Halotolerant-Koccuria rhizophila (14asp)-Induced Amendment of Salt Stress in Pea Plants by Limiting Na⁺ Uptake and Elevating Production of Antioxidants

Round 1

Reviewer 1 Report

The idea of the experiment described in the article seems to me interesting and actual. But the article itself requires serious revision. I have the following comments to the article.

1. In the introduction, please state the scientific novelty of your work among other works devoted to the study of the effects of bacteria on plant resistance to drought.
2. In the introduction, you do not need to write twice about the importance of peas in people's nutrition (lines 49 and 106). Once is enough.
3. In Figure 1, the differences between the experience variants are most likely not statistically significant. When describing the presence of differences, it is better to write less categorically as an assumption.
4. Please check again in the statistical program how the letters are arranged in Table 2. I believe that you made several mistakes when filling it out. For example, (15.83±0.16 a) and (15.67±0.66 b) cannot differ significantly. Also (11.67±0.66 e) and (11±0.57 f). And a few more numbers. If you find errors, revise the description of the results taking into account the corrections.
5. Line 263. The description of the results does not correspond to Fig.2. «The proline content in inoculated T3 plants was not enhanced in both varieties as compared to control». The pea variety 9800-10 increases the proline content relative to the control. «However, there was a net increase in 9800-10 inoculated plants at 150mM NaCl compared to un-inoculated 150mM treated T2 plants». According to the placed letters, T2 and T5 do not differ. Although I'm not sure that the letters are placed correctly.
6. Line 249. The description of the results does not correspond to Fig.3. «The leaf RWC is decreased in salt-treated plants». In some cases, it increases or remains at the same level. The further stated degree of increase in RWC under the action of bacteria does not correspond to the columns in the figure.
7. Line 240. The description of the results does not correspond to Fig.3. «Leaf chlorophyll contents i-e chlorophyll a, chlorophyll b, and carotenoids contents decreased in salt-treated plants». Only chlorophyll b decreases. "However, inoculation of K. rhizophilla 14asp enhanced the chlorophyll content in both varieties". Only in a few cases. Further statements in this paragraph are also not supported by graphs.
8. The results of statistical data analysis make up a significant part of the presented results (two figures out of five). Therefore, more attention should be paid to their discussion in the article. How the components were formed in the PCA. What do the three equally named points in Fig.4(a) and 4(c) mean? What initial data was used to calculate correlations. They were obtained by combining data on the variants of the experiment 75 mM and 150 mM? What value of the correlation coefficient indicated a strong correlation and why? Between which parameters the strongest correlation was obtained, how do you explain it?
9. The reaction of pea varieties to treatment with bacteria is different. This is evidenced by most of the tables and figures. This obvious difference is often not mentioned in the description of the results. In the discussion of the results, the reason for the differences between the varieties is not explained.
10. The article is carelessly framed. Please correct the numbering of the sections. Write the full generic name of the bacterium in the name, abstract and at its first mention in the text. Decipher the abbreviations when they are first mentioned. To indicate the significance of statistical differences throughout the text, use only lowercase letters or only uppercase letters, and not a mixture of them. The name of the section "Leaf Electrolyte content" apparently meant "Leaf chlorophyll content". In line 127, write the unit of measurement "0 to 15".

Author Response

Please see the attached file

Author Response File: Author Response.pdf

Reviewer 2 Report

In the manuscript entitled "Halotolerant; K.rhizophila (14asp)-induced amendment of salt stress in pea plants by limiting Na+ uptake and elevated production of antioxidants" the authors investigated the effect of K.rhizophila on the physiology parameters of pea plants in salt stress conditions. The research work is well-planed and performed. In general, the manuscript is written in good manner and order. However, it needs to be improved a lot before publication.

Further questions and comments:

•  There are many typos and grammatical mistakes in the text, so English proofreading is necessary (just some example: Line #21: Halotolerant plant, Line #38: affects, Line #61: leads, Line #118: were->was, Line #119: greenhouse, Line #124: Bacteria salt, Line #127: LB, Line #126: Bacteria antibiotic, Line #139: National Agriculture Research Center, Line #216: ACC, Line #219: Plant growth parameters, Line #227: in peas2009, Line #233: 9800-10, respectively, Line #234: FW, Line #238: DW, Line #256: peroxidase, Fig. 2 legend and Line #544, 221, 241: K. rhizophila)
• K. rhizophila is not always in italic format, correct it. (Table 3. legend, Fig. 4 legend, Line #231)
• Line #26: PGPE, at the first pace in the text, explain the abbreviation.
• Line #57: After nascent oxygen what is that "2-"?
• Line #57: Correct H₂O₂
• Line #85: alleviate stress induced ...(what?)....due to high
• When citing with names in the text, year is not necessary (Line #100, 127, 176, 179, 179)
• Line #106-107: I would suggest: either eaten as raw or boiled.
• The numbering in the Materials and Methods are not correct (Line #124: 2.1 Bacteria salt stress analysis, Line #128: 2.2 Bacteria antibiotic stress resistance, Line #134: 2.3 Bacteria growth conditions..., Line #146: 2.4 Experimental setup, Line #164: 2.5 Plant growth parameters, Line #168: 2.6 Leaf relative water content (RWC), Line #174: 2.7 Estimation of leaf chlorophyll content, Line #177: 2.8 Antioxidants assay in pea, Line #180: 2.9 Proline assay in pea, Line #183: 2.10 Mineral analysis in pea shoots, Line #192: 2.11 Recovery of inoculated bacteria, Line #201: 2.12: Statistics)
• Line #158, 199: use numbered citation instead of name and year format
• Line #159: write the photoperiod as 10h:14h (light:dark)
• Line #168-173: To determine the relative water content, weight with maximum turgor is also required (TW). After measuring fresh weight, did the authors incubate the samples in water for 24 H to measure TW, before drying out the samples? If not, only actual water content (AWC) can be determined.
• In case of sections 2.7, 2.8 and 2.9 more details required about the sample preparation, and more information about the used formulas.
• Line #185: format HNO₃ and HClO₄
• Put the tables and the figure closer to the place, where these are mentioned in the text.
• The numbering in the Results are not correct (Line #211: 3.1 Bacteria salt tolerance, Line #219: 3.2 Plant growth parameters, Line #239: 3.3 Leaf chlorophyll content, Line #248: 3.4 Leaf relative water contents (RWC), Line #254: 3.5 Pea leaves antioxidants, Line #262: 3.6 Proline, Line #267: 3.7 Inorganic osmolytes, Line #275: 3.8 Principal component analysis and Pearson's correlation)
• Be consistent, do not use in three different format: peas 2009, peas2009 or peas-2009
• Correct Table 1 legend
• Correct Table 2 legend (means ± standard error; no full stop after "replicates"; delete "Where", write K. rhizophila instead of Kocuria)
• Table 2: The statistic is not correct!
• Correct Figure 2. legend (instead of Catalase write CAT; start a sentence with capital letter; in leaves of two pea varieties)
• Figure 2: Use the same format of the diagramm in case of SOD also, like in the case of the other three diagramms. Please, try to use similar letter size in all four cases. The statistic is not correct in any cases!
• Figure 3: The statistic is not correct! The format is not adequate, correct the diagramms.
• Table 3: The statistic is not correct!
• Table 4 is not necessary in the text, better in the Supplementary
• Delete Line #629-630.
• The reference list is double-numbered

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

There are still shortcomings in the manuscript. Here is the main one. The errors that I pointed out in the first review were not corrected in table 2. The significance of the differences between the values in some columns is determined incorrectly. If I use standard errors written by the authors in the test, I get a different result. There are problems with the placement of letters in Table 3. (15.31±0.01 cd) probably should be (15.31±0.01 d). (20.26±0.09d) and (20.32±0.05c) cannot differ significantly. Contact a specialist in statistical analysis methods to arrange the letters.

Less important shortcomings.

Line 260. Specify the salt concentration, chlorophyll b decreases only at a salinity of 150mM (T2). The Line 263. 150mM NaCl instead of 75mM NaCl.

Line 265. "But in inoculated plants exposed to 150mM concentration, i-e the leaf RWC is significantly higher leaf water potential with a net increase of 73% and 70% was found in inoculated plants exposed to high salinity levels (Figure 3)". The sentence is written unsuccessfully. It is unclear which experience options are being compared. And, perhaps it meant "up to 73% and 70%" instead of "of 73% and 70%".

I noticed a few typos.

Line 117 and several other places in the text.  Acc → ACC

Line 163. 80 μM S−1 m−2 → 80 μM S⁻¹ m⁻²

Line 307. shhot → shoot

Line 256. Dw → DW

It also seems to me that the specific name of the microorganism in the title of the article should be written in full Kocuria rhizophila.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript improved a lot after following the instructions of the other reviewer also. The authors corrected the suggested points, however, still there are some parts, what can be corrected for the better quality:

• According to the answers of the authors, they determined the TW values for RWC calculation. In this case, also complete the text in Section 2.6.
• According to the answers of the authors, they decided to use "peas2009" format in the text. In this case, also correct Figure 2 labels of data.
• In Figure 2, only on the 4. graph (SOD) the labels of data is missing, and on the 2. graph (POD) labels of data is placed down, while on the other two it is placed on the top of the graph. Please, try to unify.
• Figure 3 legend is a bit far from the figure. On this figure, the graph (d) Y title format is different from the other three graphs. Please, try to unify.

Author Response

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Author Response File: Author Response.pdf