Characterising the Influence of First-Year Wheat Cultivar on Pseudomonas Selection and Function in a Take-All Infected Field

Round 1

Reviewer 1 Report

This paper presents analysis the population diversity, Gt suppressive capacity, the presence or absence of gene loci associated with rhizosphere colonisation and in planta assays with cultivars Hereward and Cadenza of 411 Pseudomonas isolates in the second year of wheat cultivation.

The results indicated that wheat variety grown in the first year impacts selection of root associated Pseudomonas isolates in the second year of wheat cultivation.

 This is a well-structured research paper. I have just a few comments to consider during revision, below.

1）There are some errors in the table of this paper, which should be shown in a standard way. For example, the table should be displayed in a three-line table and cannot be displayed across pages. The primer sequence in Table 1 has no title. 

2）"Gt" in lines 80 and 326 and "pseudomonads" in line 336 should be indicated in italics.

3）The data in Figure 1, Figure 2, Figure 4 etc. in the paper be statistically analyzed as necessary? 

4）In Figure 2, the presence rates of fecB and wsm in Class 1, Class 2 and Class 3 are compared in a single graph, can provides a better explanation of the correlation between fecB and wsm incidence correlate with fungal inhibition? Similarly, in Figure 4, the number of infected roots of wheat varieties Cadenza and Hereward can be compared on the same horizontal coordinate under different Pseudomonas isolates.

5）In this paper, 6 Pseudomonas isolates were identified as having strong Gt antagonism, which were verified by in vitro antagonism studies and in planta Take-all disease control assays. However, the author only shows the statistical data, and does not show the important result pictures, which I think is necessary.

Author Response

Thank you for your comments that our manuscript is a well-structured research paper.

1）There are some errors in the table of this paper, which should be shown in a standard way. For example, the table should be displayed in a three-line table and cannot be displayed across pages. The primer sequence in Table 1 has no title. 

We are not clear what the reviewer means here. We believe the table presented here is in a standard format. However, we have now added 5' and 3' to each primer sequence and added in a title.

2）"Gt" in lines 80 and 326 and "pseudomonads" in line 336 should be indicated in italics. We have now italicised Gt in line 80 and 326. pseudomonads should not be italicised.

3）The data in Figure 1, Figure 2, Figure 4 etc. in the paper be statistically analyzed as necessary?

We presented the significant differences among the data within the manuscript to make it scientifically and statistically easier for the readers to understand (e.g. see line 237 of the original file). 

4）In Figure 2, the presence rates of fecB and wsm in Class 1, Class 2 and Class 3 are compared in a single graph, can provides a better explanation of the correlation between fecB and wsm incidence correlate with fungal inhibition? Similarly, in Figure 4, the number of infected roots of wheat varieties Cadenza and Hereward can be compared on the same horizontal coordinate under different Pseudomonas isolates.

We appreciate your comment; however, our aim was to present the data as simply as possible and this is why we presented them in separate figures.

5）In this paper, 6 Pseudomonas isolates were identified as having strong Gt antagonism, which were verified by in vitro antagonism studies and in planta Take-all disease control assays. However, the author only shows the statistical data, and does not show the important result pictures, which I think is necessary.

We have now added pictures of antagonist assay in Table 2.

Reviewer 2 Report

Dear Authors, 

I find your work as very good and important for the science and agricultural practice, however, I have some technical remarks. You can find them as comments in the main text of the article. They concern mostly Methods, which I suggest to describe more precise. There are also some technical mistakes. 

Comments for author File: Comments.pdf

Author Response

Thank you for highlighting that our work was very good and important for the science and agricultural practice.

Line 18, Word ‘crop’ is now un-italicised

Line 63, ‘and’ was added in

Line 85, please add the procedure of bacteria isolation from both sites.

In lines 94-96 we already explained that we followed Mauchline [15] methods for bacterial isolation

Line 113, for how long?

We have now added ‘until dry’ to the sentence

Line 136, the source of primers??? The source of primers is now explained

Line 147, titles do not contain full stop. We have now removed the full stop

Line 148, this sholud be added in the maim text.

We believe information on the source of primers should be part of tha table legend, but the same text has also been added to the materials and methods.

Line 153, not included in the references.

Altschul et al. has now ben added into the reference.

Line 183, as I understand, they were placed in the Falcon 50ml? Five plugs is a lot of pathogen inoculum, so the disease pression was extremaly high. Am I right?

The amswers/comments please include in the main text, improving the clarity of the procedure.

Our initial analysis showed that 5 plugs are sufficient to cause disease symptoms while 3 plugs or less did not cause any disease (data not shown). We have now added a sentence explaining this.

Line 226, please correct the error bar for "h", there is only half of it.

The error bar in Figure 1 is now corrected.                     

Line 228, Figure 1, which microorganism was inhibited? the title suggests taht Pseudomonas bacteria were inhibited. And the work was done to inhibit the fungus. so should be: ".... inhibited by ......".

Thanks for spotting this. We have now corrected Figure 1 legend.

Line 308, change italics. ‘In Planta’ is not in italics here because the heading should be italic.

Line 357, MazzolaGu [22] now changed to Mazzola and Gu [22],

Reviewer 3 Report

The article covers a topic of wide interest and is generally well written, however I have some important concerns to submit to the authors:

 -lines 125-126  “2.4. DNA extraction DNA template for PCR was prepared from bacterial colonies “ How many colonies? The authors wrote that they collected “411 Pseudomonas isolates”…Was the PCR (for identifying the bacterial wsm and fecB loci) performed on all the 411 isolates? Please specify

 -From 411 isolates the in vitro trial brought to select ”the six most antagonistic isolates to control Gt” for the subsequent in planta assay. However, this part has been poorly discussed, and lacks of a statistical analysis leading to more robust statements.

Lines 263-266 “3.2. fecB and wsm incidence correlate with fungal inhibition. When comparing the presence of fecB and wsm and pathogen suppression, we found that a positive correlation existed with presence of fecB and the ability of isolates to supress  Gt. The opposite was found to be the case with the presence of wsm (Figure 2)” These sentences are too generic, which were the values of  Pearson’s r ? Was this analysis strong enough to suggest a clear role in fungal suppression/inhibition (two different concepts)?  

 - Line 308  3.4. In planta Take-all disease control assays. In this paragraph the authors wrote they also tested “a mixture of all 6 antagonistic isolates” ;wouldn't it have been better to test mixtures of isolates from each of the four planting combinations, to highlight the actual differences (and the eventual real synergistic effect of the differentially found isolates) of the employed cultivars? Accordingly, the 6 isolates were from different thesis, representing thus just one of the hypothetical combinations.

 -Figure 4 This figure is poorly described in the text and somewhat misleading. Given that the “sterile” treatment was the “no Pseudomonas” one (please specify it in the text), it seem that the 24E/4, the 30R/11 and 44R/4 alone were as effective as the MIX in both cultivars, thus contradicting the otherwise interesting finding that “For all other test isolates it was found that the inoculant could prevent Gt disease occurrence for only one cultivar”. In any case, this last statement should be more discussed.

-The authors wrote that “A subset of 25 Pseudomonas isolates were genotypically characterised using a fragment of the DNA gyrase B subunit (gyrB) gene”. How -and why- did they choose this subset?

Author Response

Thank you for recognising that our manuscript covers a topic of wide interest and is generally well written.

lines 125-126  “2.4. DNA extraction DNA template for PCR was prepared from bacterial colonies “ How many colonies? The authors wrote that they collected “411 Pseudomonas isolates”…Was the PCR (for identifying the bacterial wsm and fecB loci) performed on all the 411 isolates? Please specify.

We have now added ‘prepared from a single colony’ to line 126. In line 254-255 we already specified that ‘PCR screening of the 411 isolates using the wsm and fecB degenerate primers was performed and isolates were scored for the presence or absence of these genes’.

 -From 411 isolates the in vitro trial brought to select ”the six most antagonistic isolates to control Gt” for the subsequent in planta assay. However, this part has been poorly discussed, and lacks of a statistical analysis leading to more robust statements.

We explained the rationale in selecting the six most antagonistic isolates in line 223 to 227 and data are presented in supplementary Table S3.

Lines 263-266 “3.2. fecB and wsm incidence correlate with fungal inhibition. When comparing the presence of fecB and wsm and pathogen suppression, we found that a positive correlation existed with presence of fecB and the ability of isolates to supress  Gt. The opposite was found to be the case with the presence of wsm (Figure 2)” These sentences are too generic, which were the values of  Pearson’s r ? Was this analysis strong enough to suggest a clear role in fungal suppression/inhibition (two different concepts)?  

We have now added the Pearson data analysis in.

 - Line 308  3.4. In planta Take-all disease control assays. In this paragraph the authors wrote they also tested “a mixture of all 6 antagonistic isolates” ;wouldn't it have been better to test mixtures of isolates from each of the four planting combinations, to highlight the actual differences (and the eventual real synergistic effect of the differentially found isolates) of the employed cultivars? Accordingly, the 6 isolates were from different thesis, representing thus just one of the hypothetical combinations.

We appreciate your comment, although, the main hypothesis tested was that the wheat variety grown in the first year impacts selection of root-associated Pseudomonas isolates in the second year of wheat cultivation, we also wanted to find Pseudomonas strains with biocontrol potential, therefore we only selected the six most antagonistic Pseudomonas strains. The testing of the mixtures of isolates from each of the four planting combinations is beyond the scope of this study.

 -Figure 4 This figure is poorly described in the text and somewhat misleading. Given that the “sterile” treatment was the “no Pseudomonas” one (please specify it in the text), it seem that the 24E/4, the 30R/11 and 44R/4 alone were as effective as the MIX in both cultivars, thus contradicting the otherwise interesting finding that “For all other test isolates it was found that the inoculant could prevent Gt disease occurrence for only one cultivar”. In any case, this last statement should be more discussed.

We have added the word ‘sterile’ into the text to clarify. We have now removed the last sentence.

-The authors wrote that “A subset of 25 Pseudomonas isolates were genotypically characterised using a fragment of the DNA gyrase B subunit (gyrB) gene”. How -and why- did they choose this subset?

We have now removed ‘a fragment of the DNA’ and referred to table 1 (primer used in the study). In line 278-282 we already explained the rationale in choosing the 25 isolates.

Round 2

Reviewer 3 Report

The authors responded satisfactorily to my comments, so I have no further doubts.