The Root Microbiome of Salicornia ramosissima as a Seedbank for Plant-Growth Promoting Halotolerant Bacteria

Round 1

Reviewer 1 Report

The manuscript tries to describe with lot of detailed experiments the root microbiome of Salicornia ramosissima and claims that this information may serve as a seedbank for plant-growth promoting bacteria. The manuscript is quite elaborate and well written. However, I have some queries/concerns.

The work is not exclusively novel; similar work describing cultivable bacteria associated with the rhizosphere of halophyte Salicornia ramosissima and its application have been already published: (Mesa-Marín et al., 2019c; Mesa-Marín et al., 2020)

Whether the halophyte(s) are indeed colonized and further benefited from the PGP traits from inoculation of some of these rhizosphere isolates, is not authenticated by additional plant experiments. Perhaps beyond the scope of this research, this is important to prove it before one builds up a seedbank for plant-growth promoting bacteria.

Introduction: How do you define salt tolerance? what are the threshold’s that define the salt tolerance? 

Interesting is also to know about Microbial composition of bulk soil? Is it different from Rhizosphere?

More information about the difference or similarity (if any) between the isolates from wild and crop (cultivated) plants.

Interesting is to know that only 5 isolates (4%) evidenced nitrogen fixation capacity although functions strongly coupled to nitrogen fixation like phosphate solubilization capacity (61%) and siderophore production (58%) were strongly represented by the isolates. Do you have any explanation?

Author Response

Reviewer 1 Comments:

The manuscript tries to describe with lot of detailed experiments the root microbiome of Salicornia ramosissima and claims that this information may serve as a seedbank for plant-growth promoting bacteria. The manuscript is quite elaborate and well written. However, I have some queries/concerns.

The work is not exclusively novel; similar work describing cultivable bacteria associated with the rhizosphere of halophyte Salicornia ramosissima and its application have been already published: (Mesa-Marín et al., 2019c; Mesa-Marín et al., 2020)

Whether the halophyte(s) are indeed colonized and further benefited from the PGP traits from inoculation of some of these rhizosphere isolates, is not authenticated by additional plant experiments. Perhaps beyond the scope of this research, this is important to prove it before one builds up a seedbank for plant-growth promoting bacteria.

 

Author response: Thank you for your comments. Regarding to the last suggestion, we are conducting a factorial-designed experiment to evaluate the interaction between selected PGPB consortia and plants under different abiotic conditions both in microcosm and field conditions.

• Introduction: How do you define salt tolerance? what are the threshold’s that define the salt tolerance? 

Authors response: Thank you for your comment. Salt tolerance is the capacity to grow and reproduce under high salt concentrations. Halophytes are able to grow and reproduce under salinities >200 mM NaCl [1]. Most plants, on the other hand, can withstand moderate salinities but begin to express stress signals at salinities higher than 80mM of NaCl [2].

Consequently, we can assume that the stress threshold for plants is ~ 80mM.

So, as suggested by the reviewer, we have now added the following paragraph to the introduction (lines 41 to 43 ).

“These plants are able to grow and reproduce under salinities >200 mM NaCl [1] whereas most non-halophytes can withstand moderate salinities, but begin to express stress signals at salinities higher than 80mM of NaCl [2]. “

• Interesting is also to know about Microbial composition of bulk soil? Is it different from Rhizosphere?

Authors response: Thank you for pointing this out. Our objective was to find bacteria that had already established a symbiotic relation with a plant and displayed PGP traits. We would not find that in bulk soil microbial communities. However, although we did not analyze the microbial community of bulk soil, some studies found that it was closely related to that of the rhizosphere [3] and that rhizosphere soil was found to contain higher microbial biomass compared to bulk soil [4]. Nevertheless, in a separate study, significant differences were found between rhizosphere and bulk soil microbial composition [5]. As soil is the primary source of microbes inhabiting the rhizosphere, similarities are expected.  

Please, let us know if you would like this information to be included in the manuscript.

• More information about the difference or similarity (if any) between the isolates from wild and crop (cultivated) plants.

Authors response: As shown in Table 2, Av1 (crop site) rendered the highest number of OTUs (17). Four of these OTU (Kocuria, Virgibacillus, and Pseudomonas) were found exclusively associated with plants from that site (manuscript lines 322-324 of the revised manuscript). Although Kocuria was only isolated from Av1 site, Virgibacillus and Pseudomonas were also isolated from Al and Tg, respectively. All other OTUs present crop plants were also detected in wild plants from other sites. That is the case of OTUs corresponding to Micrococcus (also in Al), Bacillus (in all other sites), Stenotrophomonas (also in Tg), Pseudomonas (also in Tg) and Oceanobacillus (also in Av3). Therefore, there is no evidence that cultivated plant are different from wild plants in terms of culturable populations represented in the microbiome (this information was included in the manuscript, lines 488-497).

In unpublished results from our group we have explored bacterial diversity from rhizosphere and endosphere of S. ramosissima in Av1 and Av3 sites (crop vs wild sites) using culture independent approaches and we have found no statistical differences in the structure of these communities, such as observed in this study of the culturable fraction.

• Interesting is to know that only 5 isolates (4%) evidenced nitrogen fixation capacity although functions strongly coupled to nitrogen fixation like phosphate solubilization capacity (61%) and siderophore production (58%) were strongly represented by the isolates. Do you have any explanation?

Authors response: Thank you for pointing this out. We were also surprised by the low numbers of N-fixing bacteria in the collection. Nitrogen fixation capacity is represented in several bacterial phyla, namely Proteobacteria, Actinobacteria, Firmicutes and Cyanobacteria. In shallow coastal water and illuminated sediments, marine cyanobacteria are major players in N-fixation. N-fixing cyanobacteria have also been detected in the epiphytic communities associated with halophytes ([6,7]. In Salicornia dominated areas, cyanobacteria have a very important role in Nitrogen fixation, especially in warmer months and low plant density areas  [8]. Therefore, our explanation for these results is that cyanobacteria are important in the diazotroph communities associated with Salicornia but were not well covered by the strategy of cultivation and isolation. It is also possible that the method we used to screen nitrogen fixation was not fully adequate for our isolates. In addition to these factors, there is the possibility of loss of the ability to express N-fixation genes during successive cultivations in TSA medium.

 This explanation has been included in the manuscript (lines 567-574).

References

1. Flowers, T.J.; Colmer, T.D. Salinity tolerance in halophytes. New Phytol. 2008, 179, 945–963, doi:10.1111/j.1469-8137.2008.02531.x.
2. Volkov, V.; Beilby, M.J. Editorial: Salinity tolerance in plants: Mechanisms and regulation of ion transport. Front. Plant Sci. 2017,8, 1795, doi:10.3389/fpls.2017.01795.
3. De Ridder-Duine, A.S.; Kowalchuk, G.A.; Klein Gunnewiek, P.J.A.; Smant, W.; Van Veen, J.A.; De Boer, W. Rhizosphere bacterial community composition in natural stands of Carex arenaria (sand sedge) is determined by bulk soil community composition. Soil Biol. Biochem. 2005, 37, 349–357, doi:10.1016/j.soilbio.2004.08.005.
4. Chaudhary, D.R.; Saxena, J.; Lorenz, N.; Dick, L.K.; Dick, R.P. Microbial Profiles of Rhizosphere and Bulk Soil Microbial Communities of Biofuel Crops Switchgrass (Panicum virgatum L.) and Jatropha (Jatropha curcas L.). Appl. Environ. Soil Sci.2012, 2012, 906864, doi:10.1155/2012/906864.
5. Shi, J.-Y.; Yuan, X.-F.; Lin, H.-R.; Yang, Y.-Q.; Li, Z.-Y. Differences in soil properties and bacterial communities between the rhizosphere and bulk soil and among different production areas of the medicinal plant Fritillaria thunbergii. Int. J. Mol. Sci. 2011, 12, 3770–3785, doi:10.3390/ijms12063770.
6. Elmerich, C.; Newton, W.E. Associative and Endophytic Nitrogen-fixing Bacteria and Cyanobacterial Associations. Assoc. Endophytic Nitrogen-fixing Bact. Cyanobacterial Assoc. 2007, doi:10.1007/1-4020-3546-2.
7. Currin, C.A.; Paerl, H.W. Environmental and physiological controls on diel patterns of N2 fixation in epiphytic cyanobacterial communities. Microb. Ecol. 1998, 35, 34–45, doi:10.1007/s002489900058.
8. Jones, K. Nitrogen Fixation in a Salt Marsh. J. Ecol. 1974, 62, 553–565, doi:10.2307/2258998.

 

 

Reviewer 2 Report

The article provides an interesting survey of the root microbiome associated with the halophyte Salicornia ramosissima and its' potential as a PGPR.   The authors have conducted the relevant experiments to support their research and have provided a well-written informative article which could benefit the readers of Applied Sciences.   Just some minor comments:   An important aspect to consider when working on culture dependent amplicon sequence analysis is that not all organisms can be grown in a specific media. Have the authors noticed any organisms that were identified in the culture independent studies conducted by [19] on S. europaea but were not found in the present study?   Line 127 & Line 152: After reading the methods section, it is evident that the authors are selecting for halotolerant bacterial species (Line 137, Line 144-146). However the title mentioned in these methods is not appropriate, since it would refer to all bacterial species, not the halotolerant ones. Please explain.   Line 461: Citation [19[21] is misplaced.    - Information on technology utilized for sequencing is missing. Did the authors use Miseq,  Hiseq or other sequencing platform for amplicon sequencing?    - Number of reads obtained after sequencing is missing? What kind of quality check was performed on the obtained reads?   - Have the authors looked at the different genotypes of S. ramosissima and the influence of genotypes on the microbiome?   Line 130 -At which growth stages were the S. ramosissima plants harvested. How old were the plants? Have the authors found a correlation between the different growth stages of the S. ramossimma and a change in the microbiome associated with it?

Author Response

Comments from Reviewer 2

The article provides an interesting survey of the root microbiome associated with the halophyte Salicornia ramosissima and its' potential as a PGPR.   The authors have conducted the relevant experiments to support their research and have provided a well-written informative article which could benefit the readers of Applied Sciences. 

Authors response: Thank you for the encouraging comments.

Just some minor comments:  

1. An important aspect to consider when working on culture dependent amplicon sequence analysis is that not all organisms can be grown in a specific media. Have the authors noticed any organisms that were identified in the culture independent studies conducted by [19] on S. europaea but were not found in the present study?  

Authors response: We thank the reviewer for a very pertinent comment. Culture independent approaches as performed by [19] contribute to better resolution of the bacterial communities associated with plants. However, the culture dependent studies allow the access to the metabolic capabilities of the bacterial communities which are later useful in testing their activity in host-microbiota interactions. From the total of 110 families reported by [19], in our study we shared in common 9 of these families and were able to recover five other families not detected in [19] being Micrococcaceae, Brevibacteriaceae, Morganellaceae, Micrococcaceae and Lysobacteraceae. Therefore, for the aims of this study which included the isolation of bacterial communities associated with S. ramossisima to test their potential for PGP, our approach achieved the access of the bacterial members that would actually be useful for the development of better strategies in sustainable agriculture.

This information was included in the manuscript, lines 464-474.  Note that, in the revised manuscript, reference 19 is now reference [21].

2. Line 127 & Line 152: After reading the methods section, it is evident that the authors are selecting for halotolerant bacterial species (Line 137, Line 144-146). However, the title mentioned in these methods is not appropriate, since it would refer to all bacterial species, not the halotolerant ones. Please explain.

Authors response: We agree with the reviewer’s assessment. As stated in the abstract we aimed at the isolation of halotolerant bacteria. We have now added the term halotolerant to the title of the manuscript and the methods section referring to the isolation of bacteria (line 3 and line 129).

3. Line 461: Citation [19[21] is misplaced.

Authors response: The reviewer is correct, and we have made the appropriate correction (line 480). Note that these references are now #21 and #23, respectively.

4. Information on technology utilized for sequencing is missing.

Authors response: The reviewer’s assessment is correct. The sequencing of the 16S rRNA fragments was performed with Sanger technology and this information was added to the manuscript (line 163).

5. Did the authors use Miseq,  Hiseq or other sequencing platform for amplicon sequencing?    - Number of reads obtained after sequencing is missing? What kind of quality check was performed on the obtained reads?  

Authors response: Thank you for pointing this out. The sequencing of the 16S rRNA fragments was performed with Sanger technology. We obtained 120 sequences that were subjected to chimera removal, alignment and clustering following the tutorial for 16S rRNA data of the unsupervised method available in the Ribosomal Database Project. We have now complemented this information in the manuscript (lines 166-167).

6. Have the authors looked at the different genotypes of S. ramosissima and the influence of genotypes on the microbiome?  

 Authors response: Thank you for pointing this out. We have not check for the plant genotype effects but we agree it is an important question. One of our goals was to determine whether sediment properties could explain the composition of the plants associated microbiome and the prevailing PGP mechanisms in different populations of the same plant species. Both the rhizosphere and the endosphere isolates were obtained from the same plant species in different locations.

As for different genotypes, Szymánska et al. [1] found that the culturable fraction of endosphere and rhizosphere bacteria of Salicornia europaea belonged to the same phyla as our isolates (Proteobacteria, Firmicutes and Actinobacteria). However, at the genera level, only 5 genera (Bacillus, Thalassospira, Pseudomonas, Salinicola, Kushneria) are common; S. europaea and S. ramosissima both provided for 16 different genera each. Therefore, similarities in bacterial taxa can be found in closely related plant genotypes, however, at species level there can be found differences that could be explained by plant genotype and/or geographical location.

Please let us know if you would like this comment to be included in the manuscript.

7. Line 130 -At which growth stages were the  ramosissima plants harvested. How old were the plants? Have the authors found a correlation between the different growth stages of the S. ramosissimaand a change in the microbiome associated with it?

Authors response: Plants were harvested in Late summer - autumn (September-October) of 2019. The last sampled site was Almargem (Algarve), where we found the plants collected already had their color changed from green to red, indicating that they were either  experiencing abiotic stress or senescence. This could explain the lower taxonomic diversity of the cultivable bacterial community found in this site. This information was added to the revised manuscript, lines 440-443.

Environmental stress, such as drought, temperature, salinity and plant development stage, can change the interactions between the plant and its microbiome [2,3]. However, one report  [4] observed significant differences between the distribution of bacterial and fungal endophytes in plant organs of S. europaea due to salinity but not to seasons (spring and autumn). Still, we could not find literature linking the microbiome and the growth or life cycle of the plant for S. ramosissima. It is another important aspect to explore in future, such as the genotype, for a deeper understanding of plant drivers of the microbiome structure.

Please let us know if you would like more information to be included in the manuscript.

 

References

1. Szymańska, S.; Płociniczak, T.; Piotrowska-Seget, Z.; Hrynkiewicz, K. Endophytic and rhizosphere bacteria associated with the roots of the halophyte Salicornia europaea L. - community structure and metabolic potential. Microbiol. Res. 2016, 192, 37–51, doi:10.1016/j.micres.2016.05.012.
2. Jones, P.; Garcia, B.J.; Furches, A.; Tuskan, G.A.; Jacobson, D. Plant Host-Associated Mechanisms for Microbial Selection. Front. Plant Sci. 2019, 10, 862, doi:10.3389/fpls.2019.00862.
3. Chaparro, J.M.; Badri, D. V.; Vivanco, J.M. Rhizosphere microbiome assemblage is affected by plant development. ISME J. 2014,8, 790–803, doi:10.1038/ismej.2013.196.
4. Furtado, B.U.; Gołebiewski, M.; Skorupa, M.; Hulisz, P.; Hrynkiewicz, K. Bacterial and fungal endophytic microbiomes of Salicornia europaea. Appl. Environ. Microbiol. 2019, 85, 1–18, doi:10.1128/AEM.00305-19.

 

 

Round 2

Reviewer 1 Report

The Manuscript may be accepted for publication including the information about the microbial composition of bulk soil compared to that of Rhizosphere as described and answered by the authors to one of my queries.

Also avoid using the word "symbionts" in line 18 as it mostly  refers to the association of individuals of two different species, which is beneficial for both partners.

Author Response

We would like to thank you for the comments that allowed us to improve our work.

We have included the information required in lines 497-502. The changes are highlighted within the manuscript.

We also replaced the words “bacterial symbionts”, line 18 with “microorganisms” in the same line. 

We very much hope the revised manuscript is accepted for publication in Applied Sciences.

Sincerely yours,