Moss Biochar Facilitates Root Colonization of Halotolerant Halomonas salifodinae for Promoting Plant Growth Under Saline–Alkali Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study " Moss biochar facilitates root colonization of halotolerant Halo-2 monas salifodinae for promoting plant growth under saline-al-3 kali stress" provides valuable insights into biochar-halotolerant bacterium joint strategy to enhance Medicago tolerance under saline-alkali soils. However, some issues were identified in the manuscript.

Introduction-Should include more related review to have strong background of the study and methods.

A section about saline-alkali stress response mechanisms including mainly antioxidant and osmoprotectant (sugar and proline) responses is missed.

Please explain how biochar promote the phytoremediation of heavy metals.

L69-71- It is hypothesized that specific kinds of biochar ……….: Could you identify these specific kinds of biochar

In the discussion section author should incorporate more references to support their present findings. Authors also incorporated the saline tolerance mechanisms of other related plant species having corresponding data of the present study. It could provide good insights to draw comparative statement on tolerance mechanisms.
Discussion-

Discussion lacks important references in the subject matter.

The section about Improvement of plant physiological and biochemical indicators needs more elaboration

POD and CAT activities were not discussed.

Methods- If authors could use more than one saline-alkali stress treatment then it would be interesting and systematic to monitor the parameters/attributes well.

L343-344 : The plants were cultured under room temperature and free sunlight irradiation for 30 days …… : The culture conditions are not enough clear, is it under controlled or field conditions ?? Why did the authors select 30 days of plant culture? What stage of plant development does this correspond to? For plant growth and care appropriate reference should be included.

Why antioxydant enzymes were measured in leaves while proline and MDA in roots ??

L360-362 : The activities of S-POD, S-AKP, S-UE and ….. this sentence was repeated in L377-381.

 

 

 

 

Author Response

Response to Reviewer 1

1. Introduction-Should include more related review to have strong background of the study and methods.

A section about saline-alkali stress response mechanisms including mainly antioxidant and osmoprotectant (sugar and proline) responses is missed.

Response:

Thank you for your valuable suggestions. Here, we revised and refined saline-alkali stress response mechanisms in the Instruction:

“Saline-alkali stress, a combined abiotic stress involving high salinity (ionic toxicity) and elevated pH (alkaline toxicity), disrupts plant homeostasis through four primary mechanisms: osmotic imbalance, sodium hyperaccumulation-induced ionic toxicity, oxidative stress, and pH-mediated metabolic dysfunction [10, 11]. Notably, the synergistic effects of excessive Na⁺ and alkaline conditions amplify reactive oxygen species (ROS) overproduction—including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (·OH)—which induce structural damage to cellular membranes, proteins, and nucleic acids. To counteract this, plants employ a dual-defense strategy integrating enzymatic (SOD, CAT, APX) and non-enzymatic (ascorbate, glutathione) antioxidant systems with osmoprotectant synthesis. The ROS signaling cascade is mechanistically linked to calcium-mediated pathways. Experimental evidence indicates that transient Ca²⁺ flux activates the MAPK cascade and downstream transcription factors (e.g., NAC, WRKY), thereby upregulating antioxidant enzyme genes [10, 12]. Osmoprotectant-mediated osmotic homeostasis, utilizing metabolites such as soluble sugars and proline. Notably, these solutes function not merely as osmotic balancers but also as multifunctional regulators—directly neutralizing ROS, modulating redox-sensitive signaling cascades, and preserving macromolecular integrity under oxidative damage. Emerging evidence highlights their synergistic roles in stress adaptation: proline stabilizes subcellular structures while enhancing NADPH-dependent antioxidant regeneration, whereas soluble sugars participate in ROS metabolism through the pentose phosphate pathway, concurrently buffering cytoplasmic pH under alkaline conditions.”

 

2. Please explain how biochar promote the phytoremediation of heavy metals.

Response:

We sincerely appreciate the valuable comments. Our group previous research found that the fungal hypha-based biochar and moss biochar may promote the phytoremediation of heavy metals by regulating of rhizosphere microbiome. The mechanism as follows:

“The robust bacterial-binding capacity of biochar facilitates stable colonization of exogenous STBs on roots and in rhizosphere soils by cross-linking these inoculants with indigenous microbiota. Notably, the biochar-STB synergy further enhances rhizospheric biofilm formation, creating a protective niche that shields microorganisms from environmental stressors while concurrently amplifying cadmium uptake in plants. This dual mechanism—microbial stabilization coupled with heavy metal mobilization—systematically elevates phytoremediation efficiency through coordinated biotic-abiotic interactions [29,30].”

 

3. L69-71- It is hypothesized that specific kinds of biochar ……….: Could you identify these specific kinds of biochar.

Response:

In this study, we used prepared moss biochar, which is a known material. The preparation process is as follows:

“The moss biochar was prepared by our previous study [31]. Briefly, the Brachythecium plumosum plants were selected as the primary raw material. Fresh moss plants were subjected to a baking process at 80 °C for a duration of 6 h, resulting in the acquisition of dried moss. The dried moss was subsequently placed in a Muffle furnace (Tianjin Muffle Technology Co. Ltd., China) and heated under nitrogen flow at 500 °C for 1 h. Following this process, the cooled sample was ground to yield moss biochar, which was then utilized in subsequent experiments. The mor-phology of the prepared biochar was characterized through scanning electron microscopy (TESCAN MIRA LMS, Czech).”

 

4. In the discussion section author should incorporate more references to support their present findings. Authors also incorporated the saline tolerance mechanisms of other related plant species having corresponding data of the present study. It could provide good insights to draw comparative statement on tolerance mechanisms.
   Discussion lacks important references in the subject matter.

The section about Improvement of plant physiological and biochemical indicators needs more elaboration. POD and CAT activities were not discussed.

Response:

Thank you for your suggestion. We have fully revised the discussion section, and added references to provide a favorable basis for the experimental results:

“In this study, the moss-derived biochar significantly stimulated biofilm formation in the halotolerant bacterium Halomonas salifodinae strain 9106 by enhancing extracellular polymeric substance (EPS) biosynthesis. Biochemical quantification revealed a 4.9-fold increase in the EPS produced by the strain 9106 under biochar amendment, which consequently promoted EPS matrix accumulation and facilitated the assembly of biofilm structures. Therefore, this kind of biochar could be used for regulation of biofilm formation during microbial remediation.”

“Most microbes could not only live freely as dispersed cells, but also interact with each other to form biofilms. These microbial cells within biofilms reside in a self-produced matrix of EPS, which are predominantly composed of polysaccharides and endow the cells high tolerance to environmental stresses [32].”

“Our findings are consistent with the results of previous studies. For instance, the biochar facilitates the rhizosphere colonization of the functional bacteria, including Bacillus, Azospirillum, and Rhizobium [35. In saline-alkali soil, the biochar has an impact on the relative abundance of bacterial communities, modifies the bacterial community structure, and further stimulates the growth of plants [36].”

“4.2 The moss biochar improves plant physiological and biochemical indicators

Plant height is one of the most important morphological characteristics of plants, which reflects the growth rate and health status of the plants [37]. The results of this study indicated that the addition of the prepared biochar significantly increased the heights of the plants. Consequently, the combination of 9106 and biochar enhanced plant growth under the stress. Both biochar alone and 9106+biochar increased the content of chlorophyll a and soluble sugars in plant leaves, which indicated that the two treatments promoted the synthesis of chlorophyll a, and thus enhanced the photosynthetic efficiency of the plants for plant growth under the saline-alkali condition [38].

The plant root is the first organ suffering from saline-alkali stress in the soil. The proline and malondialdehyde contents, which reflect the stress degree [39,40]. After 30 days of culturing, the proline content in plant roots markedly increased, serving as an indicator of stress tolerance. The experimental results demonstrated that the proline concentration in the three experimental groups was significantly higher compared to the control group. The root malondialdehyde levels of the 9106 + biochar group was lower than that of the 9106 group and the biochar group. This confirmed that the biochar and 9106 in combination drastically alleviated oxidative stress caused by the saline-alkali stress. This is consistent with existing results of Nielsen’s research, who demonstrated that the Halomonas strain stimulates the growth of Alfalfa in salty soil [41]. ”

 

5. L343-344: The plants were cultured under room temperature and free sunlight irradiation for 30 days ……: The culture conditions are not enough clear, is it under controlled or field conditions? Why did the authors select 30 days of plant culture? What stage of plant development does this correspond to? For plant growth and care appropriate reference should be included.

Response:

Thank you for the valuable advice. The culture conditions are under controlled. We have implemented the following revisions to enhance the clarity and flow:

“The plants were cultured under room temperature and free sunlight irradiation for 30 days at the branching stage. During this process, to induce saline-alkali stress, irrigation water was substituted with saline-alkali solution once (10 mL, pH = 8.2) a week, and this regimen was continued until harvest. Followed by plant sampling (collecting the leaves, stems and roots of each plant respectively) for further analysis.”

 

6. Why antioxydant enzymes were measured in leaves while proline and MDA in roots?

Response:

Thank you for the valuable suggestion. The measurement of antioxidant enzymes in leaves versus proline and malondialdehyde (MDA) in roots is typically driven by the distinct roles and stress responses of these plant organs, as well as methodological considerations. (1) Antioxidant Enzymes in Leaves: Primary Site of ROS Production: Leaves are major sites of photosynthesis, where chloroplasts generate reactive oxygen species (ROS) under stress (e.g., high light, drought). Antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are critical for neutralizing ROS in these metabolically active tissues. Stress Focus: Stressors affecting leaves (e.g., UV radiation, temperature extremes) often directly impact photosynthetic machinery, making antioxidant activity a key defense mechanism measured here. (2) Proline and MDA in Roots: Osmotic Adjustment: Roots, exposed to soil-related stresses (e.g., salinity, drought, heavy metals), accumulate proline as an osmoprotectant to maintain cellular water balance and membrane integrity. MDA as a damage marker: roots are prone to lipid peroxidation due to abiotic stressors like waterlogging or nutrient toxicity. MDA, a byproduct of this process, serves as a reliable indicator of oxidative membrane damage in root tissues. (3). Tissue-Specific Responses: Leaves and roots often exhibit divergent stress adaptation strategies. While leaves prioritize enzymatic ROS scavenging, roots may rely more on osmotic adjustment (proline) and exhibit stress severity via MDA levels. (4). Biological Relevance: Proline in roots may reflect early stress responses to soil conditions, while leaf antioxidants address downstream effects of systemic stress. MDA in roots directly correlates with root health under soil-borne stressors. In summary, this partitioning aligns with the functional roles of each organ: leaves, as photosynthetic hubs, prioritize enzymatic ROS detoxification, while roots, facing direct soil stressors, emphasize osmotic balance and membrane integrity markers. The approach reflects both biological rationale and experimental practicality. The reasons were described in the Method:

“The antioxidant enzyme activity of the leaves, which reflects the capacity of the plants against oxidant stress, was assayed by using commercial kits. The bicinchoninic acid assays was employed to determine the protein content in the leaves. The root tissues of each plant were sampled from each treatment. The proline and malomdialdehvde (MDA) levels of the roots, which indicate the stress degree of the roots in the saline-alkaline soil, were determined by using the proline content detection kits and the MDA detection kits (Beijing Solarbio, China), respectively.”

 

7. L360-362: The activities of S-POD, S-AKP, S-UE and ….. this sentence was repeated in L377-381.

Response:

We are grateful for the suggestion. To address the issues mentioned above, we have refined, optimized, and adjusted the content accordingly. The duplicate content has been deleted:

“For soil enzyme analysis, root-associated soil fractions were collected from individual plants and stabilized through ambient drying protocols. Processed substrates were mechanically homogenized using ceramic mortars followed by gravimetric standardization. Enzymatic profiles were quantified respectively using commercial assay kits (Solarbio, Beijing) for soil peroxidase (S-POD), soil alkaline phosphatase (S-AKP), soil urease (S-UE), and soil sucrase (S-SC) activities through spectrophotometric determination at characteristic wavelengths.”

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The main question addressed by the authors in this study is the effect of moss biochar on root colonization by halotolerant bacteria, in this article. They also performed and discussed obtained results about plant-bacteria interactions that could benefit plant growth in stress conditions, especially on saline soils.

The antioxidant enzymes like SOD and Catalase related to coping with oxidative stress and cell adaptive mechanisms are also examined in the leaves of different plants exposed to saline stress conditions in the presence of biochar and halotolerant bacterium Halomonas salifodinae.

The main outcome is a new strategy for increasing the yield of forage grasses on saline-alkaline soils using biochar and exogenous halotolerant bacteria. The new knowledge will help address challenges such as remediation and continued use of saline soils for crop production, as well as better adaptation to climate change associated with rising temperatures and increasing area of saline soils.

The topic is original and relevant. Saline-alkali lands are widely distributed worldwide and their proportion is constantly growing because of climate changes. This article provides information how Halomonas salifodinae, an extremophile bacteria which can effectively remove nitrogen at 15% salinity, can colonised plant roots in presence of biochar and could promote plant growth. An interesting moment here is that the authors have studied a colonization capabilitie of a bacterial strain with different plant growth-promoting benefits such as nitrogen-fixing and salinity tolerance.

In my knowledge there is no another article describing complicated mechanisms of root colonisation of model plant (grass Medicago sativa) by Halomonas salifodinae in presence of different biochars.

There is another articles related to root colonisations in stress conditions, but the using model plants and bacteria are different.

Also in this study the authors have perform a set of biochemical analyses describing some adaptive mechanisms. Their study also reveals biofilm formation on the biochar surface. Experiments in greenhouses were also presented, in which the development of plants was analyzed. The biochemical analyses performed revealed a higher rate of photosynthesis, accompanied by the accumulation of chlorophyll, an increase in the size of plants, and the accumulation of carbohydrates and other substances.

I have no suggestions related to methodology. The experimental design is logical and well-structured. The methodology and scientific approaches used are relevant to the scientific tasks.

The conclusion is consistent with the evidence and arguments presented and do they address the main question posed.

The results are visualized with figures and then discussed.

The references are appropriate and major numbers of them are articles published during the last 5 years.

I highly recommended the publication of your article. 

I suggest to included a visual abstract, although this is only a recommendation, not a requirement.

The last sentence in the abstract (row33) and in the conclusion (row 404) should  be checked. They are similar and need some revision.

References 1, 2 and 4 should be corrected - there is a comma before the article title that should be removed.

In the discussion section, the authors must compare the results obtained with those published in other articles on using biochar to improve plant growth under stressful conditions or to enhance root system colonization. I also have a question: are the results of this study consistent with other publications, or are there some results that contradict previous studies? The authors also must provide some examples and add them to the discussion.

 

Author Response

Response to Reviewers 2

1. The last sentence in the abstract (row33) and in the conclusion (row 404) should be checked. They are similar and need some revision.

Response:

Thank you for your suggestion. We have revised the conclusion as follow, please see the lines 31-33 and 421-423: This study demonstrates a novel biochar-microbe integrated approach synergistically enhancing forage crop productivity in saline-alkaline soils through rhizosphere microbiome modulation and stress mitigation mechanisms:

“This study developed the biochar-halotolerant bacterium joint strategy to improve the yield of forage grasses in saline-alkali soil.”

“This study demonstrates a novel biochar-microbe integrated approach synergistically enhancing forage crop productivity in saline-alkaline soils through rhizosphere microbiome modulation and stress mitigation mechanisms.”

 

2. References 1, 2 and 4 should be corrected - there is a comma before the article title that should be removed.

Response:

Thank you for your valuable feedback. We are sorry for our carelessness. We have removed the comma to the manuscript, please see the lines 443-450:

“1. Negacz, K.; Malek, Ž.; de Vos, A.; Vellinga, P. Saline soils worldwide: Identifying the most promising areas for saline agriculture. J. Arid Environ. 2022, 203, 104775.

2. Chen, L.; Zhou, G.; Feng, B.; Wang, C.; Luo, Y.; Li, F.; Shen, C.; Ma, D.; Zhang, C.; Zhang, J. Saline-alkali land reclamation boosts topsoil carbon storage by preferentially accumulating plant-derived carbon. Sci. Bull. 2024, 69, 2948-2958.
3. Wang, G.; Gang, N.; Feng, G.; Burrill, H.; LI, J.; Zhang, J.; Zhang, F. Saline-alkali soil reclamation and utilization in China: progress and prospects. Front. Agric. Sci. Eng. 2024, 11, 216-228.”

 

3. In the discussion section, the authors must compare the results obtained with those published in other articles on using biochar to improve plant growth under stressful conditions or to enhance root system colonization. I also have a question: are the results of this study consistent with other publications, or are there some results that contradict previous studies? The authors also must provide some examples and add them to the discussion.

Response:

We thank the reviewer for pointing this out. We have added comparation and discussion in manuscript. For the functions of biochar, we compared with Zhuang’s research, which noted that biochar facilitates the colonization of the functional bacteria including Bacillus, Azospirillum, and Rhizobium; Jin’s research noted that biochar significantly impacted the abundance of bacterial communities and modified the bacterial community structure, which stimulates the growth of plants in saline-alkali soil. For Halomonas salifodinae, we compared with Nielsen’s research, who demonstrated that Halomonas stimulates growth of Alfalfa in salty soil. These results are the same as our findings. Please see the lines 330-334:

“Our findings are consistent with the results of previous studies. For instance, the biochar facilitates the rhizosphere colonization of the functional bacteria, including Bacillus, Azospirillum, and Rhizobium [35. In saline-alkali soil, the biochar has an impact on the relative abundance of bacterial communities, modifies the bacterial community structure, and further stimulates the growth of plants [36].”

Reviewer 3 Report

Comments and Suggestions for Authors

In the manuscript titled "Moss biochar facilitates root colonization of halotolerant Halomonas salifodinae for promoting plant growth under saline-alkali stress" the authors investigate the effect of moss biochar on the root colonization of the exogenous halotolerant Halomonas salifodinae isolated from saline lake sediments. Several observations are required in the current version of the manuscript for it to be reconsidered for publication.

Materials and Methods section: This section should be restructured to detail all aspects necessary to replicate the results of the present study. All information related to Halomonas must be specified, including the isolation method, the methodology followed for species-level identification, and any molecular biology analyses performed, these should be described in detail, including phylogenetic and morphological aspects. Finally, this section should be placed immediately after the introduction for better comprehension of the manuscript.

L327-328: What methods were used to detect biofilm formation and polysaccharide production? Even if the authors reference the methodology from another article, the specific methods performed and the justification for their use should be mentioned, without going into step-by-step procedural details.

L359-362: The full names of the enzymatic activities should be written out the first time they are mentioned.

Results section: At the beginning (L86-89), it seems that the results and discussion sections have been merged; the citation included caused confusion. The authors should carefully review the journal's author guidelines, as citations should not appear in the results section, that is the purpose of the discussion section.

L89-92: The writing here seems to describe the methodology used rather than presenting results. The authors need to pay close attention to these details, as they are unacceptable in a submission to an international, indexed journal. They should review how a results section should be structured and focus on clearly describing the observed phenomena.

L99-100: Again, the authors are speculating on the results. This type of writing does not belong in the results section but should be part of the discussion, supported by scientific references.

L110-112: The same issue persists, the authors need to completely rewrite this section and clearly distinguish between results, methodology, and discussion, placing each element in its appropriate section. Similar problems are observed throughout the section, for example, in lines 136-137, 143-144, and so on.

Discussion section: This entire section should be reformulated after properly organizing the results section. Additionally, speculation should be strictly avoided here; the authors may propose hypotheses about what could be happening in the evaluated microenvironment. This issue appears in lines 234-250. Moreover, an "in summary" statement should not be placed at the beginning of the discussion, the summary should be presented at the end.

L255-268: There are no citations supporting the authors’ claims, and most of the content is merely an explanation of the results rather than a proper scientific discussion.

 

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Response to Reviewers 3

1. Materials and Methods section: This section should be restructured to detail all aspects necessary to replicate the results of the present study. All information related to Halomonas must be specified, including the isolation method, the methodology followed for species-level identification, and any molecular biology analyses performed, these should be described in detail, including phylogenetic and morphological aspects. Finally, this section should be placed immediately after the introduction for better comprehension of the manuscript.

Response:

We thank the reviewer for this insightful comment. We have placed the materials and methods section immediately after the introduction. Moreover, the methods of the Halomonas salifodinae 9106 isolation and identification were supplied in the Method. Please see lines:104-108:

“The halotolerant bacterium 9106 was isolated from the Saline Lake in Yuncheng, Shanxi Province, China, by using the solid Dmn medium. The compositions of this medium included: Dnm medium: glucose 0.5% (W/V), peptone 0.25%, KH₂PO₄ 0.025%, K₂HPO₄·3H₂O 0.1%, MgSO₄ 0.01%, NaCl 12%, agar 1.5%, pH = 7.7. By 16S rDNA sequencing and BLASN analysis in the NCBI system, the strain was identified as the species Halomonas salifodinae.”

 

2. L327-328: What methods were used to detect biofilm formation and polysaccharide production? Even if the authors reference the methodology from another article, the specific methods performed and the justification for their use should be mentioned, without going into step-by-step procedural details.

Response:

We have supplied the details of biofilm formation assays and polysaccharide production assays in the Method section:

“To detect biofilm formation of 9106, the bacterium was cultured in 96-well polystyrene microplates containing the liquid Dnm medium. After 24 h of incubation at 30 °C, the wells were gently washed twice with stilled water, and stained by 0.1% crystal violet (Dingguo, China) for 5 min. The dye adsorbed by the formed biofilms was extracted by 10% acetic acid. The absorbance of the dye extracts at 595 nm was measured by a UV-Vis spectrometer (Bio-Rad, USA). The polysaccharides produced by the bacterium was quantified by the phenol sulphuric acid method.”

 

3. L359-362: The full names of the enzymatic activities should be written out the first time they are mentioned.

Response:

Thanks for your careful checks. We have added full names of the enzymatic activities, please see lines168-171:

“Enzymatic profiles were quantified respectively using commercial assay kits (Solarbio, Beijing) for soil peroxidase (S-POD), soil alkaline phosphatase (S-AKP), soil urease (S-UE), and soil sucrase (S-SC) activities through spectrophotometric determination at characteristic wavelengths.”

 

4. Results section: At the beginning (L86-89), it seems that the results and discussion sections have been merged; the citation included caused confusion. The authors should carefully review the journal's author guidelines, as citations should not appear in the results section, that is the purpose of the discussion section.

Response:

We appreciate the reviewer’s insightful suggestion. We have placed this part of the discussing contents in the discussion section, please see lines: 317-329:

“In this study, the moss-derived biochar significantly stimulated biofilm formation in the halotolerant bacterium Halomonas salifodinae strain 9106 by enhancing extracellular polymeric substance (EPS) biosynthesis. Biochemical quantification revealed a 4.9-fold increase in the EPS produced by the strain 9106 under biochar amendment, which consequently promoted EPS matrix accumulation and facilitated the assembly of biofilm structures. Therefore, this kind of biochar could be used for regulation of biofilm formation during microbial remediation.

The rhizosphere microbiome, comprising the microorganisms residing within the soil surrounding plant roots, plays a pivotal role in maintaining plant health and nutrient availability. Most microbes could not only live freely as dispersed cells, but also interact with each other to form biofilms. These microbial cells within biofilms reside in a self-produced matrix of EPS, which are predominantly composed of polysaccharides and endow the cells high tolerance to environmental stresses [32]. Salt stress has been observed to reduce the rate of microbial attachment to the plant rhizosphere, thereby impeding plant growth [33].”

 

5. L89-92: The writing here seems to describe the methodology used rather than presenting results. The authors need to pay close attention to these details, as they are unacceptable in a submission to an international, indexed journal. They should review how a results section should be structured and focus on clearly describing the observed phenomena.

Response:

Thank you for the valuable advice. We have modified the description of this sentence to the following content. please see lines: 202-204:

“The biochars were fabricated via pyrolysis of the moss Brachythecium plumosum at 500 °C. Scanning electron microscopy (SEM) revealed the irregular sheet-morphology of the biochars, with moss-derived particles distributing on the surfaces (Figure 1a).”

 

6. L99-100: Again, the authors are speculating on the results. This type of writing does not belong in the results section but should be part of the discussion, supported by scientific references.

Response:

We agree with the reviewer. We have changed this part of the content and placed it in the discussion section. please see lines: 319-324:

“In this study, the moss-derived biochar significantly stimulated biofilm formation in the halotolerant bacterium Halomonas salifodinae strain 9106 by enhancing extracellular polymeric substance (EPS) biosynthesis. Biochemical quantification revealed a 4.9-fold increase in the EPS produced by the strain 9106 under biochar amendment, which consequently promoted EPS matrix accumulation and facilitated the assembly of biofilm structures. Therefore, this kind of biochar could be used for regulation of biofilm formation during microbial remediation.”

 

7. L110-112: The same issue persists, the authors need to completely rewrite this section and clearly distinguish between results, methodology, and discussion, placing each element in its appropriate section. Similar problems are observed throughout the section, for example, in lines 136-137, 143-144, and so on.

Response:

We appreciate the reviewer’s suggestion. We have re-written this section. Placed each element in its appropriate section. please see lines 241-244 and 258-261:

“The plant heights of Medicago sativa were measured after 30 days of culturing in saline-alkali soil (Figure 3a-b). The results demonstrated that the 9106 and biochar in combination led to a remarkable increase in the heights of Medicago sativa plants from 10.3 cm to 36.0 cm after 30 days of the saline-alkali stress, while 9106 or biochar alone only partially increased the heights.”

“As compared to the CK group, the SOD activity of 9106+biochar group significantly increased from 150.0 U/g leaves to 324.3 U/g leaves (Figure 3f). On the contrary, the CAT and POD activities of 9106+biochar group decreased from 1361 U/g leaves to 736.8 U/g leaves, 79557 U/g leaves to 64530 U/g leaves, respectively (Figure 3g-h).”

 

8. Discussion section: This entire section should be reformulated after properly organizing the results section. Additionally, speculation should be strictly avoided here; the authors may propose hypotheses about what could be happening in the evaluated microenvironment. This issue appears in lines 234-250. Moreover, an "in summary" statement should not be placed at the beginning of the discussion, the summary should be presented at the end.

Response:

We thank the reviewer for this insightful comment. We have reformulated the results and discussion sections after properly organizing them. Please see the Result and Discussion sections as described above.

 

 

9. L255-268: There are no citations supporting the authors’ claims, and most of the content is merely an explanation of the results rather than a proper scientific discussion.

Response:

We thank the reviewer for this insightful comment. We have reformulated the discussion section. Please see the lines 348-364:

“4.2 The moss biochar improves plant physiological and biochemical indicators

Plant height is one of the most important morphological characteristics of plants, which reflects the growth rate and health status of the plants [37]. The results of this study indicated that the addition of the prepared biochar significantly increased the heights of the plants. Consequently, the combination of 9106 and biochar enhanced plant growth under the stress. Both biochar alone and 9106+biochar increased the content of chlorophyll a and soluble sugars in plant leaves, which indicated that the two treatments promoted the synthesis of chlorophyll a, and thus enhanced the photosynthetic efficiency of the plants for plant growth under the saline-alkali condition [38].

The plant root is the first organ suffering from saline-alkali stress in the soil. The proline and malondialdehyde contents, which reflect the stress degree [39,40]. After 30 days of culturing, the proline content in plant roots markedly increased, serving as an indicator of stress tolerance. The experimental results demonstrated that the proline concentration in the three experimental groups was significantly higher compared to the control group. The root malondialdehyde levels of the 9106 + biochar group was lower than that of the 9106 group and the biochar group. This confirmed that the biochar and 9106 in combination drastically alleviated oxidative stress caused by the saline-alkali stress. This is consistent with existing results of Nielsen’s research, who demonstrated that the Halomonas strain stimulates the growth of Alfalfa in salty soil [41].”

 

10. Comments on the Quality of English Language. The English could be improved to more clearly express the research.

Response:

We thank the reviewer for this insightful comment. We tried our best to improve the Quality of English Language.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments for Authors

In this manuscript, the authors conducted a useful study with the aim of   provide valuable insights on the effect of moss biochar  on the root colonization of the exogenous halotolerant Halomonas salifodinae isolated from  saline lake sediments.

The authors found  that after 30 days of culturing, the biochar and the bacterium in  combination increased the plant height of 250 % over control.

The morphometric increase among different plant groups reflects an improvement in biochemical and physiological parameters such as increased chlorophyll a levels, reducing sugars, soluble proteins, and superoxide dismutase in  the leaves etc. that provide the plant with water and salt stress defense capabilities.

The experimentation was well performed and the methods used are in accordance with analytical criteria.

The results exhibited and commented by the authors allow highlighting the ability of moss biochar in facilitating root colonization of halotolerant Halomonas salifodinae microbial inoculants, enhancing nutrient assimilation, and in bringing significant changes in metabolic and transcriptomic profiles, particularly by increasing key secondary metabolites that enable Medicago sativa to thrive under adverse conditions by activating stress adaptation pathways.

The work is original and can be accepted if the authors respond to the following reviewer's comments:

• In the line 122 Biochar concentration is correctly expressed in mg/Kg soil. Why does the term soil disappear at line 343? Also the content of the various cations in the soil should be referred to Kg soil.
• I recommend that the authors also indicate the size of the moss biochar used.
• I ask the authors to indicate up to what limit of ionic strength of the circulating soil solution, the biochar still retains adsorptive capacity with the root.
• I ask the authors whether biofilm formation also invades root hairs.
• I ask the authors whether biofilm formation can hinder the colonization of nitrogen-fixing bacteria.
• I ask the authors if they have calculated the colonization rate of nitrogen-fixing bacteria after moss biochar treatment. As the authors well know, colonization of roots by nitrogen-fixing bacteria (mainly Rhizobium) greatly alleviates salt stress and they are able to tolerate NaCl concentrations between 100 and 150 mM.
• Since the authors point out a polysaccharide secretion of roots, which benefits microbial colonization, which in turn defends against salt stress and improves the assimilative capacity of micro and macronutrients, if they believe it is possible to consider this condition as a condition of mutualistic exosymbiosis.
• I ask the authors what irrigation system they used when the plants were kept in pots for 30 days and also later when they were transferred to the open field. Did they use natural or brackish water?
• I ask the authors if at the end of the production cycle have they tried to calculate the yield increase of treated plants compared to control plants?

 

 

 

 

Author Response

Response to Reviewer 4

1. In the line 122 Biochar concentration is correctly expressed in mg/Kg soil. Why does the term soil disappear at line 343? Also the content of the various cations in the soil should be referred to Kg soil.

Response:

Thanks for your careful checks. We have changed the symbols in the text to keep it consistent throughout.

 

2. I recommend that the authors also indicate the size of the moss biochar used.

Response:

Thank you for your suggestion. In this study, biochars were fabricated via pyrolysis of the moss Brachythecium plumosum at 500 °C. Morphological characterization using scanning electron microscopy (SEM) revealed irregularly structured moss-derived biochar particles with a diameter of range10–60 μm. We have added the description in the Result:

“The biochars were fabricated via pyrolysis of the moss Brachythecium plumosum at 500 °C. Scanning electron microscopy (SEM) revealed the irregular sheet-morphology of the biochars with a diameter of 10~60 μm, and with moss-derived particles distributing on the surfaces. (Figure 1a).”

 

3. I ask the authors to indicate up to what limit of ionic strength of the circulating soil solution, the biochar still retains adsorptive capacity with the root.

Response:

We are grateful for the suggestion. The limitation of ionic strength and the effect of biochar on salinity were supplied in the introduction:

“Meanwhile, high ionic strengths contribute to soil salinity, the biochar with the capacity of ion adsorption may attenuate the saline stress to plant roots. Therefore, it is hypothesized that specific kinds of biochar could be developed as the regulator of root bacterial dynamic, soil salinity and plant growth when the plants suffer from soil saline-alkaline stress.”

 

4. I ask the authors whether biofilm formation also invades root hairs.

Response:

Thank you for the valuable suggestions. We performed scanning electron microscopy (SEM) to observe the distribution of biofilms around the roots. The SEM images in Figure 2a showed that the biofilms formed around the roots in the group of the 9106+biochar. The result was described in the Results:

“In contrast, the 9106+biochar group had the roots with the bacterial biofilm densely surrounding the roots, suggesting the biochar facilitated the bacterial biofilm formation on the root surface (Figure 2a).”

 

5. I ask the authors whether biofilm formation can hinder the colonization of nitrogen-fixing bacteria.

Response:

Thank you very much for the important point raised by the expert. We have conducted colony forming unit (CFU) assays. Nitrogen-fixing bacteria (NFB) increased from 10^4.7 CFU/g soil to 10^6.3 CFU/g soil. Biofilm formation did not hinder the colonization of nitrogen-fixing bacteria. The result was discussed in the text:

“Consequently, the moss biochar promoted colonization of 9106 on the plant roots and also enhanced the colonization of NFBs, which was not impaired by biofilm formation.”

 

6. I ask the authors if they have calculated the colonization rate of nitrogen-fixing bacteria after moss biochar treatment. As the authors well know, colonization of roots by nitrogen-fixing bacteria (mainly Rhizobium) greatly alleviates salt stress and they are able to tolerate NaCl concentrations between 100 and 150 mM.

Response:

Thank you for your valuable feedback. We have conducted colony forming unit (CFU) assays.

The result revealed that the biochar+9106 group exhibited a significantly higher bacterial numbers on the roots compared to other three groups, the numbers of total bacteria, halotolerant bacteria (STB), and nitrogen-fixing bacteria (NFB) increased from 10^5.5 CFU/g soil to 10^7.2 CFU/g soil, from 10^4.5 CFU/g soil to 10^6.1 CFU/g soil and from 10^4.7 CFU/g soil to 10^6.3 CFU/g soil, respectively. The moss biochar promoted colonization of 9106 on the plant roots and also enhanced the colonization of NFBs. Furthermore, as you kindly pointed out, the root colonization of NFBs may alleviates salt stress and promotes the plants to tolerate NaCl. We added the discussion in the text:

“Moreover, CFU assays revealed that the biochar increased root colonization of NFBs. The root colonization of NFBs may further alleviate salt stress and promote the plants to tolerate high-level NaCl.”

 

 

7. Since the authors point out a polysaccharide secretion of roots, which benefits microbial colonization, which in turn defends against salt stress and improves the assimilative capacity of micro and macronutrients, if they believe it is possible to consider this condition as a condition of mutualistic exosymbiosis.

Response:

We are grateful for the suggestion. We found that the secretion of polysaccharides and the formation of biofilm was by Halomonas salifodinae 9106 not by roots, biochar is attached to roots, it can promote the secretion of polysaccharides by the strain 9106, which results in the colonization of 9106 on the root surface. The interaction between the root surface-colonized bacterium and the plants indicates the mutualistic exosymbiosis. We added the discussion in the text:

“The interaction between the root surface-colonized bacterium and the plants indicates the mutualistic exosymbiosis.”

 

8. I ask the authors what irrigation system they used when the plants were kept in pots for 30 days and also later when they were transferred to the open field. Did they use natural or brackish water?

Response:

Thank you for your valuable feedback. During the plants were kept in pots for 30 days, saline-alkali solution (pH = 8.2) was used for irrigation a week, and this regimen was continued until harvest. The details were supplied in the Method:

“During this process, to induce saline-alkali stress, irrigation water was substituted with saline-alkali solution (10 mL/week, pH = 8.2, NaCl 1%, NaHCO₃ 0.1%, W/V) a week, and this regimen was continued until harvest. Followed by plant sampling (collecting the leaves, stems and roots of each plant respectively) for further analysis.”

 

9. I ask the authors if at the end of the production cycle have they tried to calculate the yield increase of treated plants compared to control plants?

Response: Thank you for the valuable advice. In this study, the plant weights of Medicago sativa were measured in saline-alkali soil after 30 days of culturing. The results demonstrated that the addition of the prepared biochar and 9106 significantly increased the weights of the plants (Figure S1). For instance, after 30 days of the saline-alkali stress, the 9106 and biochar in combination led to a remarkable increase in the weights of Medicago sativa plants from 0.26 g to 1.46 g, which was 4.6-fold higher than the control. In contrast, 9106 and biochar alone only increased the weights to 1.02 g and 0.65 g, respectively. These results indicated that the combination of 9106 and biochar enhanced plant growth under the stress.

The result was supplied in the Supporting Information and described in the text:

“Plant weight quantification further demonstrated that the addition of the prepared biochar and 9106 significantly increased the weights of the plants (Figure S1). For instance, after 30 days of the saline-alkali stress, the 9106 and biochar in combination led to a remarkable increase in the weights of Medicago sativa plants from 0.26 g to 1.46 g, which was 4.6-fold higher than the control. In contrast, 9106 and biochar alone only increased the weights to 1.02 g and 0.65 g, respectively.”

Figure S1. The plant weights of Medicago sativa after 30 days of cultivation in the saline-alkaline soil. The different letters indicate significant difference between the groups (P<0.05).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully revised the manuscript according to the comments, and the current version have been significantly improved. Therefore, I believe the manuscript is now ready for publication in the journal.

Author Response

The authors have carefully revised the manuscript according to the comments, and the current version have been significantly improved. Therefore, I believe the manuscript is now ready for publication in the journal.

Response: Thank you very much for your positive comments and great help to our manuscript.