Black Knot Unraveled: Phenotypic Characterization of Disease Resistance in Japanese Plums

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author

Please find my general comments on the manuscript:

 

-For the trees that show high resistance to the disease, it is necessary to confirm their resistance. This resistance may be due to uneven inoculum distribution in the field or the edge effect, as the trees may have been exposed to different environmental conditions.

-Additionally, a molecular test is needed to determine which protein expressions related to resistance may be induced in the trees that show resistance as evidence of resistance on molecular level.

 

-Re-test the varieties that show resistance in greenhouse or nursery on small trees, 1-2 years old to confirm its resistance or re inoculated the old trees with the pathogen to confirm its resistance.

-Providing a visual disease scale would be helpful. Additionally, including pictures that compare resistant and susceptible infection types on trees would make it easier to understand the differences.

 

Author Response

Dear Author

Please find my general comments on the manuscript:

 We thank the reviewer for the comments and suggestions. Although the comments are valid and would help in general, some of the points raised are not possible in black knot due to the nature of the disease. Further it is the lack of such repeatable in vitro assay, probably precluded many researchers to work on this disease, as it is a very time-consuming process and that too without a firm answer.

-For the trees that show high resistance to the disease, it is necessary to confirm their resistance. This resistance may be due to uneven inoculum distribution in the field or the edge effect, as the trees may have been exposed to different environmental conditions.

The reasoning has been explained in the manuscript both in introduction (65-78). The trees were there for well over 10 years without much disturbance increasing the chances of infection on any escapes. The inoculum spread is beyond doubt as there were trees side by side with highly susceptible nature and resistant.

-Additionally, a molecular test is needed to determine which protein expressions related to resistance may be induced in the trees that show resistance as evidence of resistance on molecular level.

 Yes, that would be interesting but that is outside the scope of this study. It requires a lot more in-depth analyses and that is ongoing and will be published later as a separate manuscript.

-Re-test the varieties that show resistance in greenhouse or nursery on small trees, 1-2 years old to confirm its resistance or re inoculated the old trees with the pathogen to confirm its resistance.

Again, we have clearly indicated that this is not possible as consistent re-inoculation has never been done successfully. If such a mechanism is available the current study would never have taken place. Lack of such, successful and repeatable controlled inoculation tests only lead to the current study.

-Providing a visual disease scale would be helpful. Additionally, including pictures that compare resistant and susceptible infection types on trees would make it easier to understand the differences.

I am not sure what is suggested here. If the reviewer is asking for pictures of the whole trees with the infection scale- it is provided in Fig 1. In that itself, we have provided the resistant and susceptible genotype pictures, which should address the second point.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is devoted to an important phytopathological problem in horticulture. Apiosporina morbosa is a dangerous pathogen causing black knot disease in Prunus species in Canada, Mexico and the continental states of the USA. It is important to breed plum trees resistant to this disease in the near future. The aim of the present work is related to this. The authors believe that it is necessary to find a method that will allow an objective assessment of the degree of susceptibility to A. morbosa. In the present work they presented a quite satisfactory proposal, because the method used provided high reproducibility. The studies were carried out on 200 trees of known origin. Minimization of differences in the assessment of the disease severity was achieved thanks to observations by multiple raters over different time. The manuscript is written carefully, and the subsequent stages of the analysis are well explained. However, there are some ambiguities in some places. After minor revision the manuscript should be published in Horticulturae.

Remarks

Line 1 it should be Apiosporina morbosa (Schwein.) v. Arx instead of ‘Apiosporina morbosa (Schwein.)’

Line 7-8  unclear text

Line 35 it should be Apiosporina morbosa (Schwein.) v. Arx instead of ‘Apiosporina morbosa Schwein.’

Line 36-37 The term ‘disease’ should be distinguished from the term ‘pathogen’. This sentence is incorrect: ‘The lifecycle of BK disease begins with the release of ascospore…’ The lifecycle refers to the pathogen, not the disease. The pathogen releases ascospores, not the disease.

Line 92 ‘since the combination of the fungal invasion with the plant’s immune system causes hypertrophy of the tissues [13].’ – Unfortunately, in the publication [13] such information is not provided. The problem of hypertrophy is more complex and should be distinguished from ‘callus formation’. Tissue ‘hypertrophy’ is often the result of plant cells growing excessively or dividing excessively under the influence of a pathogen. This is hyperplasia or hypertrophy (see Tainter & Baker, Principles of Forest Pathology). It is advisable to generalize the content.

Figure 1, Figure 2 Prunus salicina L. – remove L. [the author is given only when the name is used for the first time, here it is on line 23

Line 145-146 this text requires a short explanation or citation of literature (the name of a German institution is given, where black knot does not occur).

Line 199-210 this text is inconsistent with the data in Figure 3, despite Figure 3 being cited. It is not clear what the percentage scale in Figure 3 represents. Perhaps instead of 1, 2, 3…, it should be 10, 20, 30….?

 

Line 260 ‘Five years later in 2023, this number dropped significantly to 4%.’ – why were the results of the black knot disease severity assessment not presented in the Results section. This information is only in the Discussion.

Line 411, 419, 421 Apiosporina Morbosa – it should be in italic

Line 414 it should be 2006 instead of 1935

Author Response

This manuscript is devoted to an important phytopathological problem in horticulture. Apiosporina morbosa is a dangerous pathogen causing black knot disease in Prunus species in Canada, Mexico and the continental states of the USA. It is important to breed plum trees resistant to this disease in the near future. The aim of the present work is related to this. The authors believe that it is necessary to find a method that will allow an objective assessment of the degree of susceptibility to A. morbosa. In the present work they presented a quite satisfactory proposal, because the method used provided high reproducibility. The studies were carried out on 200 trees of known origin. Minimization of differences in the assessment of the disease severity was achieved thanks to observations by multiple raters over different time. The manuscript is written carefully, and the subsequent stages of the analysis are well explained. However, there are some ambiguities in some places. After minor revision the manuscript should be published in Horticulturae.

We thank this reviewer profusely, as we are finally able to get someone who can understand the magnitude of this disease as well as the significance of the work. Thank you again and the comments are very useful to make the ms much better and we have addressed the comments in full as below.

Remarks

Line 1 it should be Apiosporina morbosa (Schwein.) v. Arx instead of ‘Apiosporina morbosa (Schwein.)’

It has been updated.

Line 7-8  unclear text

“Biologische Bundesanstalt, 7 Bundessortenamt and CHemical industry (BBCH) growth stage 1,” has been removed. It indeed does not contribute to enhanced understanding, with the potential of overcomplicating the description.

Line 35 it should be Apiosporina morbosa (Schwein.) v. Arx instead of ‘Apiosporina morbosa Schwein.’

This has been fixed.

Line 36-37 The term ‘disease’ should be distinguished from the term ‘pathogen’. This sentence is incorrect: ‘The lifecycle of BK disease begins with the release of ascospore…’ The lifecycle refers to the pathogen, not the disease. The pathogen releases ascospores, not the disease.

This is a very good point: it has been changed from “BK disease” to “the BK fungus”

Line 92 ‘since the combination of the fungal invasion with the plant’s immune system causes hypertrophy of the tissues [13].’ – Unfortunately, in the publication [13] such information is not provided. The problem of hypertrophy is more complex and should be distinguished from ‘callus formation’. Tissue ‘hypertrophy’ is often the result of plant cells growing excessively or dividing excessively under the influence of a pathogen. This is hyperplasia or hypertrophy (see Tainter & Baker, Principles of Forest Pathology). It is advisable to generalize the content.

“Hypertrophy of the tissues” has been changed to “abnormal growth of the tissues” to ensure generality.

Figure 1, Figure 2 Prunus salicina L. – remove L. [the author is given only when the name is used for the first time, here it is on line 23

It has been removed.

Line 145-146 this text requires a short explanation or citation of literature (the name of a German institution is given, where black knot does not occur).

I agree with the question to relevance, thus “Biologische Bundesanstalt, Bundessortenamt and CHemical industry (BCCH) growth stage 1,” has been entirely removed (as well as in the abbreviations on the Latex file)

Line 199-210 this text is inconsistent with the data in Figure 3, despite Figure 3 being cited. It is not clear what the percentage scale in Figure 3 represents. Perhaps instead of 1, 2, 3…, it should be 10, 20, 30….?

You are correct to point out that the y axis should be multiplied by a factor of 10, thus it has been updated. To further clarify, the exact percentages of each bar has also been added to the plot. Now it should be clearer that line 100-210 refers to Figure 3.

Line 260 ‘Five years later in 2023, this number dropped significantly to 4%.’ – why were the results of the black knot disease severity assessment not presented in the Results section. This information is only in the Discussion.

Thanks for pointing it out. Somehow, we missed that during revisions. We created an additional version of the revised manuscript including a table of the disease rating counts for the 2023 year.

Line 411, 419, 421 Apiosporina morbosa – it should be in italic

These have been changed.

Line 414 it should be 2006 instead of 1935

This has been amended.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors

The manuscript has been improved and looks ready for publication. Just one thing: in the Results section, page 5, line 170 (3-1: Phenotypic Evaluations), the paragraph about the data rating count should appear in the middle of the paragraph, not at the beginning.

Thanks

 

Author Response

Thanks for the comment. the sentence has been re arranged as suggested.

Jay