Unravelling the Combined Use of Soil and Microbial Technologies to Optimize Cultivation of Halophyte Limonium algarvense (Plumbaginaceae) Using Saline Soils and Water

Round 1

Reviewer 1 Report

Soil degradation, especially salinization, is currently one of the important environmental issues faced by many countries around the world. The author used AMF and PGPB bacteria to alleviate salt stress. There have been many similar studies using these two strains alone. However, this article combined the two strains and achieved good experimental results, which has potential application prospects. 

From the experimental results, there was no significant difference in the reduction of soil Na⁺content between AMF+PGPB inoculation and AMF alone inoculation. Similarly, there was no significant difference in the fresh weight of stems and roots of Limonium algarvense (Table 6C). However, PGPB-inoculation led to higher NDVI values in the TEC treatment group. The combined use of soil and microorganisms in the article has certain application prospects, but can the author further explain that the cost of large-scale use is competitive?

Author Response

Although the use of halotolerant microbial inocula in large-scale applications is still an underexplored issue, the industry of plant-growth promoting microorganisms is experiencing steady growth each year. These microorganisms are predominantly used in agriculture, as well as in reforestation and revegetation projects, which leads us to believe that their application in saline contexts could also be competitive. Additionally, the practice of using tailored soils for the recovery of polluted or degraded lands is already being implemented by several companies today, further indicating the potential cost-effectiveness of applying this approach in saline soils.

We have included this paragraph in the conclusion to acknowledge the need for further studies and research to scale up these technologies.

Now reads:

However, despite these promising findings, it is essential to acknowledge that further validation through field trials and collaboration with stakeholders are essential steps to-wards assessing the cost-effectiveness of large-scale use and for successfully applying these combined soil and microbial technologies in real-world salt-affected soil recovery and vegetation restoration efforts.

Reviewer 2 Report

The text is well written and I learned with this work. However, field validation is still necessary for practical application and mechanisms are not explored in the article. In my view, this effort is necessary, but additional studies are needed before publicaion. Additional suggestions are following:

- The underlying mechanisms related to the effect of microorganisms on plant responses could be better explored in the introduction

- Hypothesis or scientific questions should be presented

- Improve methods in order to allow reproducibility. 

- Improve discussion and conclusions (not suported by methods and results in this version). 

Author Response

The text is well written and I learned with this work. However, field validation is still necessary for practical application and mechanisms are not explored in the article. In my view, this effort is necessary, but additional studies are needed before publication.

Even if field validation is the next step, this does not disvalue the merit of the manuscript. Experiments were conducted in semi-controlled conditions, with non-sterile soil and autochthonous seeds collected from their original sites, and under such conditions, we found a positive effect of both kind of techniques. The information that we collected was highly valuable and certainly encourages to carry on with field trials. However, in our opinion, this should be conducted in a different experiment, which would lead another scientific article.

In any case, we have addressed now this issue in the Conclusion section.

Now reads:

However, despite these promising findings, it is essential to acknowledge that further validation through field trials and collaboration with stakeholders are essential steps to-wards assessing the cost-effectiveness of large-scale use and for successfully applying these combined soil and microbial technologies in real-world salt-affected soil recovery and vegetation restoration efforts.

Additional suggestions are following:

- The underlying mechanisms related to the effect of microorganisms on plant responses could be better explored in the introduction.

We have included some extra information in the introduction (Lines 66-71):

Now reads:

One of the ways they achieve this is by activating membrane transporters, such as Na+/H+ antiporters, which help the plant cope with high salt levels and contribute to improving plant water relations [15–19]. Moreover, AMF and PGPB can also lead to an increase in photosynthetic pigments and glutathione levels in their hosts [20], which, in turn, results in a decrease in ROS levels and lipid peroxidation [21–22].

 - Hypothesis or scientific questions should be presented

We have included a sentence at the end of the introduction to specify the hypothesis of this work.

Now reads:

The underlying hypothesis was that the combination of PGPB, AMF and a Technosol could synergistically improve plant growth, as well as vegetative and reproductive development.

- Improve methods in order to allow reproducibility.

We would like to ask the reviewer to be more specific about the methods that have not been explained sufficiently. Some procedures were explained in detail in our previous published articles, and therefore, we put the reference of those ones. Anyway, if there is any particular methodology that should be better explained, we will be happy to develop it further.

- Improve discussion and conclusions (not supported by methods and results in this version).

We have carefully read the discussion to see if there was any part that did not match the results that we obtained, and we have not found anything.  For this reason, we would like to kindly ask the Reviewer to be more specific and to let us know which paragraph(s) of the Discussion are in disagreement with the results.

Here is a summary of the main results that we have, and the corresponding explanation in the Discussion section:

1. Technosols were a good strategy to improve Fluvisol characteristics (as shown in Table 4) and allowed plant development even when those were not inoculated with any beneficial microorganism (see Discussion-Lines 494-497).

2. Only mycorrhizal plants were able to grow in the FLU (lines 330-333). The explanation of those results can be found in discussion section, lines 552-532.
3. A significant positive effect of mycorrhizal inoculation was found in inflorescence fresh biomass (Figure 3, Line 4142). The explanation for those results is in Discussion section Lines 552-565.

4. The possitive effect of PGPB is shown in Figure 1, Lines 341-343, Lines 369-375. This is discussed in Discussion section, lines 537-538, 542 and 567-575.

We even make a summary of the main results at the end the Discussion section (Lines 575-577):

Now reads:

In summary, AMF-inoculation promoted higher growth and reproductive development in TEC, while PGPB-inoculation led to higher NDVI values, supporting the use of these microbial-based technologies in salt-affected soils recovery.

We have improved the conclusion section in the new, revised version of the manuscript.

Reviewer 3 Report

The study is very interesting and well written, this manuscript “Unravelling the combined use of soil and microbial technologies to optimize cultivation of halophyte Limonium algarvense (Plumbaginaceae) using saline soils and water” presented very important results and good discussion, the conclusion can be improved for more clarification of the importance of this study.

This work can be published in soilsystems journal.

Author Response

The study is very interesting and well written, this manuscript “Unravelling the combined use of soil and microbial technologies to optimize cultivation of halophyte Limonium algarvense  (Plumbaginaceae) using saline soils and water” presented very important results and good discussion, the conclusion can be improved for more clarification of the importance of this study.

This work can be published in soilsystems journal.

We thank the reviewer for the nice comments.

Round 2

Reviewer 2 Report

I am grateful for the opportunity to read and learn from this manuscript. The work has been improved after the review. However, I remain convinced that it is a preliminary work that should be strengthened by the elucidation of mechanisms underlying current findings. Additionally, the statistical analysis has problems, not detected in the previous review. The experiment is a triple factorial and the analysis was performed considering a double factorial, within each soil. So, it is not possible to conclude on the effects of this factor. Additionally, with one degree of freedom, the F test of the anova is conclusive and a post hoc test is not necessary.

Author Response

I am grateful for the opportunity to read and learn from this manuscript. The work has been improved after the review.

We thank the reviewer for his/her comments.

However, I remain convinced that it is a preliminary work that should be strengthened by the elucidation of mechanisms underlying current findings.

In our opinion, a thorough examination of the mechanisms underlying the experiment's findings would necessitate complementary transcriptomic analyses in plants, at the very least. However, this would fall beyond the scope of this journal. We are of the belief that the data we have collected in our study are both sufficient and coherent for a well-constructed article.

Nonetheless, we comment on the mechanisms that could contribute to the improved performance of the plants resulting from the inoculations:

• In the introduction, lines 63-68: “In saline environments, both PGPB and AMF play a crucial role in promoting plant growth and development through various mechanisms. They improve nutrient uptake, produce phytohormones like auxins, gibberellins, and abscisic acid, and enhance the plant's tolerance to saline stress. One of the ways they achieve this is by activating membrane transporters, such as Na⁺/H⁺ antiporters, which help the plant cope with high salt levels and contribute to improving plant water relations [15–19]”.

• In the discussion, lines 578-583 “However, since AMF improve membrane integrity and permeability under saline conditions [103,104], stimulate ABA production, accumulate osmolytes), and promote the uptake of osmotic equivalents like K⁺ [76], mycorrhizal plants may have experienced a faster adaptation to the non-amended saline soil and potentially a less intense transplantation shock compared to non-mycorrhizal plants.

• Lines 624- 626 “The bacteria used in the present study possessed plant growth promoting activities like siderophore and auxin production, and was diazotrophic [49], which may have contributed to a better vegetative and reproductive performance of those plants.”.

Additionally, the statistical analysis has problems, not detected in the previous review. The experiment is a triple factorial and the analysis was performed considering a double factorial, within each soil. So, it is not possible to conclude on the effects of this factor. Additionally, with one degree of freedom, the F test of the anova is conclusive and a post hoc test is not necessary.

In our case, missing values in two experimental groups (resulting from the mortality of all non-AMF plants in the FLU condition) increase the complexity of the statistical analysis. Thus, it is not possible to analyze our soil and plant data with a three-way ANOVA. In other words, in the FLU, we have missing values in one of the levels of the AMF factor (we don’t have data for the non-AMF), which hinders the study of the effect of this factor and its interactions in a three-way ANOVA.

However, we agree that conducting a two-way ANOVA where “soil” and “microbial inoculation” are considered as main factors might not have been the most suitable way to analyze those data. For this reason, we have decided to eliminate this analysis from the manuscript, and we have removed from the Results and Discussion sections the paragraphs commenting the results of the “soil type” factor.

Instead, we have included a t-test to compare soil and plant parameters in AMF and AMF+PGPB treatments in the FLU and in the TEC.

We have addressed this matter in the Statistical Analysis section, and in Results and Discussion.

For example:

• In statistical Analysis section, lines 224-228:

At the end of the experiment data collected from different soils under various microbial inoculation treatments were analyzed separately for the TEC and FLU conditions. The soil type factor could not be included in a factorial ANOVA due to the non-survival of plants from two experimental groups in the FLU (non-inoculated and PGPB-inoculated plants).

• In results section, lines 28-292:

When L. algarvense plants were transplanted to those soils, non-mycorrhizal plants did not survive in the FLU. Therefore, the pots containing those plants were discarded and the soils were not analyzed at the end of the experiment. However, although we could not study the effect of the soil type factor in a full factorial ANOVA, several trends could be observed between FLU and TEC.