora strains from inside legume root nodules that grow in semisolid Nfree media, and of which a partial amplification of the nifH gene was accomplished. These results point in the same direction, namely, Micromonospora has the ability to fix N2. The discovery of a new, non Frankia actinobacteria, capable of nitrogen fixation in association with plants, is of potential significance both from a theoretical and practical point of view. The aim of this work was thus to establish whether Micromonospora isolated from alfalfa root nodules is also capable of fixing N2 as a free-living diazotroph or in association with alfalfa. Naturally occurring plants of Medicago sativa were collected from the regions of Castilla y León (Spain): Babilafuente (40 580
* Corresponding author. Department of Microbiology and Genetics, University of Salamanca, Doctores de la Reina s/n, 37007 Salamanca, Spain. Tel.: þ34 923294532; fax: þ34 923294611. E-mail addresses: [email protected] (P. Martínez-Hidalgo), jose.olivares@eez. csic.es (J. Olivares), [email protected] (A. Delgado), eulogio.bedmar@eez. csic.es (E. Bedmar), [email protected] (E. Martínez-Molina). http://dx.doi.org/10.1016/j.soilbio.2014.03.011 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
22.2200 Ne5 250 20.5000 W) and Palaciosrubios (41 20 36.3400 Ne5 110 49.5500 W) Micromonospora strains ALFpr18c and ALFb5 were isolated from surface sterilized alfalfa root nodules and belong to different species of the genus Micromonospora according to their rrs sequences (Fig. 1). The two strains were stab-inoculated into nitrogen-free semisolid medium to determine their growth ability as free-living diazotrophs. Yeast Carbon Base (Difco) was used for this experiment supplemented with noble agar at 0.75%. All Micromonospora strains tested grew visibly on this culture medium. These results were consistent with those obtained previously (Trujillo et al., 2010). To confirm these results, we conducted a similar assay in the same medium without agar. After two weeks of incubation a weak but clear bacterial growth was observed. However, in a second subculture in flasks inoculated with 1 mL of the preceding culture, measurable growth was not observed. We considered the possibility that nitrogen was not the only limiting factor for the growth of Micromonospora. To rule out this possibility, the ability to fix N2 by Micromonospora was analysed using the acetylene reduction technique (ARA) as described previously (Mesa et al., 2004). Nitrogenase activity of Micromonospora strains grown in stab-inoculated semisolid nitrogenfree media was assayed after 1, 6 and 24 h of incubation. Despite repeated attempts, none of the Micromonospora strains studied produced acetylene-dependent ethylene indicating that
202
P. Martínez-Hidalgo et al. / Soil Biology & Biochemistry 74 (2014) 201e203
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between Micromonospora isolates and the closest Micromonospora type strains together with L5, MIA57 and MIA32 strains. Bootstrap percentages (1000 replicates) are shown at nodes. GenBank accession numbers: ALFb5 (KF876226) and ALFpr18c (KF876230).
there was no functional nitrogenase, and consequently no N2 fixation occurred. These findings must be confirmed since a new nitrogenase has been described in Streptomyces thermoautotrophicus, which is functional and structurally different from the nitrogenase systems found in the rest of nitrogen-fixing organisms (Ribbe, 1997). The existence of nitrogenases e different to those already described e that are not detectable by ARA should also be considered. The techniques based on the use of 15N2 as tracer, give us a direct measurement of the incorporation of this element to the microbial biomass. Micromonospora strains ALFpr18c and ALFb5 were selected for this study as they are phylogenetically closely related to L5 (ALFb5) and M. saelicesensis and lupini (ALFpr18c) (Fig. 1). Selected strains were grown on solid medium and suspended in sterile distilled water, 1 mL of said bacterial suspensions (109 cells per mL) were inoculated in flasks with 25 mL of nitrogenfree liquid medium and incubated for 1 week. Then the flasks were sealed with serum stoppers and 15N gas was injected into the closed flasks. After 5 days of incubation the bacterial biomass was collected and washed to remove remnants of the culture medium. Dried cells were analysed for tracer 15N and total-N content (Boddey and Knowles, 1987). The 15N/14N ratios did not significantly differ from the negative controls, which indicates that there was no incorporation of 15N2 from the atmosphere to the bacterial biomass, and hence that the strains of Micromonospora cultivated were unable to fix N2. Thus our strains of Micromonospora were isolated from surface sterilized alfalfa nodules but were not able to induce these structures on the roots of the plant that could be essential for symbiotic N2 fixation in legumes (Martínez-Hidalgo, 2012). For this reason, we then focused on the analysis of the N2 fixing capability of Micromonospora strains ALFpr18c and ALFb5 in association with Medicago sativa. For this purpose we needed a microorganism that was capable of developing a nodule for Micromonospora with all the characteristics of the functional nodule but that could not fix N2. Ensifer meliloti strain 1354 (nif A), a mutant that cannot fix N2 but induces the development of normal nodules was used (Szeto et al., 1984). This way Micromonospora would be housed in a nodular structure that allowed N2 fixation by the actinobacteria. To perform this experiment in gnotobiotic conditions, surfacesterilized M. sativa seeds were germinated axenically and transferred aseptically to glass tubes as described by Olivares et al. (1984), with various inoculation treatments (Table 1). Plants inoculated with E. meliloti 1021 (Meade et al., 1982) served as positive controls and M. sativa plants inoculated with E. meliloti 1354 were used as negative controls. Plants were placed in a growth chamber
for 30 days. The whole plant system was used to assay 15N incorporation by plants. Test tubes were sealed with rubber stoppers and 15 N gas was injected into the closed test tube. After 48 h in a growth chamber the whole plants were dried and then powdered. Tracer 15 N and total-N were analysed. In these experiments the results for nitrogenase activity were identical to those obtained for 15N incorporation to the plant biomass (Table 1). When E. meliloti 1021 was inoculated, alone or co-inoculated with Micromonospora, 15N was incorporated to the plant biomass indicating N2 fixation. For the treatments in which alfalfa was inoculated with Micromonospora or with E. meliloti 1354 or coinoculated with both micro-organisms, the results of incorporation of 15N2 to the plant were negative. This fact further supports that Micromonospora has no ability to fix N2. The presence of nodules induced by E. meliloti mutant (nif A) does not affect the fixation of N2 by Micromonospora, which remains negative. ARA of nodulated plants was also assayed on detached root systems excised at the cotyledonary node as described by Mesa et al. (2004). Nitrogenase activity was not detected by ARA in the treatments inoculated with E. meliloti 1354, ALFpr18c, ALFb5, and any of the coinoculations with this mutant of Ensifer. Positive results only appeared in the treatments that had been inoculated with E. meliloti 1021. To conclude this work, essays were conducted for amplification of the gene nifH using Frankia alni ACN14a as positive control, following the protocols designed by Valdés et al. (2005) and Trujillo et al. (2010). Our results were also negative for our two selected
Table 1 Nitrogenase activity based on 15N content and ethylene measurements in each treatment. Since negatives test are in the natural abundance range “d” has been included. Treatment
ARA
d15N & (AIR)
15
Ensifer Sm 1354 Ensifer Sm 1021 Micromonopora ALFb5 Micromonopora ALFpr18 Sm 1354 þ M. ALFb5 Sm 1354 þ M. ALFpr18 Sm 1021 þ M. ALFb5 Sm 1021 þ M. ALFpr18
þ þ þ
9.64 620* 10.1 10.2 10.1 9.09 427* 534*
0.371 0.614* 0.371 0.371 0.371 0.371 0.525* 0.564*
(1.20) (244) (0.65) (0.41) (0.40) (0.86) (225) (270)
N in % (0.0004) (0.089) (0.0002) (0.0001) (0.0001) (0.0003) (0.094) (0.099)
d ¼ (Rsample/Rstandard 1)*1000, where R ¼ 15N/14N. The standard for reporting nitrogen measurements is atmospheric nitrogen (AIR). Dunnett’s test for multiple comparisons was used to identify treatments with means significantly different from the control treatment at P 0.05. Asterisks indicate treatments different from the negative control (Ensifer Sm 1354). Standard deviation is enclosed in brackets.
P. Martínez-Hidalgo et al. / Soil Biology & Biochemistry 74 (2014) 201e203
strains; we could not amplify this gene with any of the protocols used. These results are in accord with the rest of the observations reported above. Currently two strains of genus Micromonospora (Alonso-Vega et al., 2012; Hirsch et al., 2013) have been sequenced. We have not been able to find nif genes in the Micromonospora strains that have their genome sequenced. When we tracked the presence of the three structural genes for N2 fixation in these strains, using PSIBLAST (Altschul et al., 1997), we did not find evidence for them, as also stated by Hirsch et al. (2013). Although it has been reported that different Micromonospora strains fix N2 (Valdés et al., 2005) we could not find, even with the use of diverse techniques, any data that agree with those reported results. Our data here are clear: under the conditions tested Micromonospora does not fix N2 either as free-living diazotrophs or in association with alfalfa plants. Given the importance of the topic, the results obtained and because the number of potential N2-fixing Actinobacteria is growing very fast over the last decade (Gtari et al. 2012), a list of suggestions should be outlined that allows clear establishment that N2 fixation is genuinely occurring: (i) Growth in nitrogen free media is a technique that could be used as a preliminary screening for N2 fixing strains. However, Micromonospora may be an oligotrophic bacterium that grows in extremely nutritionally deficient conditions to which a very small amount of nitrogen is sufficient to allow proliferation and thus, to appreciate a visible growth in the culture medium. The nitrogen source could be provided by the agar in the semi-solid medium or also by the components of recycled dead cells, which could lead to false positives. (ii) Identification of nifH gene via PCR amplification is an important tool to detect potential N2 fixers. However, this approach is limited to a single gene and we can obtain false positives, specially using partial nifH sequences. This is the reason why it would be prudent to sequence at least two of the three structural genes from the nif operon. (iii) The ARA technique is also a valuable tool but with the qualification that some nitrogenases have been described which do not reduce acetylene to ethylene, leading to false negatives in some cases. (iv) Incorporation of 15N2 from the atmosphere to the bacterial or plant biomass is the most reliable technique to assess N2 fixation, since the only means by which an organism can increase the ratio in 15N is to via fixation of atmospheric nitrogen when in a 15N2 artificially enriched atmosphere.
203
This technique should be the method of choice wherever possible, in these types of studies.
Acknowledgements This work was supported by Junta de Castilla y León (Ref: SA306A11-2) and MICINN (Ref: AGL2010-17380) P.M-H was supported by a fellowship from CSIC JAE-PRE. We thank three anonymous reviewers for their constructive comments and recommendations.
References Alonso-Vega, P., Normand, P., Bacigalupe, R., Pujic, P., Lajus, A., Vallenet, D., et al., 2012. Genome sequence of Micromonospora lupini Lupac 08, isolated from root nodules of Lupinus angustifolius. Journal of Bacteriology 194, 4135. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.H., Zhang, Z., Miller, W., et al., 1997. Gapped BLAST and PSI-Blast: a new generation of protein database search programs. Nucleic Acids Research 25, 3389e3402. Boddey, R.M., Knowles, R., 1987. Methods for quantification of nitrogen fixation associated with gramineae. Critical Reviews in Plant Sciences 6, 209e266. Gtari, M., Ghodhbane-Gtari, F., Nouioui, I., Beauchemin, N., Tisa, L.S., 2012. Phylogenetic perspectives of nitrogen fixing actinobacteria. Archives of Microbiology 194, 3e11. Hirsch, A.M., Alvarado, J., Bruce, D., Chertkov, O., De Hoff, P.L., Detter, J.C., et al., 2013. Complete genome sequence of Micromonospora strain L5, a potential plantgrowth-regulating actinomycete, originally isolated from Casuarina equisetifolia root nodules. Genome Announcements 1 e00759-13. Martínez-Hidalgo, P., 2012. Actinobacterias endofíticas aisladas de nódulos de Medicago sativa: Análisis de su biodiversidad y su potencial agronómico como PGPR (Ph.D. thesis). Universidad de Salamanca, Spain. http://hdl.handle.net/ 10366/121386. Meade, H.M., Long, S.R., Ruvkun, G.B., Brown, S.E., Ausubel, F.M., 1982. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. Journal of Bacteriology 149, 114e122. Mesa, S., Alché, J., de, D., Bedmar, E., Delgado, M.J., 2004. Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules. Physiologia Plantarum 120, 205e211. Ribbe, M., 1997. N2 fixation by Streptomyces thermoautotrophicus involves a molybdenum-dinitrogenase and a manganese-superoxide oxidoreductase that couple N2 reduction to the oxidation of superoxide produced from O2 by a molybdenum-Co dehydrogenase. Journal of Biological Chemistry 272, 26627e 26633. Szeto, W.W., Zimmerman, J.L., Sundaresan, V., Ausubel, F.M., 1984. A Rhizobium meliloti symbiotic regulatory gene. Cell 36, 1035e1043. Trujillo, M.E., Alonso-Vega, P., Rodríguez, R., Carro, L., Cerda, E., Alonso, P., et al., 2010. The genus Micromonospora is widespread in legume root nodules: the example of Lupinus angustifolius. ISME Journal 4, 1265e1281. Valdés, M., Pérez, N.-O., Santos, P.E.-D.L., Caballero-Mellado, J., Peña-Cabriales, J.J., Normand, P., et al., 2005. Non-Frankia actinomycetes isolated from surfacesterilized roots of Casuarina equisetifolia fix nitrogen. Applied and Environmental Microbiology 71, 460e466.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio
Short communication
Endophytic Micromonospora from Medicago sativa are apparently not able to fix atmospheric nitrogen Pilar Martínez-Hidalgo a, d, José Olivares c, Antonio Delgado b, Eulogio Bedmar c, Eustoquio Martínez-Molina a, d, * a
Department of Microbiology and Genetics, University of Salamanca, Doctores de la Reina s/n, 37007 Salamanca, Spain Instituto Andaluz de Ciencias de la Tierra, CSIC-Universidad de Granada, Avda. de las Palmeras. 4, 18100 Armilla, Granada, Spain Estación Experimental del Zaidín, CSIC, Profesor Albareda, 1, 18008 Granada, Spain d Unidad Asociada USAL-CSIC “Interacción Planta-Microorganismo”, Salamanca, Spain b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 December 2013 Received in revised form 10 March 2014 Accepted 16 March 2014 Available online 31 March 2014
It has been reported that Micromonospora strains isolated from root nodules of Casuarina and Lupinus could be capable of fixing atmospheric nitrogen (N2) both inside root nodules and as free-living bacteria. Given the potential significance of such findings, we performed similar studies using isolates from Medicago root nodules in order to confirm if this phenomenon was a universal characteristic in nodulederived Micromonospora isolates. Our results showed that of the Micromonospora strains studied: (i) they did not grow in N-free culture media, (ii) nitrogenase activity or incorporation of N2 to the biomass was not detected, neither in trials with free-living bacteria, nor in symbiosis with plants and (iii) the presence of genes for nitrogen fixation in these strains was impossible to confirm. It can be concluded that, under the conditions tested and with the variety of techniques used, Micromonospora strains employed do not necessarily fix N2 as free-living diazotrophs nor in symbiosis with alfalfa plants. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Actinobacteria Micromonospora Biological N2 fixation Plantemicrobe interactions
Over the past decade, several new micro-organisms capable of fixing N2 in symbiosis with legumes have been found. Valdés et al. (2005) described an actinobacteria isolated from Casuarina nodules, that belong to the genus Micromonospora that could fix N2. In subsequent works (Trujillo et al., 2010) isolated Micromonospora strains from inside legume root nodules that grow in semisolid Nfree media, and of which a partial amplification of the nifH gene was accomplished. These results point in the same direction, namely, Micromonospora has the ability to fix N2. The discovery of a new, non Frankia actinobacteria, capable of nitrogen fixation in association with plants, is of potential significance both from a theoretical and practical point of view. The aim of this work was thus to establish whether Micromonospora isolated from alfalfa root nodules is also capable of fixing N2 as a free-living diazotroph or in association with alfalfa. Naturally occurring plants of Medicago sativa were collected from the regions of Castilla y León (Spain): Babilafuente (40 580
* Corresponding author. Department of Microbiology and Genetics, University of Salamanca, Doctores de la Reina s/n, 37007 Salamanca, Spain. Tel.: þ34 923294532; fax: þ34 923294611. E-mail addresses: [email protected] (P. Martínez-Hidalgo), jose.olivares@eez. csic.es (J. Olivares), [email protected] (A. Delgado), eulogio.bedmar@eez. csic.es (E. Bedmar), [email protected] (E. Martínez-Molina). http://dx.doi.org/10.1016/j.soilbio.2014.03.011 0038-0717/Ó 2014 Elsevier Ltd. All rights reserved.
22.2200 Ne5 250 20.5000 W) and Palaciosrubios (41 20 36.3400 Ne5 110 49.5500 W) Micromonospora strains ALFpr18c and ALFb5 were isolated from surface sterilized alfalfa root nodules and belong to different species of the genus Micromonospora according to their rrs sequences (Fig. 1). The two strains were stab-inoculated into nitrogen-free semisolid medium to determine their growth ability as free-living diazotrophs. Yeast Carbon Base (Difco) was used for this experiment supplemented with noble agar at 0.75%. All Micromonospora strains tested grew visibly on this culture medium. These results were consistent with those obtained previously (Trujillo et al., 2010). To confirm these results, we conducted a similar assay in the same medium without agar. After two weeks of incubation a weak but clear bacterial growth was observed. However, in a second subculture in flasks inoculated with 1 mL of the preceding culture, measurable growth was not observed. We considered the possibility that nitrogen was not the only limiting factor for the growth of Micromonospora. To rule out this possibility, the ability to fix N2 by Micromonospora was analysed using the acetylene reduction technique (ARA) as described previously (Mesa et al., 2004). Nitrogenase activity of Micromonospora strains grown in stab-inoculated semisolid nitrogenfree media was assayed after 1, 6 and 24 h of incubation. Despite repeated attempts, none of the Micromonospora strains studied produced acetylene-dependent ethylene indicating that
202
P. Martínez-Hidalgo et al. / Soil Biology & Biochemistry 74 (2014) 201e203
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationship between Micromonospora isolates and the closest Micromonospora type strains together with L5, MIA57 and MIA32 strains. Bootstrap percentages (1000 replicates) are shown at nodes. GenBank accession numbers: ALFb5 (KF876226) and ALFpr18c (KF876230).
there was no functional nitrogenase, and consequently no N2 fixation occurred. These findings must be confirmed since a new nitrogenase has been described in Streptomyces thermoautotrophicus, which is functional and structurally different from the nitrogenase systems found in the rest of nitrogen-fixing organisms (Ribbe, 1997). The existence of nitrogenases e different to those already described e that are not detectable by ARA should also be considered. The techniques based on the use of 15N2 as tracer, give us a direct measurement of the incorporation of this element to the microbial biomass. Micromonospora strains ALFpr18c and ALFb5 were selected for this study as they are phylogenetically closely related to L5 (ALFb5) and M. saelicesensis and lupini (ALFpr18c) (Fig. 1). Selected strains were grown on solid medium and suspended in sterile distilled water, 1 mL of said bacterial suspensions (109 cells per mL) were inoculated in flasks with 25 mL of nitrogenfree liquid medium and incubated for 1 week. Then the flasks were sealed with serum stoppers and 15N gas was injected into the closed flasks. After 5 days of incubation the bacterial biomass was collected and washed to remove remnants of the culture medium. Dried cells were analysed for tracer 15N and total-N content (Boddey and Knowles, 1987). The 15N/14N ratios did not significantly differ from the negative controls, which indicates that there was no incorporation of 15N2 from the atmosphere to the bacterial biomass, and hence that the strains of Micromonospora cultivated were unable to fix N2. Thus our strains of Micromonospora were isolated from surface sterilized alfalfa nodules but were not able to induce these structures on the roots of the plant that could be essential for symbiotic N2 fixation in legumes (Martínez-Hidalgo, 2012). For this reason, we then focused on the analysis of the N2 fixing capability of Micromonospora strains ALFpr18c and ALFb5 in association with Medicago sativa. For this purpose we needed a microorganism that was capable of developing a nodule for Micromonospora with all the characteristics of the functional nodule but that could not fix N2. Ensifer meliloti strain 1354 (nif A), a mutant that cannot fix N2 but induces the development of normal nodules was used (Szeto et al., 1984). This way Micromonospora would be housed in a nodular structure that allowed N2 fixation by the actinobacteria. To perform this experiment in gnotobiotic conditions, surfacesterilized M. sativa seeds were germinated axenically and transferred aseptically to glass tubes as described by Olivares et al. (1984), with various inoculation treatments (Table 1). Plants inoculated with E. meliloti 1021 (Meade et al., 1982) served as positive controls and M. sativa plants inoculated with E. meliloti 1354 were used as negative controls. Plants were placed in a growth chamber
for 30 days. The whole plant system was used to assay 15N incorporation by plants. Test tubes were sealed with rubber stoppers and 15 N gas was injected into the closed test tube. After 48 h in a growth chamber the whole plants were dried and then powdered. Tracer 15 N and total-N were analysed. In these experiments the results for nitrogenase activity were identical to those obtained for 15N incorporation to the plant biomass (Table 1). When E. meliloti 1021 was inoculated, alone or co-inoculated with Micromonospora, 15N was incorporated to the plant biomass indicating N2 fixation. For the treatments in which alfalfa was inoculated with Micromonospora or with E. meliloti 1354 or coinoculated with both micro-organisms, the results of incorporation of 15N2 to the plant were negative. This fact further supports that Micromonospora has no ability to fix N2. The presence of nodules induced by E. meliloti mutant (nif A) does not affect the fixation of N2 by Micromonospora, which remains negative. ARA of nodulated plants was also assayed on detached root systems excised at the cotyledonary node as described by Mesa et al. (2004). Nitrogenase activity was not detected by ARA in the treatments inoculated with E. meliloti 1354, ALFpr18c, ALFb5, and any of the coinoculations with this mutant of Ensifer. Positive results only appeared in the treatments that had been inoculated with E. meliloti 1021. To conclude this work, essays were conducted for amplification of the gene nifH using Frankia alni ACN14a as positive control, following the protocols designed by Valdés et al. (2005) and Trujillo et al. (2010). Our results were also negative for our two selected
Table 1 Nitrogenase activity based on 15N content and ethylene measurements in each treatment. Since negatives test are in the natural abundance range “d” has been included. Treatment
ARA
d15N & (AIR)
15
Ensifer Sm 1354 Ensifer Sm 1021 Micromonopora ALFb5 Micromonopora ALFpr18 Sm 1354 þ M. ALFb5 Sm 1354 þ M. ALFpr18 Sm 1021 þ M. ALFb5 Sm 1021 þ M. ALFpr18
þ þ þ
9.64 620* 10.1 10.2 10.1 9.09 427* 534*
0.371 0.614* 0.371 0.371 0.371 0.371 0.525* 0.564*
(1.20) (244) (0.65) (0.41) (0.40) (0.86) (225) (270)
N in % (0.0004) (0.089) (0.0002) (0.0001) (0.0001) (0.0003) (0.094) (0.099)
d ¼ (Rsample/Rstandard 1)*1000, where R ¼ 15N/14N. The standard for reporting nitrogen measurements is atmospheric nitrogen (AIR). Dunnett’s test for multiple comparisons was used to identify treatments with means significantly different from the control treatment at P 0.05. Asterisks indicate treatments different from the negative control (Ensifer Sm 1354). Standard deviation is enclosed in brackets.
P. Martínez-Hidalgo et al. / Soil Biology & Biochemistry 74 (2014) 201e203
strains; we could not amplify this gene with any of the protocols used. These results are in accord with the rest of the observations reported above. Currently two strains of genus Micromonospora (Alonso-Vega et al., 2012; Hirsch et al., 2013) have been sequenced. We have not been able to find nif genes in the Micromonospora strains that have their genome sequenced. When we tracked the presence of the three structural genes for N2 fixation in these strains, using PSIBLAST (Altschul et al., 1997), we did not find evidence for them, as also stated by Hirsch et al. (2013). Although it has been reported that different Micromonospora strains fix N2 (Valdés et al., 2005) we could not find, even with the use of diverse techniques, any data that agree with those reported results. Our data here are clear: under the conditions tested Micromonospora does not fix N2 either as free-living diazotrophs or in association with alfalfa plants. Given the importance of the topic, the results obtained and because the number of potential N2-fixing Actinobacteria is growing very fast over the last decade (Gtari et al. 2012), a list of suggestions should be outlined that allows clear establishment that N2 fixation is genuinely occurring: (i) Growth in nitrogen free media is a technique that could be used as a preliminary screening for N2 fixing strains. However, Micromonospora may be an oligotrophic bacterium that grows in extremely nutritionally deficient conditions to which a very small amount of nitrogen is sufficient to allow proliferation and thus, to appreciate a visible growth in the culture medium. The nitrogen source could be provided by the agar in the semi-solid medium or also by the components of recycled dead cells, which could lead to false positives. (ii) Identification of nifH gene via PCR amplification is an important tool to detect potential N2 fixers. However, this approach is limited to a single gene and we can obtain false positives, specially using partial nifH sequences. This is the reason why it would be prudent to sequence at least two of the three structural genes from the nif operon. (iii) The ARA technique is also a valuable tool but with the qualification that some nitrogenases have been described which do not reduce acetylene to ethylene, leading to false negatives in some cases. (iv) Incorporation of 15N2 from the atmosphere to the bacterial or plant biomass is the most reliable technique to assess N2 fixation, since the only means by which an organism can increase the ratio in 15N is to via fixation of atmospheric nitrogen when in a 15N2 artificially enriched atmosphere.
203
This technique should be the method of choice wherever possible, in these types of studies.
Acknowledgements This work was supported by Junta de Castilla y León (Ref: SA306A11-2) and MICINN (Ref: AGL2010-17380) P.M-H was supported by a fellowship from CSIC JAE-PRE. We thank three anonymous reviewers for their constructive comments and recommendations.
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