Physiological and Biochemical Behaviors of Date Palm Vitroplants Treated with Microbial Consortia and Compost in Response to Salt Stress

Round 1

Reviewer 1 Report

This publication presents interesting results of the physiological and biochemical behaviors of date palm vitroplants when they are subjected to salt stress through the use of a conjunction of biofertilizers or alone. The proposal presented by treating the plants with microbial consortia and compost showed a promising way to overcome the big negative effect that salinity cause in plants.

Many studies deal with similar topic, the originality relies in the use of the different biofertilizers and the analysis of the best group.

Overall, the manuscript is well written, presented and discussed. With too many results authors presented their findings in an easy and understandable manner. I recommend its publication after minor revision;

L32_Please define growth parameters in the beginning, such as SDW. The rest are also missing in methods.

L241_Please include % for PGPR+AMF1 and PGPR+AMF2

263_Include the % for bipartite and tripartite

L322 and L423_ please verify, enzyme activities increase or deacrease?

Author Response

Dear Editor,

Thank you for your email, please find bellow revision of the paper entitled "Physiological and biochemical behaviors of date palm vitroplants treated microbial consortia and compost supplemented to salt stress".

We really appreciated your time and efforts to evaluate our manuscript and we thank the reviewers for their interesting comments, which have been immensely helpful.

We have included the reviewer comments immediately after this letter and respondedto them individually, indicating exactly how we addressed each concern or problemand describing the changes we have made. The revisions have been approved by all13 authors. The changes are indicated using "track changes".

Sincerely,

Pr. Abdelilah Meddich

Response to Reviewer 1 Comments

Comments and Suggestions for Authors

This publication presents interesting results of the physiological and biochemical behaviors of date palm vitroplants when they are subjected to salt stress through the use of a conjunction of biofertilizers or alone. The proposal presented by treating the plants with microbial consortia and compost showed a promising way to overcome the big negative effect that salinity cause in plants.

Response 1: Thank you for your comment.

Many studies deal with similar topic, the originality relies in the use of the different biofertilizers and the analysis of the best group.

Overall, the manuscript is well written, presented and discussed. With too many results authors presented their findings in an easy and understandable manner. I recommend its publication after minor revision;

Response 2: Thank you for the comment.

L32_Please define growth parameters in the beginning, such as SDW. The rest are also missing in methods.

Response 3: Done as requested (Page 1, Line 38). For the rest of the manuscript SDW and RDW were cited for the first time in line 292. Abbreviations SDW was defined in page 8 line 295 and in line 306 for RDW. After that we use the abbreviation.

L241_Please include % for PGPR+AMF1 and PGPR+AMF2

Response 4: The percentages for PGPR+AMF₁ and PGPR+AMF₂ were added as requested (Page 7, Lines 277 and 278).

263_Include the % for bipartite and tripartite

Response 5: The percentages for bipartite and tripartite were added as requested (Page 8, Lines 312 and 313).

L322 and L423_ please verify, enzyme activities increase or decrease?

Response 6: Salt stress increased significantly enzyme activities. The statement was corrected (Page 13, Line 519).

Reviewer 2 Report

The manuscript applsci-989853 from Toubali et al., collects many growth factors of physiological, physicochemical and phenotypical changes of Morocco’s date palm (P. dactylifera) in response to salinity stress and biofertilizer treatment. Authors found that tripartite combination of biofertilizers could significantly alleviate the negative growth caused by salinity stress. The clear background, informative methods, properly presented results and open discussion of the current version provides a fundamental understanding of how biofertilizer enhances plant performance. I believe this manuscript could meet the standard of Applied Sciences, if authors could address some concerns as listed below.

Major concerns:

1) Authors provided organised results in graphs and tables. Could authors show representative plant photos with shoot and root structures in 36 treatments?

2) Authors clearly know the reasons of why each parameters were chosen. In the result parts, authors may briefly explain the reasons behind the experimental design. For example, in result 3.2, why SDW and RDW are critical growth parameters?

Minor concerns:

3) Line 32 (L32), what is SDW? Shoot dry weight?

4) L59-61, citations of previous studies.

5) L69, reference 30 is for IAA not GA.

6) L77, authors would like to briefly talk about the principle of the combination method. And would it become a popular way for many other crops. Are there limitations in application?

7) L144, cite Table 2.

8) L143, is one pot one plant? How many plants were considered as one replication?

9) L147, necessary information of F and I calculation is required.

10) L166, what are Fv and Fm?

11) L180, Specify the fresh tissue.

12) L218, for the statistical analysis in Fig 1 to 4, authors would like to provide the test values.

13) Could subtitles in result include conclusive information?

14) In result 3.1, salinity and comp are two major negative factors on F and I. Could authors pinpoint those conclusive messages?

15) In the legend of Fig 2 to 4, n=3. According to the method (L143), authors prepared 8 replications. How 3 out 8 was chosen? Do I misunderstand it?

Author Response

Dear Editor,

Thank you for your email, please find bellow revision of the paper entitled "Physiological and biochemical behaviors of date palm vitroplants treated microbial consortia and compost supplemented to salt stress".

We really appreciated your time and efforts to evaluate our manuscript and we thank the reviewers for their interesting comments, which have been immensely helpful.

We have included the reviewer comments immediately after this letter and respondedto them individually, indicating exactly how we addressed each concern or problemand describing the changes we have made. The revisions have been approved by all13 authors. The changes are indicated using "track changes".

Sincerely,

Pr. Abdelilah Meddich

Response to Reviewer 2 Comments

Comments and Suggestions for Authors

The manuscript applsci-989853 from Toubali et al., collects many growth factors of physiological, physicochemical and phenotypical changes of Morocco’s date palm (P. dactylifera) in response to salinity stress and biofertilizer treatment. Authors found that tripartite combination of biofertilizers could significantly alleviate the negative growth caused by salinity stress. The clear background, informative methods, properly presented results and open discussion of the current version provides a fundamental understanding of how biofertilizer enhances plant performance. I believe this manuscript could meet the standard of Applied Sciences, if authors could address some concerns as listed below.

Response: Thank you for your comment.

Major concerns:

1) Authors provided organised results in graphs and tables. Could authors show representative plant photos with shoot and root structures in 36 treatments?

Response 1: Thank you for your comment. You will find some photos as attached file, on the other hand, we will not have photos showing the roots or all the tests.

2) Authors clearly know the reasons of why each parameter were chosen. In the result parts, authors may briefly explain the reasons behind the experimental design. For example, in result 3.2, why SDW and RDW are critical growth parameters?

Response 2: The accumulation of biomass (shoot dry weight and root dry weight) are critical growth parameters since the impact of salt stress on plant growth and development is a consequence of the effects of this constraint on physiological, biochemical and nutritional aspects. This explanation was added in the main text (Page 8, Lines 292-295).

Minor concerns:

3) Line 32 (L32), what is SDW? Shoot dry weight?

Response 3: Yes, of course. The full name of the abbreviation was added (Page 1, Line 38) and other clarifications of this parameter are added in the manuscript.

4) L59-61, citations of previous studies.

Response 4: The citations of previous studies were added (Page 2, Line 71-73).

5) L69, reference 30 is for IAA not GA.

Response 5: The statement was corrected (Page 3, Lines 82 and 83).

6) L77, authors would like to briefly talk about the principle of the combination method. And would it become a popular way for many other crops. Are there limitations in application?

Response 6: In this study, we investigated the effects of AMF and/or PGPR combined with compost on date palm vitroplants. Yes, it will become a popular way for many other crops since the individual and the combined application of these biofertilizers showed spectacular results on many crops. The used biofertilizers are known to improve the crops tolerance to various abiotic and biotic constraints with the greatest improvement in the presence of the combination of these biofertilizers (Ortiz et al., 2015; Hidri et al., 2016; Meddich et al., 2018; Anli et al., 2020; Ben-Laouane et al., 2020). In principle, the biofertilizers used compost-AMF-PGPR interact positively in synergy to strengthen their common actions qualified as biofertilizers, bioprotectors, bioregulators and biostabilisers.

In our knowledge, there are some limitations in their application:

i) Microbial biotechnologies are little used in the open field. One of the reasons for this lack of interest from farmers is the poor reproducibility of the effect of the inoculant on the plant (Antunes et al., 2011; Duponnois et al., 2013). This finding results mainly from the lack of compatibility of the inoculant with the edaphic characteristics of the planting soil which results in the disappearance of the introduced strain and its potential impact on the plant. It is therefore necessary to select native strains (CMA and PGPR) adapted to the soil conditions and to the constraints of the environment. (mineral deficiencies, water and salt stress) encountered in the regions where these plantings will be carried out. Thus, it is important, as in the case of the present work, to select suitable mycorrhizal and bacterial consortia with a high infectivity power from the rhizosphere of date palms.

ii) The better functioning of the selected biostimulants applied in a three-part combination is dependent on the soil quality. It is important to ensure that the culture medium is poor in mineral elements, mainly P for AMF and N for PGPR. Thus, for the choice of low doses of compost applied, it is advantageous to maintain a soil poverty in mineral elements and organic matter which may be mineralized.

iii) Limitation in terms of large-scale application given the necessary equipment (greenhouses, refrigerators, etc.) to be installed to produce large quantities of mycorrhizal and bacterial inocula to treat large areas (thousands of hectares).

References

Anli, M., Baslam, M., Tahiri, A.-I., Raklami, A., Symanczik, S., Boutasknit, A., et al. (2020). Biofertilizers as Strategies to Improve Photosynthetic Apparatus, Growth, and Drought Stress Tolerance in the Date Palm. Front. Plant Sci. 11, 1–21. doi:10.3389/fpls.2020.516818.

Antunes PM, Koch AM, Morton JB, Rillig MC and Klironomos JN. 2011. Evidence for functional divergence in arbuscular mycorrhizal fungi from contrasting climatic origins. New Phytologist. 189 : 507-514.

Ben-Laouane, R., Baslam, M., Ait-El-Mokhtar, M., Anli, M., Boutasknit, A., Ait-Rahou, Y., et al. (2020). Potential of Native Arbuscular Mycorrhizal Fungi, Rhizobia, and/or Green Compost as Alfalfa (Medicago sativa) Enhancers under Salinity. Microorganisms 8, 1695. doi:10.3390/microorganisms8111695.

Duponnois R, Hafidi M, N’Doye I, Ramanankierana H and Bà A.M. 2013. Des champignons symbiotiques contre la desertification. Ecosystèmes méditerranéens, tropicaux et insulaires. IRD Editions, ISBN : 978-2-7099-1827-5. 511p.

Hidri, R., Barea, J. M., Mahmoud, O. M.-B., Abdelly, C., and Azcón, R. (2016). Impact of microbial inoculation on biomass accumulation by Sulla carnosa provenances, and in regulating nutrition, physiological and antioxidant activities of this species under non-saline and saline conditions. J. Plant Physiol. 201, 28–41. doi:10.1016/j.jplph.2016.06.013.

Meddich, A., Ait El Mokhtar, M., Bourzik, W., Mitsui, T., Baslam, M., and Hafi, M. (2018). “Optimizing growth and tolerance of date palm (Phoenix dactylifera L.) to drought, salinity, and vascular fusarium-induced wilt (Fusarium oxysporum) by application of arbuscular mycorrhizal fungi (AMF),” in Root Biology, eds. B. Giri, R. Prasad, and A. Varma, 239–258. doi:10.1007/978-3-319-75910-4.

Ortiz, N., Armada, E., Duque, E., Roldán, A., and Azcón, R. (2015). Contribution of arbuscular mycorrhizal fungi and/or bacteria to enhancing plant drought tolerance under natural soil conditions: Effectiveness of autochthonous or allochthonous strains. J. Plant Physiol. 174, 87–96. doi:10.1016/j.jplph.2014.08.019.

7) L144, cite Table 2.

Response 7: Table 2 reported the effect of the applied biofertilizers on leaves numbers and plant height. However, in line 144 we give explanations on the number of treatments and repetitions per treatment.

8) L143, is one pot one plant? How many plants were considered as one replication?

Response 8: One plant per pot is considered as a replicate. The statement was edited to be more clear (Page 4, Lines 164 and 165). Please see also L126 and L127 (page 4).

9) L147, necessary information of F and I calculation is required.

Response 9: The formula of F and I calculation was provided (Page 5, Lines 175-177).

10) L166, what are Fv and Fm?

Response 10: The signification of Fv and Fm were provided in the main text (Page 5, Lines 195-196).

11) L180, Specify the fresh tissue.

Response 11: Specification added. We used leaves to determine the photosynthetic pigment content (see Page 6, Line 206).

12) L218, for the statistical analysis in Fig 1 to 4, authors would like to provide the test values.

Response 12: We carried out a one way ANOVA using the Student-Newman-Keuls test at 5% threshold level. The P and F values were added in the text (L263, L275, L286, L296, L307, L320, L333, L378, L393, L396, L398-399, L404, L409).

13) Could subtitles in result include conclusive information?

Response 13: The results subtitles were edited to be conclusive as requested.

14) In result 3.1, salinity and comp are two major negative factors on F and I. Could authors pinpoint those conclusive messages?

Response 14: A statement was added in themain text to pinpoint those conclusive messages (Page 8, Lines 283-284).

15) In the legend of Fig 2 to 4, n=3. According to the method (L143), authors prepared 8 replications. How 3 out 8 was chosen? Do I misunderstand it?

Response 15: The three analyzed replications were randomly chosen.