Sodium Selenate: An Environmental-Friendly Means to Control Tomato Bacterial Speck Disease

Round 1

Reviewer 1 Report

Work is very important. The methodologies used were really adequate and allow obtaining accurate results. However, the manuscript is based only on the response of a cultivar and early stage evaluations. This may have greatly limited the potential of the manuscript.

Author Response

Work is very important. The methodologies used were really adequate and allow obtaining accurate results.

We thank the Reviewer, we are really glad you appreciated our work.

However, the manuscript is based only on the response of a cultivar and early stage evaluations. This may have greatly limited the potential of the manuscript.

Cultivar (cv.) RioGrande is a very widespread cv. currently cultivated in Europe. The aim of our work was to find out how selenate is able to protect plants against P. syringae pv. tomato attacks. Further studies will be carried out in order to verify whether this protection can be applied in field; and other cvs. and evaluation of the protective effect throughout the vegetative cycle of tomato will be investigated.

Are the methods adequately described?

Methods have been improved (e.g. qPCR description).

Reviewer 2 Report

Protecting plants from infectious and fungal diseases such as Pseudomonas syringae pv. (…) is an urgent problem. The unique role of selenate as an environmentally friendly and effective bacterial control agent may be important for direct antimicrobial activity protecting not only tomatoes but many other plants from Ps(...) attacks. The results obtained in this work may be of interest to many readers. The study was carried out at a high level using a wide range of research instruments and methods. The experimental results are described in detail. However, there are some remarks to the analysis and discussion of the obtained results. The manuscript is well written, with almost no typos. The manuscript may be published after a significant revision of the discussion paragraph and minor changes listed below.

Notes:

l.53 “UV-B”, l.175 “qRT-PCR”, l.215 “DAPP-PG 215”, l.344 “ROS “ - abbreviations should be explained

l.253 It would be interesting to know the details of how the authors obtained the Ec75, Ec90 values. Is it possible to draw two more curves in Fig. 3?

l.294-298(l.182-188) Not every reader is familiar with the methods described in refs. [41],[42] in detail. It is necessary to briefly describe how figures 6A and 6B were obtained, as well as the use of data from table 1.

l.309-353 The authors try to show the importance of selenium in protecting plants from diseases, referring both to their own research and to the results of the cited studies. As a comparative standard, they chose ASM - a sulfur-containing compound. It is known that sulfur deficiency can play an equally important role in the defense mechanism of plants against Ps(...), for example, the plant itself can generate sulfur-containing compounds (see Camalexin), as a reaction to Ps(...). It is necessary to describe the influence of sulfur-containing compounds in the discussion of protection mechanisms. A simple Internet search reveals several review papers on the effect of sulfur on plant metabolism, for example: The Versatile Roles of Sulfur-Containing Biomolecules in Plant Defense—A Road to Disease Resistance (doi:10.3390/plants9121705), Sulfur and sulfur compounds in plant defence. Nat. Prod. Commun. 2012 Mar;7(3):395-400. Is it possible that selenium simply replaces sulfur in case of its deficiency?

Author Response

Protecting plants from infectious and fungal diseases such as Pseudomonas syringae pv. (…) is an urgent problem. The unique role of selenate as an environmentally friendly and effective bacterial control agent may be important for direct antimicrobial activity protecting not only tomatoes but many other plants from Ps(...) attacks. The results obtained in this work may be of interest to many readers. The study was carried out at a high level using a wide range of research instruments and methods. The experimental results are described in detail. However, there are some remarks to the analysis and discussion of the obtained results. The manuscript is well written, with almost no typos. The manuscript may be published after a significant revision of the discussion paragraph and minor changes listed below.

 We thank the reviewer

 

Notes:

 

l.53 “UV-B”

Abbreviation was explained.

 

l.175 “qRT-PCR”

Abbreviation was explained.

 

l.215 “DAPP-PG 215”

In paragraph 2.2 line 96 it was specified that DAPP-PG 215 is the Pseudomonas syrinage pv tomato strain used in the present work.

 

l.344 “ROS “

Abbreviation was explained.

 

l.253 It would be interesting to know the details of how the authors obtained the Ec75, Ec90 values. Is it possible to draw two more curves in Fig. 3?

The Fig. 3 has been changed as suggested by Reviewer.

 

l.294-298(l.182-188) Not every reader is familiar with the methods described in refs. [41],[42] in detail. It is necessary to briefly describe how figures 6A and 6B were obtained, as well as the use of data from table 1.

More details have been added.

In TableS1 (not in Table 1) the sequences of primers used for qRT-PCR are reported as mentioned in the text (lines 176-177 and 184).

 

l.309-353 The authors try to show the importance of selenium in protecting plants from diseases, referring both to their own research and to the results of the cited studies. As a comparative standard, they chose ASM - a sulfur-containing compound. It is known that sulfur deficiency can play an equally important role in the defense mechanism of plants against Ps(...), for example, the plant itself can generate sulfur-containing compounds (see Camalexin), as a reaction to Ps(...). It is necessary to describe the influence of sulfur-containing compounds in the discussion of protection mechanisms. A simple Internet search reveals several review papers on the effect of sulfur on plant metabolism, for example: The Versatile Roles of Sulfur-Containing Biomolecules in Plant Defense—A Road to Disease Resistance (doi:10.3390/plants9121705), Sulfur and sulfur compounds in plant defence. Nat. Prod. Commun. 2012 Mar;7(3):395-400. Is it possible that selenium simply replaces sulfur in case of its deficiency?

The mechanism of action of the ASM is fully described: ASM is a functional analogue of salicylic acid and its action is mediated by the activation of pathogenesis proteins. However, it cannot be excluded that the presence of sulfur may also have a protective effect. We believe that the presence of sulfur in the molecule can contribute to the activation of resistance but it is not the main mechanism.

A sentence has been added in Discussion section.

 

Are the results clearly presented?

Are the conclusions supported by the results?

We thank the Reviewer for these suggestions and we have improved the result and conclusion sections.

 

Reviewer 3 Report

The authors provide results that demonstrate that a treatment with harmless forms of selenate can protect tomato plants against a severe bacterial disease caused by Pseudomonas syringae pv. tomato.

The topic is of high novelty, although it has been already investigated before in other pathosystems.

The methodology is well designed and all methodological details are covered, making the research perfectly reproducible.

The results obtained are clearly presented and are of importance for tomato producers worldwide.

The discussion is concise, clear and powerfully applied to solve both the bacterial speck disease problem and the selenium fortification of tomato with this treatment.

I consider that the manuscript is of high quality.

Congratulations to the authors for their work!

Author Response

We thank the Reviewer and we are glad you appreciated our work.

Author Response File: Author Response.docx

Reviewer 4 Report

In this manuscript authors aimed to assess the effect of sodium selenate on bacterial speck disease severity.

I've found some aspects that deserve a closer look.

The authors explained the use of selenate and its advantages to mitigate bacterial infections in tomato crops. However, I wonder to know the relevance of selenate when compared with other chemicals showing similar effects. Is it possible to clarify this point?

In lines 89-90: the authors refer to data not shown. In my perspective, all data is important, in this case, preliminary data should be shown at least in the supplementary material.

It seems that ASM treatment showed better results when compared to the other experimental groups.

Please revise figures 6 A and B. Relative expression of what and to what? There seems to be a lack of information in the graphics. It would be instructive if the authors could present the fold-variation expression relatively to control.

Line 314: This information must be supported with literature.

Regarding qPCR were these primers designed by the authors? Can you please provide the accession number?

Reference 35 needs to be revised.

Author Response

In this manuscript authors aimed to assess the effect of sodium selenate on bacterial speck disease severity.

I've found some aspects that deserve a closer look.

The authors explained the use of selenate and its advantages to mitigate bacterial infections in tomato crops. However, I wonder to know the relevance of selenate when compared with other chemicals showing similar effects. Is it possible to clarify this point?

Similar results were obtained with zinc phosphate soil treatment (Quaglia et al., 2021 [32]). Zinc phosphate inorganic salt protects tomato plants against P. syringae pv tomato attack, through the induction of morphological and biochemical plant defence responses and direct antimicrobial activity. The antimicrobial activity of zinc phosphate is greater than that of selenate, and in zinc phosphate treated plants, at the early treatement, a reduction of PIN2 gene expression was observed. In sodium selenate treated plants, the PIN2 gene was not expressed.

A sentence has been added in Discussion section.

In lines 89-90: the authors refer to data not shown. In my perspective, all data is important, in this case, preliminary data should be shown at least in the supplementary material.

Using the concentration ranging from 2 to 8 ppm we observed that 4 ppm was not phytotoxic and was able to protect tomato plants against P. syringae pv. tomato attack.

It seems that ASM treatment showed better results when compared to the other experimental groups.

It has been reported in many references that the resistance inducer ASM is very effective in controlling bacterial speck disease, therefore it has been used as control in our experiments. Of note, ASM is allowed in integrated but not organic management of plant diseases while selenate is allowed in both therefore we belive that selenate is a promising tool for controlling P. syringae pv. tomato.

Please revise figures 6 A and B. Relative expression of what and to what? There seems to be a lack of information in the graphics. It would be instructive if the authors could present the fold-variation expression relatively to control.

Figures 6A-B have been revised. In particular, the label of y-axes have modified with “relative expression ratio (log+1)”. Basically, the relative expression ratio is the ratio of the expression of a target gene in a control (in our case Control at 24 hpt, as specified in L186-187 of Materials and Methods) versus a sample and the expression of reference (housekeeping) gene(s) (in our case, two reference genes ) in control versus sample. This ratio is eventually log transformed for better downstream statistical analysis and graphical display. Here, we have decided to adopt a log2 rather than a log10 transformation so the average values in the pictures for control at 24 hpt is 1.

We have added this information in L186-189.

We have followed method and terminology in Pfaffl, 2001, doi:10.1093/nar/29.9.e45 (ref [41] cited in the manuscript) and modified the label of y-axis “relative expression” with “relative expression ratio (log+1)”. In details, the formula in Pfaffl, 2001 is modified as in Vandesompele et al 2002, doi:10.1186/gb-2002-3-7-research0034 (ref [42] in the manuscript) to account for two reference genes.

With such mathematical approach, we think that terms such as “fold variation” or “fold changes” may be confusing as these often refer to the ratio of the expression level of a gene in the target sample to the ratio of the same gene in the control sample. Clearly, this is not our case as we have calculated the ratio between the target gene and the references genes.

Line 314: This information must be supported with literature.

References have been added.

Regarding qPCR were these primers designed by the authors? Can you please provide the accession number?

As reported in the text and in Table S1 primers used for qPCR are those from Goyal et al., 2016 [39], Castagna et al., 2007 [40] and Fowler et al., 2009 [62].

Reference 35 needs to be revised.

Reference has been revised.

Round 2

Reviewer 1 Report

The manuscript has scientific merit. However, the results are limited by using few treatments. Evaluation of few plants to obtain averages. Evaluation techniques were important.

Author Response

The manuscript has scientific merit. However, the results are limited by using few treatments. Evaluation of few plants to obtain averages. Evaluation techniques were important.

A comment has been added in the Discussion section.

Reviewer 2 Report

Thanks to the authors.

The manuscript has been improved and may be published.

Author Response

Thanks to the authors.

The manuscript has been improved and may be published.

We thank the Reviewer and we are glad you appreciated our work.

Reviewer 4 Report

The authors took into consideration all comments. 

Author Response

The authors took into consideration all comments.

We thank the Reviewer and we are glad you appreciated our work.