Poaceae with PGPR Bacteria and Arbuscular Mycorrhizae Partnerships as a Model System for Plant Microbiome Manipulation for Phytoremediation of Petroleum Hydrocarbons Contaminated Agricultural Soils
, Mario Marziano, Maria Tartaglia, Antonello Prigioniero, Alessia Postiglione, Pierpaolo Scarano and Rosaria SciarrilloRound 1
Reviewer 1 Report
The literature review should be improved, with more content in the world wide soil problematic with TPH's, also, give a little bit more details in what are the effects on soil of the TPH's and the physical chemical characteristics. Also, you mentioned that this study is focused on the rhizodegradation as a phytoremediation method, so expand a litttle bit on it.
In line 53 you mentioned that contains genes that can improve TPH's, which ones??
Line 67, no references you are indicating about the topic.
Improve that table 1. Reduce or improved the font, so the titles are clear.
In line 98. should be endomycorrhizae, with an "h"
In line 114, you need to have italics for the specie. P. setaceum.
In line 123, you mentioned a fourth month (240 days), it should be 120 days or if it is 240 days should be 8 months??
The contaminated soil, which kind of density of bacteria was present? as the basal amount?
line 250. You mention it improves plant uptake, but you didn't say of what.
Line 252-253. What is the difference between shoots and leaves??
In table 4, you used leaves, be consistent, or leaves or shoots.
Table 6. It is missing the control group for P. miliaceum
Lines 302-304. In your discussion, you talk about that the endomycrobes improved plant nutrient status, did you measure elemental content? (Nitrogen, Carbon)? otherwise, if you are talking about other authors experiences, you should add a references for each statement. Line 309, as well.
These poaceae plants after uptaken and rhizodegrade TPH's what will be their fate?
Line 341-342. There are some studies, in organics. https://doi.org/10.1590/1807-1929/agriambi.v21n8p573-578
References like 10.3389/fmicb.2016.01836
could be important for your discussion
Author Response
RESPONSE TO REVIEWERS
Title: “Poaceae with PGPR bacteria and arbuscular mycorrhizae partnerships as model system for plant microbiome manipulation for phytoremediation of a petroleum hydrocarbons contaminated agricultural soils."
Ref.: agronomy-743858
Agronomy
OUR RESPONSES IN RED
Reviewer #1:
1. The literature review should be improved, with more content in the world wide soil problematic with TPH's, also, give a little bit more details in what are the effects on soil of the TPH's and the physical chemical characteristics. Also, you mentioned that this study is focused on the rhizodegradation as a phytoremediation method, so expand a little bit on it. Since TPH contamination in the soil is a worldwide problem and also very studied, the AA. were considered more interesting to focus mainly on the activities carried out and the expected results.
2. In line 53 you mentioned that contains genes that can improve TPH's, which ones?? Expression of one or more catabolic genes in either one or both partners in plant-endophyte interaction is well-reported for enhanced removal of pollutants in mixed as well as single contamination scenarios. Catabolic genes against organic compounds: CYP2E1 gene belongs to a family of genes involved in xenobiotic tolerance and is reported to be efficiently expressed under suitable promoters in plants and microbes for reduction of trichloroethylene (TCE) in contaminated site. Another set of genes effective in detoxification of aromatic xenobiotic compounds in microbes is GST genes, which are often part of operons responsible for degradative metabolic pathways. Bacterial bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase enzyme (BphC), which metabolizes PCBs by cleaving their aromatic ring.
3. Line 67, no references you are indicating about the topic. Pignatti, S.; Guarino, R.; La Rosa, M. Flora d’Italia; Ed. Agricole: Italy, 2017; Vol. 1; ISBN 9788850652426.
4. Improve that table 1. Reduce or improved the font, so the titles are clear. Table 1 has been improved and the titles are clear.
5. In line 98. should be endomycorrhizae, with an "h". Correct
6. In line 114, you need to have italics for the specie. P. setaceum. Correct
7. In line 123, you mentioned a fourth month (240 days), it should be 120 days or if it is 240 days should be 8 months?? Correct: Eighth month (TF: 240 days).
8. The contaminated soil, which kind of density of bacteria was present? as the basal amount? The basal amount of bacteria in the contaminated soil was 2.0x1011- CFU ml-1g of soil.
9. line 250. You mention it improves plant uptake, but you didn't say of what. The consortium also plays an important role in increasing the uptake of TPHs in the plant.
10. Line 252-253. What is the difference between shoots and leaves?? There is a writing error, only the leaves have been used.
11. In table 4, you used leaves, be consistent, or leaves or shoots. Leaves have been used.
12. Table 6. It is missing the control group for P. miliaceum. The control group for P. miliaceum is the same for Pennisetum setaceum. Now, it has been introduced for homogeneity of the table 6.
13. Lines 302-304. In your discussion, you talk about that the endomycrobes improved plant nutrient status, did you measure elemental content? (Nitrogen, Carbon)? otherwise, if you are talking about other authors experiences, you should add a references for each statement. The authors measured the elemental content of Nitrogen and carbon before and after the introduction of the endophytic consortium and verified that there is an increase compared to the controls (data not shown).
14. Line 309, as well. Correct
15. These Poaceae plants after uptaken and rhizodegrade TPH's what will be their fate? After having attacked and rhizodegraded the TPH, these Poaceae plants will be disposed of in authorized plants according to the Italian legislation in force.
16. Line 341-342. There are some studies, in organics. https://doi.org/10.1590/1807-1929/agriambi.v21n8p573-578. This study has been cited [54].
17. References like 10.3389/fmicb.2016.01836 could be important for your discussion. This study [55] had been considered by the authors but by pure forgetfulness it had not been mentioned. It has now been introduced into the text of the manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
- The research title and the abstract recommended to shortening appropriately to clearly expressing the research focus.
- The introduction should introduce the general treatment methods for remediating the TPH, and the advantages and disadvantages of these methods.
- This study lacks novelty except for the decreased of remediation costs mentioned in the introduction. I suggest that the innovative points of the manuscript provide in a supplement.
- The reasons for choosing Poaceae with two plants (Piptatherum miliaceum and Penensetum setaceum) must explain in this content.
- According to the results of this study, it is known that the combination of different methods can obtain favorable treatment efficiency. Have there been similar studies in the past? If have, it is recommended to discuss the main mechanism of cooperative effect in this article.
- This study has a detailed experimental design and explanation. However, the reason for some operation parameters have not explained, so it is not clear what the idea of these conditions. Besides, it is recommend that some sections can be combine to describe.
- Section 3.2 appears also on line 197; please confirm whether it is repeat.
- In summary, the experimental data analysis of this study is sufficient. However, the relative correlation between each analysis data is lack. It is recommended that it be comprehensively discussed. Besides, the content of chlorophyll whether that has a significant effect on the degradation of TPH? There is no relevant explanation.
- Does there is a regulatory maximum allowable value for TPH in the soil, it is recommended to add a description in this content. Whether the soils had met the regulatory standard after 240 days remediation?
- This article does not explain the difference between the two plants in the remediation effect.
- The effect of the Integration effect has explained in the discussion section of this study. However, the optimal operating parameters have not given in this manuscript.
- Whether have any restrictions factor in the future when the technique is widely used in such contaminated soil? For example, contaminated concentration, soil characteristics, operation time, etc.
Author Response
RESPONSE TO REVIEWERS
Title: “Poaceae with PGPR bacteria and arbuscular mycorrhizae partnerships as model system for plant microbiome manipulation for phytoremediation of a petroleum hydrocarbons contaminated agricultural soils."
Ref.: agronomy-743858
Agronomy
OUR RESPONSES IN RED
Reviewer #2:
1. The research title and the abstract recommended to shortening appropriately to clearly expressing the research focus. The abstract has been shortened. To tell the truth the title pleases the authors therefore they believe they will not change it.
2. The introduction should introduce the general treatment methods for remediating the TPH, and the advantages and disadvantages of these methods. The general treatment methods for TPH repair are very well known therefore the authors found it unnecessary to focus on general speeches and to focus the introduction on a plant-endophyte association because this partnership can generate new phytoremediation targets for polluted land.
3. This study lacks novelty except for the decreased of remediation costs mentioned in the introduction. I suggest that the innovative points of the manuscript provide in a supplement. In the introduction, the authors have made explicit the innovation points of their study. Indeed, the possibility to obtain synergistic effects between plants and microorganisms useful for the growth and development has increased the possibility of alternative techniques. Therefore, bacteria associated with plant such as endophytic bacteria and fungi that live in cortical tissue of roots of plants and rhizosphere bacteria that live on, near the roots of plants, contribute to biodegradation of organic contamination in soil, and improve phytoremediation. These endophytes, because of intimate contact with their host plants, play vital roles in plant development, growth, and fitness, as well as decontaminating polluted soil. Endophytic microorganisms are highly varied groups that stay within the tissues of plants for at least part of their life cycle. Endophytic bacteria and fungi host a large amount of genes that degrade pollutants. These benefits from endophytic microorganisms could improve the usage of endophytes in the phytoremediation of organic.
4. The reasons for choosing Poaceae with two plants (Piptatherum miliaceum and Penensetum setaceum) must explain in this content. In the introduction, the authors have made explicit the choose of two plants “Oloptum miliaceum and Pennisetum setaceum are bushy plants with numerous upright or ascending culms, wrapped in sheaths and with the appearance of tight tufts. They are perennial plants cultivated by means of gems placed at ground level. They have strong ecological value as they resist drought and live in poor and exposed land. They live well even on disturbed soils. This species are very resistant to dryness because it does not need any special cultural practices neither is subject to plant diseases “.
5. According to the results of this study, it is known that the combination of different methods can obtain favorable treatment efficiency. Have there been similar studies in the past? If have, it is recommended to discuss the main mechanism of cooperative effect in this article. The authors have experience on the combination of different methods; in fact they recently published a highly cited article in this regard (Chemosphere. 2017 Mar; 170:10-16. doi: 10.1016/j.chemosphere.2016.11.165. Assessment of three approaches of bioremediation (Natural Attenuation, Landfarming and Bioagumentation - Assistited Landfarming) for a petroleum hydrocarbons contaminated soil); but the aim of this manuscript isn’t the combination of different methods but rather the plant endophyte microorganism association. Precisely, the plant endophyte microorganism association should not be seen in a perspective of summation of effects but a rearrangement of co-metabolism to consider together one big organism that reorganizes itself through signals and metabolic adjustments. In fact, more effective degradation of contaminants requires interactions to support growth and catabolic collaboration between plants and hypothetically thousands of degradative microbial taxa that certainly colonize the host. This ultimately controlled to the concept of "Meta-organism" or Microbiome vegetable, mentioning to the total of a host plant and its connected microbial population.
6. This study has a detailed experimental design and explanation. However, the reason for some operation parameters have not explained, so it is not clear what the idea of these conditions. Besides, it is recommend that some sections can be combine to describe. The parameters chosen serve to demonstrate that Poaceae are a good candidate to obtain synergistic effects between plants and microorganisms useful to generate new phytoremediation targets for polluted land. Therefore, the authors do not find it useful to combine the description of the parameters.
7. Section 3.2 appears also on line 197; please confirm whether it is repeat. There are no repetitions.
8. In summary, the experimental data analysis of this study is sufficient. However, the relative correlation between each analysis data is lack. It is recommended that it be comprehensively discussed. Besides, the content of chlorophyll whether that has a significant effect on the degradation of TPH? There is no relevant explanation. Leaf chlorophyll content of both plants was physiological parameter. The level of reduction in chlorophyll content in plants has been an indication of environmental contamination. At the end of experiment, the chlorophyll content was highest in plants grown on control soil, slightly lower in plants grown on soil amended with inoculum of autochthonous bacteria (Group 2) and again significantly less in plants grown without the inoculum of autochthonous bacteria (Group 1). TPH is a mixture of aliphatic, aromatic, and high molecular weight organic compounds which inhibit the enzymes necessary for synthesizing chlorophyll (Anthony 2001; Al-Hawas et al., 2012; Njoku et al.,2012; Baruah et al., 2014). Decrease in the total chlorophyll content in the leaves is perhaps due to the alkaline condition created by dissolution of chemicals present in the oil in the cell sap which was responsible for chlorophyll degradation (Hussain et al., 2019).
9. Does there is a regulatory maximum allowable value for TPH in the soil, it is recommended to add a description in this content. Whether the soils had met the regulatory standard after 240 days remediation? The Italian legislation (Legislative Decree 152/06) provide two threshold limits for TPH: 50 and 750 mg/kg for recreational and commercial land use, respectively .After 240days the soils had met the limit for TPH: 750 mg/kg commercial land use.
10. This article does not explain the difference between the two plants in the remediation effect. Analysis of hydrocarbons was performed in all the variants, but the removal percentage, calculated by a formula as explained in Guarino et al. (2017): 100 × [(Ci − Cf)/Ci], where Ci stands for the TPH concentration at the initial stage and Cf shows the TPH concentration after 240 days of experiment, was the same of two plants; therefore, there isn’t significant difference between the two plants in the remediation effect.
11. The effect of the Integration effect has explained in the discussion section of this study. However, the optimal operating parameters have not given in this manuscript. The optimal operating parameters have been indicated in the materials and methods.
12. Whether have any restrictions factor in the future when the technique is widely used in such contaminated soil? For example, contaminated concentration, soil characteristics, operation time, etc. This laboratory experimentation has already been successfully used in the field without any limitation since the laboratory experimentation took place using the soils of the site to be reclaimed.
Author Response File: Author Response.docx
Reviewer 3 Report
- I think this paper gives an advancement in the phytoremediation of soil contaminated with petroleum hydrocarbons. The authors of the paper have written a very interesting paper in which the use of a consortium of indigenous bacterial selected from rhizosphere soil and mycorrhizae strains is used to remove aliphatic hydrocarbons and they have published a lot of results to demonstrate the conclusions.
- However, I suggest that the authors do major revisions of the manuscript, so specific comments can be find below:
Abstract: this section is too long. This should be a brief summary to the readers can understand the relevant of this paper. The authors have written in the abstract part of the introduction or the materials, which are not necessary in the abstract section.
Materials and methods: from my point of view I will added a first section only for the soil. In this section should be explain all the characteristics of the soil used in this study and for this reason the table S1 would be mentioned in this new section and not in the section related with the mesocosm experiment. If the authors will do it, in other sections they don’t need to repeat the explication of the soil used in the experiments. I recommended you do that because the authors have repeated in the line 88-89 the same words that in the lines 78-79.
In the section 2.2 only should be explain how the mesocosm experiment have been done, but the results of the table 1 should not be exposed in this section because this table is part of the section of the results and in addition the authors have repeated the explication of this table in the section 3.1.
The explanation of the experimental design is too long and it is difficult to understand by the readers, for this reason I proposed to do a figure summarizing the experimental design. From my point of view when plants are using in phytoremediation experiments, controls without plants are very important and these are necessary to demonstrate that the relevant results are only obtained in presence of the plants. The authors need to explain very good why these controls were not carried out.
In the section 2.6 and 2.7.1 the authors repeated the same sentence to explain the method used to quantification of petroleum hydrocarbons because this method is the same for the parts of plants and for the soil, although the extraction of plants and soil is different. For this reason it is beater to added a section only to explain the quantification of petroleum in general (for plants and for soils) and with that the authors don’t need repeated the same 8line 150 and 156). In addition they should explain in brief this method to quantification because it is not enough with the mention of the paper Guarino and Sciarrillo 2017.
Results: the most important thing that in this section should be improve, from my point of view, it is the presentation of all tables.
Tables are too big and it is difficult to get conclusions for the readers. For example in the section of materials and methods should be explain the meaning of T1, T2, T3 and TF and with this action it isn’t necessary to repeat in every tables this meaning of these.
The table 5 is very important because in that the most important conclusions of the paper are represented: the reduction of concentration TPHs that is the objective of phytoremediation. For this reason, the dates of this table could be represented in one or two graphics in which the readers could see important conclusions immediately. The same action could be done with table 4.
Author Response
RESPONSE TO REVIEWERS
Title: “Poaceae with PGPR bacteria and arbuscular mycorrhizae partnerships as model system for plant microbiome manipulation for phytoremediation of a petroleum hydrocarbons contaminated agricultural soils."
Ref.: agronomy-743858
Agronomy
OUR RESPONSES IN RED
Reviewer #3:
1. Abstract: this section is too long. This should be a brief summary to the readers can understand the relevant of this paper. The authors have written in the abstract part of the introduction or the materials, which are not necessary in the abstract section. The abstract has been re-written.
2. Materials and methods: from my point of view I will added a first section only for the soil. In this section should be explain all the characteristics of the soil used in this study and for this reason the table S1 would be mentioned in this new section and not in the section related with the mesocosm experiment. If the authors will do it, in other sections they don’t need to repeat the explication of the soil used in the experiments. I recommended you do that because the authors have repeated in the line 88-89 the same words that in the lines 78-79. The sub-paragraphs have been divided in relation to the activities that have taken place and not in relation to the matrices used. repeated phrases have been eliminated (line 88-89).
3. In the section 2.2 only should be explain how the mesocosm experiment have been done, but the results of the table 1 should not be exposed in this section because this table is part of the section of the results and in addition the authors have repeated the explication of this table in the section 3.1. The table 1 has been moved to the section of the results.
4. The explanation of the experimental design is too long and it is difficult to understand by the readers, for this reason I proposed to do a figure summarizing the experimental design. From my point of view when plants are using in phytoremediation experiments, controls without plants are very important and these are necessary to demonstrate that the relevant results are only obtained in presence of the plants. The authors need to explain very good why these controls were not carried out. The controls without plants were not included in the experimental design as the aim of the experimentation was to demonstrate the possibility to obtain synergistic effects between plants and microorganisms useful for the possibility of alternative techniques. . These endophytes, because of intimate contact with their host plants, play vital roles in plant development, growth, and fitness, as well as decontaminating polluted soil.
5. In the section 2.6 and 2.7.1 the authors repeated the same sentence to explain the method used to quantification of petroleum hydrocarbons because this method is the same for the parts of plants and for the soil, although the extraction of plants and soil is different. For this reason it is beater to added a section only to explain the quantification of petroleum in general (for plants and for soils) and with that the authors don’t need repeated the same (line 150 and 156). In addition they should explain in brief this method to quantification because it is not enough with the mention of the paper Guarino and Sciarrillo 2017. The authors preferred to keep two sub-paragraphs because although the method of quantification of TPH in the soil and in plant tissues is the same but the two extraction methods for the two matrices are different. Furthermore the authors considered the activities for the two matrices.
6. Results: the most important thing that in this section should be improve, from my point of view, it is the presentation of all tables. The tables were been improved.
7. Tables are too big and it is difficult to get conclusions for the readers. For example in the section of materials and methods should be explain the meaning of T1, T2, T3 and TF and with this action it isn’t necessary to repeat in every table this meaning of these. In the section of material and methods are explain the meaning of T1,T2 T3 and TF (lines 120-124).
8. The table 5 is very important because in that the most important conclusions of the paper are represented: the reduction of concentration TPHs that is the objective of phytoremediation. For this reason, the dates of this table could be represented in one or two graphics in which the readers could see important conclusions immediately. The same action could be done with table 4. The tables 4 and 5 are been represented in two graphics. Therefore, Table 4 has become Figure 3 and Table 5 has become Figure 4.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
No other comment.
Author Response
The work was reviewed by a native speaker.
Reviewer 3 Report
from my point of view the authors have responded favourably to all the reviewers' proposals and they have managed to improve the quality of this paper.
I think that this paper could be accept in this new format.
Author Response
The work was reviewed by a native speaker.
