Inoculated Seed Endophytes Modify the Poplar Responses to Trace Elements in Polluted Soil
, Svetoslav Staykov 2, Jan D’Haen 4,5, Luca Sebastiani 1, Jaco Vangronsveld 2,6 and Sofie Thijs 2,*Round 1
Reviewer 1 Report
Most of the previous concerns I raised were addressed by the authors. However, I suggest clarifying the following points to enhance clarity, before publication.
Here below are the previous comments and the new suggestions.
In the material and methods section, lines 142-144, I wonder why the authors did not assess the soil physico-chemical properties, especially since the plant nutritional status was later evaluated in the paper (esp. in the discussion on Mg status) as well as the macro-nutrients’ levels in plant aerial parts. In my opinion, it would be beneficial to add this feature. If it is was previously reported on the same soil, I suggest mentioning it with the appropriate reference.
Answer: We reported the element concentrations of soil as result of soil characterization in lines 313-315: the nutrient concentrations in soil were: 1611 ± 160.5 mg kg-1 Ca; 409.7 ± 46.9 mg kg-1 K; 695 ± 83.1 mg kg-1 Mg; 66 ± 6.0 mg kg-1 Cu
In most studies studying TE mobility and phytoextraction potential, soil pH is considered as a key element to include, since it may explain most of the TE bioavailability. I hence strongly suggest to include this feature to the manuscript.
Section 2.5, I wonder why were only Cd and Zn concentrations assessed? In the vicinity of smelters, the presence of several TE is often assessed. In addition, in the introductory section, the authors stressed the fact that the area was often polluted by Cd, Pb and Zn.
Answer: After analysing the TE concentrations in the soil used for the inoculation experiment, we selected the TEs above the threshold for Flanders legislation (lines 312-313).
This point should be clarified, either in the methods or the results, or even both.
Author Response
Most of the previous concerns I raised were addressed by the authors. However, I suggest clarifying the following points to enhance clarity, before publication.
Here below are the previous comments and the new suggestions.
In the material and methods section, lines 142-144, I wonder why the authors did not assess the soil physico-chemical properties, especially since the plant nutritional status was later evaluated in the paper (esp. in the discussion on Mg status) as well as the macro-nutrients’ levels in plant aerial parts. In my opinion, it would be beneficial to add this feature. If it is was previously reported on the same soil, I suggest mentioning it with the appropriate reference.
Answer: We reported the element concentrations of soil as result of soil characterization in lines 313-315: the nutrient concentrations in soil were: 1611 ± 160.5 mg kg-1 Ca; 409.7 ± 46.9 mg kg-1 K; 695 ± 83.1 mg kg-1 Mg; 66 ± 6.0 mg kg-1 Cu
In most studies studying TE mobility and phytoextraction potential, soil pH is considered as a key element to include, since it may explain most of the TE bioavailability. I hence strongly suggest to include this feature to the manuscript.
Answer: We reported the soil pH values in lines 175 -176: “the soil has a sandy texture with a pH of 4.6-5”
Section 2.5, I wonder why were only Cd and Zn concentrations assessed? In the vicinity of smelters, the presence of several TE is often assessed. In addition, in the introductory section, the authors stressed the fact that the area was often polluted by Cd, Pb and Zn.
Answer: After analysing the TE concentrations in the soil used for the inoculation experiment, we selected the TEs above the threshold for Flanders legislation (lines 312-313).
This point should be clarified, either in the methods or the results, or even both.
Answer: We clarified this point in lines 135 – 136: “The TE with values above the threshold for Flanders legislation were selected for bacterial tolerance”.
Reviewer 2 Report
The manuscript has been improved taking into account my comments and suggestions.
Author Response
The manuscript has been improved taking into account my comments and suggestions
Answer: We thank the Reviewer for the comments and suggestions.
Reviewer 3 Report
- Molecular data are still not very informative and do not shed light on the functions involved. Still too many speculations.
- The duration of the experiment is a major hindrance.
Author Response
Molecular data are still not very informative and do not shed light on the functions involved. Still too many speculations.
The duration of the experiment is a major hindrance.
Answer: We agree that deeper investigations, such as transcriptomics, are needed. However, the genetic characterization can reveal the genetic backgrounds involved in TE tolerance for the tested endophytes. We thank the Reviewer for the comments.
Round 2
Reviewer 3 Report
The manuscript suffers from serious shortcomings which the authors clearly cannot answer;
Author Response
We thank the Reviewer for their detailed comments and suggestions.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors describe the benefits of inoculating two poplar cultivars with seed bacterial endophytes, to improve their growth and tolerance to TE on polluted soils.
The manuscript topic is adequate for the aims and scope of the journal. The problem is well stated. The abstract is appropriate, the purpose of this study is clear, appears experimentally sound and the manuscript contains interesting ideas. The paper is adequately written with a good introduction, precise structure, and nice discussion.
In my opinion however, some minor corrections or precisions should be provided before the paper is acceptable for publication. Please find below an itemized list of comments:
- Line 49: it is not clear what the intended valorisation channel for the biomass is. I suggest clarifying this point, especially considering the phytoremediation method that is targeted (phytoextraction / phytostabilization …).
- In the material and methods section, lines 142-144, I wonder why the authors did not assess the soil physico-chemical properties, especially since the plant nutritional status was later evaluated in the paper (esp. in the discussion on Mg status) as well as the macro-nutrients’ levels in plant aerial parts. In my opinion, it would be beneficial to add this feature. If it is was previously reported on the same soil, I suggest mentioning it with the appropriate reference.
- Section 2.5, I wonder why were only Cd and Zn concentrations assessed? In the vicinity of smelters, the presence of several TE is often assessed. In addition, in the introductory section, the authors stressed the fact that the area was often polluted by Cd, Pb and Zn.
- Section 2.7, in accordance with the biomass valorisation channel which is intended and the reported results, highlighting a limited transfer in the plant aerial parts, should the section be named “phytoextraction efficiency”?
- In tables 3 and 4, the results of the ANOVA yield a p-value above 0.05 for the K concentration in both roots and leaves of both poplar cultivars. Hence, I find it quite surprising that different statistical groups are observed between the experimental conditions for K in leaves.
- In lines 371-375, the authors highlighted the absence of Methylbacterium sp. CP3, while Kineococcus endophyticus CP19 was possibly present. However, these facts were not further integrated in the discussion, which is in my opinion could be an interesting addition, especially regarding the potential direct/undirect effects that have been mentioned at the end of the introduction section.
Line 62, “to help their host to tolerate better high TE concentrations” seems confusing to me. I suggest rephrasing the end of the sentence.
Line 175, is the abbreviation “ISF” appropriately written, since the short form for Internal Transcribed Spacer is often "ITS"?
Line 389, the sentence “In addition, Methylobacterium sp. CP3 showed able to” looks erroneous. Please rewrite.
I congratulate the authors on the extensive work and the manuscript.
Author Response
The authors describe the benefits of inoculating two poplar cultivars with seed bacterial endophytes, to improve their growth and tolerance to TE on polluted soils. The manuscript topic is adequate for the aims and scope of the journal. The problem is well stated. The abstract is appropriate, the purpose of this study is clear, appears experimentally sound and the manuscript contains interesting ideas. The paper is adequately written with a good introduction, precise structure, and nice discussion. In my opinion however, some minor corrections or precisions should be provided before the paper is acceptable for publication. Please find below an itemized list of comments:
Line 49: it is not clear what the intended valorisation channel for the biomass is. I suggest clarifying this point, especially considering the phytoremediation method that is targeted (phytoextraction / phytostabilization …).
Answer: We clarified this point in the introduction (Lines 45-62).
In the material and methods section, lines 142-144, I wonder why the authors did not assess the soil physico-chemical properties, especially since the plant nutritional status was later evaluated in the paper (esp. in the discussion on Mg status) as well as the macro-nutrients’ levels in plant aerial parts. In my opinion, it would be beneficial to add this feature. If it is was previously reported on the same soil, I suggest mentioning it with the appropriate reference.
Answer: We reported the element concentrations of soil as result of soil characterization in lines 313-315: the nutrient concentrations in soil were: 1611 ± 160.5 mg kg-1 Ca; 409.7 ± 46.9 mg kg-1 K; 695 ± 83.1 mg kg-1 Mg; 66 ± 6.0 mg kg-1 Cu
Section 2.5, I wonder why were only Cd and Zn concentrations assessed? In the vicinity of smelters, the presence of several TE is often assessed. In addition, in the introductory section, the authors stressed the fact that the area was often polluted by Cd, Pb and Zn.
Answer: After analysing the TE concentrations in the soil used for the inoculation experiment, we selected the TEs above the threshold for Flanders legislation (lines 312-313).
Section 2.7, in accordance with the biomass valorisation channel which is intended and the reported results, highlighting a limited transfer in the plant aerial parts, should the section be named “phytoextraction efficiency”?
Answer: The heading has been changed to “Bioaccumulation and translocation factors”.
In tables 3 and 4, the results of the ANOVA yield a p-value above 0.05 for the K concentration in both roots and leaves of both poplar cultivars. Hence, I find it quite surprising that different statistical groups are observed between the experimental conditions for K in leaves.
Answer: We checked the ANOVA results and we wrongly reported the Tukey post-doc tests. It was corrected.
In lines 371-375, the authors highlighted the absence of Methylbacterium sp. CP3, while Kineococcus endophyticus CP19 was possibly present. However, these facts were not further integrated in the discussion, which is in my opinion could be an interesting addition, especially regarding the potential direct/undirect effects that have been mentioned at the end of the introduction section.
Answer: We added a discussion about this point in line 513-520.
Line 62, “to help their host to tolerate better high TE concentrations” seems confusing to me. I suggest rephrasing the end of the sentence.
Answer: We rephrased the sentence as follow (Lines 74-75): Seed endophytes can provide benefits to their host plants through the growth promotion and increasing their tolerance to abiotic stresses, as TE presence in soil.
Line 175, is the abbreviation “ISF” appropriately written, since the short form for Internal Transcribed Spacer is often "ITS"?
Answer: We corrected the abbreviation.
Line 389, the sentence “In addition, Methylobacterium sp. CP3 showed able to” looks erroneous. Please rewrite.
Answer: We corrected it (Line 438).
I congratulate the authors on the extensive work and the manuscript.
Answer: We thanks to the reviewer for the useful suggestions and comments.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript brings very interesting results for phytoremediation. However, in my opinion some issues need to be address for the manuscript to be acceptable for publication.
Questions and comments
What is the main finding of this study?
Line 101
Could authors explain in detail how the phytate mineralization was evaluated? The refereed protocol appears to be to determine phosphate solubilization from inorganic forms. I have personally evaluated phosphate solubilization using this method. I have no experience in evaluating phytate mineralization, but from what I have searched I have found, for instance, the molybdenum blue method. Please clarify.
Line 117
These strains were chosen compared to which strains?
Line 132
Important steps are missing. For instance, how were samples sequenced?
Line 151
108 CFU? This value is rather low. Is this a value corresponding to a dilution? What was the procedure used count CFUs? This value seems to be the result of counting a certain dilution.
Figure 7
It is important to have the PC1 and PC2 axis represented
Minor suggestions
The discussion could use a few paragraphs to improve readability.
Line 19
trace element -> trace element (TE)
Line 32
Why is cadmium included in the keywords but not zinc?
Line 36
Trace Elements -> trace elements
Line 93
Indole-3-Acetic Acid -> indole-3-acetic acid
Line 108
Chroom-Azurol S -> chrome azurol S
Line 141
What are the concentrations of Zn and Cd in this soil?
Line 159
Was there a physical barrier separating the two poplar cultivars?
Why did the authors not used replicate containers, using a randomized design in the greenhouse?
When was the study conducted? Year and time of year?
Line 162
Inoculation set up -> Inoculation setup
Line 322
Kineococcus. endophyticus CP19 -> Kineococcus endophyticus CP19
Line 456
which -> whose
Author Response
This manuscript brings very interesting results for phytoremediation. However, in my opinion some issues need to be address for the manuscript to be acceptable for publication.
Questions and comments
What is the main finding of this study?
Answer: This study demonstrated that the bioaugmentation with Methylobacterium sp. CP3 and Kineococcus endophyticus CP19 increased the shoot length in TE polluted soil after 6 weeks of Populus ‘Marke’ growth. In addition, the inoculation with Methylobacterium sp. affected the bioaccumulation of Zn in ‘Marke’, conferring it an excluder strategy. Methylobacterium sp. and K. endophyticus seemed to improve the plant nutritional status which can contribute to alleviate abiotic stress. These findings contribute to the valorisation of TE polluted soil in agricultural area through the cultivation of TE-tolerant non-food crops.
Line 101
Could authors explain in detail how the phytate mineralization was evaluated? The refereed protocol appears to be to determine phosphate solubilization from inorganic forms. I have personally evaluated phosphate solubilization using this method. I have no experience in evaluating phytate mineralization, but from what I have searched I have found, for instance, the molybdenum blue method. Please clarify.
Answer: We reported in the manuscript the correct reference for the phytate mineralization method.
Jorquera, M. A., Hernández M. T., Rengel Z., Marschner P. and de la Luz Mora M. (2008). Isolation of culturable phosphobacteria with both phytate-mineralization and phosphate-solubilization activity from the rhizosphere of plants grown in a volcanic soil. Biology and Fertility of Soils 44(8): 1025-1034.
Line 117
These strains were chosen compared to which strains?
Answer: Methylobacterium sp. and Kineococcus endophyticus are ones of most representative seed endophytes in the C. pumila, for almost three consecutive generations of seeds. Particularly, Methylobacterium has a potential role in improving the nutrient uptake and TE tolerance of the host plant. In addition, the sequenced DNA of bacteria was available for gene annotation analysis. The bacteria were chosen comparing the strains in Crotalaria pumila core microbiome, previously isolated and studied in the following papers:
- Sánchez-López, A.S.; Pintelon, I.; Stevens, V.; Imperato, V.; Timmermans, J.P.; González-Chávez, C.; Carrillo-González, R.; Van Hamme. J.; Vangronsveld, J. Thijs, S. Seed endophyte microbiome of Crotalaria pumila unpeeled: Identification of plant-beneficial Methylobacteria. Int. J. Mol. Sci. 2018, 19(1), 291.
- Sánchez-López, A.S., Thijs, S.; Beckers, B.; González-Chávez, M.C.; Weyens, N.; Carrillo-González, R.; Vangronsveld, J. Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of Crotalaria pumila growing on metal mine residues. Plant Soil. 2018, 422(1), 51-66.
Line 132
Important steps are missing. For instance, how were samples sequenced?
Answer: The DNA sequencing of bacteria was carried out in a previously work and available data were used for gene annotation analysis. We reported this information in line 156-157.
Sánchez-López, A.S., Thijs, S.; Beckers, B.; González-Chávez, M.C.; Weyens, N.; Carrillo-González, R.; Vangronsveld, J. Community structure and diversity of endophytic bacteria in seeds of three consecutive generations of Crotalaria pumila growing on metal mine residues. Plant Soil. 2018, 422(1), 51-66.
Line 151
108 CFU? This value is rather low. Is this a value corresponding to a dilution? What was the procedure used count CFUs? This value seems to be the result of counting a certain dilution.
Answer: The correct values is 108 CFU/ml have been corrected in the manuscript.
Figure 7
It is important to have the PC1 and PC2 axis represented
Answer: We agree. We have chosen a colour version of the PCA without axes, provided by the statistical program, for a better representation of the results.
Minor suggestions
The discussion could use a few paragraphs to improve readability.
Answer: Paragraphs were added to discussion.
Line 19
trace element -> trace element (TE)
Answer: It was corrected
Line 32
Why is cadmium included in the keywords but not zinc?
Answer: Zn was added.
Line 36
Trace Elements -> trace elements
Answer: It was corrected.
Line 93
Indole-3-Acetic Acid -> indole-3-acetic acid
Answer: It was corrected.
Line 108
Chroom-Azurol S -> chrome azurol S
Answer: It was corrected.
Line 141
What are the concentrations of Zn and Cd in this soil?
Answer: The Zn and Cd concentrations are reported in line 310: “The total TE concentrations of soil were: 223.4 ± 9.74 mg Zn kg-1 and 3.6 ± 0.18 mg Cd kg-1”
Line 159
Was there a physical barrier separating the two poplar cultivars?
Answer: Any physical barrier was added to split poplar cultivars. The cultivars were placed in the pots at distance of 1m.
Why did the authors not used replicate containers, using a randomized design in the greenhouse?
Answer: We decided to place the cultivar in the same container to expose plants to the same inoculation condition. However, selected poplar cuttings were uniform in terms of number of buds.
When was the study conducted? Year and time of year?
Answer: The inoculation experiment was carried out in March and April 2019. The time information was added in line 177.
Line 162
Inoculation set up -> Inoculation setup
Answer: It was corrected.
Line 322
Kineococcus. endophyticus CP19 -> Kineococcus endophyticus CP19
Answer: It was corrected.
Line 456
which -> whose
Answer: It was corrected.
Author Response File: Author Response.docx
Reviewer 3 Report
Vannucchi and collaborators present a manuscript dealing with the characterization of bacterial endophytes and their capability to improve metal tolerance by poplar and to support poplar growth. The study confirmed many earlier results with other bacteria (see the extensive lists of cited references on this topic), also performed with more metals, that demonstrate how bacterial endophytes can tolerate much higher concentrations than other eukaryotes. The presented investigation is mainly descriptive and the manuscript lacks an outcome. The limitation of the study to one soil does not allow to make sufficient generalizations or to test for the effects of environmental factors. Only minor changes are observed in the impact of inoculation, no mechanistic data is provided, nor a clear testable hypothesis. It looks more like a data report than a hypothesis-driven investigation. This limits the scope of the present manuscript which does not advance the field of microbial ecology sufficiently to be considered for publication in Agronomy.
- Lines 112-114 : the authors should justify more clearly the choice of TE concentrations for bacteria testing; is it related to TE concentrations of soils tested latter on ? (see comment below)
- Lines 271-273: the CaCl2-extractable Zn and Cd concentrations are huge (> 50% of the total concentrations); the authors should comment this point more fully;
- Line 167 : the duration of the experiment with poplar is rather short (6 wks); the authors should comment this point more fully;
- Table 2 and related comments lines 254-263 : I do not see the interest of these data, it does not contribute anything to the rest of the study (other than speculating)
- Line 368-375 : this is unfortunate that CP3 was not detected after only 6 wks. The authors have not discussed this point. “consortium” is inappropriate here (only 2 bacteria);
- Line 397-398 : these results should have been more fully compared to other available data. Are these bacteria highly tolerant to Cd and/or Zn ?
- The discussions on bacterial tolerance linked to the presence of certain genes (Lines 414-429) are pure speculation; some transcriptomic data would have been necessary to support these interpretations
- The findings of the study are somehow oversold in the abstract and the conclusion; how authors can conclude about the long-term impact while the experience only lasted 6 weeks ? how can one consider CP3 as good candidate while it is not detected on poplar after only 6 wks and having a very limited impact on root Markle BAF ?
Author Response
Vannucchi and collaborators present a manuscript dealing with the characterization of bacterial endophytes and their capability to improve metal tolerance by poplar and to support poplar growth. The study confirmed many earlier results with other bacteria (see the extensive lists of cited references on this topic), also performed with more metals, that demonstrate how bacterial endophytes can tolerate much higher concentrations than other eukaryotes. The presented investigation is mainly descriptive and the manuscript lacks an outcome. The limitation of the study to one soil does not allow to make sufficient generalizations or to test for the effects of environmental factors. Only minor changes are observed in the impact of inoculation, no mechanistic data is provided, nor a clear testable hypothesis. It looks more like a data report than a hypothesis-driven investigation. This limits the scope of the present manuscript which does not advance the field of microbial ecology sufficiently to be considered for publication in Agronomy.
Lines 112-114: the authors should justify more clearly the choice of TE concentrations for bacteria testing; is it related to TE concentrations of soils tested latter on? (see comment below)
Answer: TE concentrations in vitro had been selected to characterize and to evaluate the maximum inhibitory concentrations for selected bacteria. The experimental design description was added in line 135-136.
Lines 271-273: the CaCl2-extractable Zn and Cd concentrations are huge (> 50% of the total concentrations); the authors should comment this point more fully
Answer: We checked the data, and the correct values are reported. This point was commented in Line 311: “CaCl2-extractable Zn and Cd concentrations were 5.72 ± 0.032 and 0.13 ± 0.008 mg kg-1, respectively”.
Line 167: the duration of the experiment with poplar is rather short (6 wks); the authors should comment this point more fully
Answer: In pot studies an alteration of plant growth and physiology can occur, due to the limited growing condition. We decided the duration of the potting experiment in order to avoid a possible plant stress for growing condition. This was fully explained in line 192-193.
Table 2 and related comments lines 254-263: I do not see the interest of these data, it does not contribute anything to the rest of the study (other than speculating)
Answer: We have chosen to apply the gene annotation on bacteria genomes to better explain the TE tolerance tested in vitro.
Line 368-375: this is unfortunate that CP3 was not detected after only 6 wks. The authors have not discussed this point. “consortium” is inappropriate here (only 2 bacteria)
Answer: We discussed this point in lines 513-520 and the word “consortium” has been deleted along manuscript.
Line 397-398: these results should have been more fully compared to other available data. Are these bacteria highly tolerant to Cd and/or Zn?
Answer: The results were discussed in lines 453-458.
The discussions on bacterial tolerance linked to the presence of certain genes (Lines 414-429) are pure speculation; some transcriptomic data would have been necessary to support these interpretations
Answer: This assessment was added to the conclusions. As mentioned above, the gene annotation was applied to characterize the tested bacteria and better explain the TE tolerance tested in vitro
The findings of the study are somehow oversold in the abstract and the conclusion; how authors can conclude about the long-term impact while the experience only lasted 6 weeks? how can one consider CP3 as good candidate while it is not detected on poplar after only 6 wks and having a very limited impact on root Markle BAF?
Answer: We rephrase the conclusion section.
Author Response File: Author Response.docx
