Review Reports - Resistance of Mulberry Fruit Sclerotiniosis Pathogens to Thiophanate-Methyl and Boscalid

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This current manuscript deals with the search for susceptibility and resistance analysis to fungicides in fungi identified in fruits of Mulberry plants, in a particular region of China with a long history of growing mulberry tree previously used for harvesting their leaves for silk worm production. In recent years this activity changed to fruit production, which been affected by different fungi species with the development of resistance to fungicide treatment. This resistance had not been previously evaluated in this region. Authors isolated different strains of two fungal species: Sclerotinia sclerotiorum and Scleromitrula shiraiana from mulberry fruits. Then, they tested them for resistance/susceptibility to two fungicides, thiophanate-methyl and boscalid. Resitance to thiophanate-methyl was found in some S. sclerotiorum strains. Then, crossed resitance assays were done with different fungicides. Furthermore, the mechanism of resistance was evaluated by sequencing of the predicted β-tubulin protein variants, in single and double mutants and docking experiments with the substrate thiophanate-methyl.

Comments:

The introduction is lacking important information regarding the fungicides studied in this manuscript, Boscalid and Thiophanate-methyl and their mechanisms of resistance. Please include references and its mechanism of action, mechanisms of resistance, properties, crops in which they are used and issues with resistance.

.Figure legend figure 3 is incomplete: Frequency of What? It should say Figure 3. Frequency of sensitivity and of resistance to thiophanate-methyl and boscalid in both species…

Lane 199 explain the concept of negative Cross resistance.

. Figure legend figure 4 is not clear.It says “Figure 4. Growth performance of methyl thiophanate-methyl resistant isolate SS-4 (A) and sensitive isolate SS-2 (B) on thiophanate-methyl and diethofencarb plates. It should say “Figure 4. Growth performance of methyl thiophanate-methyl resistant isolate SS-4 (A) and sensitive isolate SS-2 (B) on thiophanate-methyl (upper panel) and diethofencarb plates (bottom panel). Also Indicate what is CK.

Figure 5. Indicate which sections correspond to A, B or C (is not written in the figure).

Lane 221 to227. Background information is missing. There are several reports indicating that resistance to Thiophanate-methyl is linked to point mutations in the beta𝛽-tubulin gene. Please include some of these references. This can explain why did the authors decided to do this assay, this was done Based on previously published research, Then cite it.

Lane 232. It says “ The results indicated that the binding pattern of the wild-type target protein to the substrate is quite different from that of the mutant type” No background information is provided regarding the binding mechanism of beta tubuline with the fungicides. However, plenty published information already exists. It is necessary to include this information here. Also the authors need to stablish why it is important to determine the binding capability of the mutants found.

Lane 260 it says “However, this group (B1, group 1) of fungicides”…please explain about the groups of fungicides, context and background information is lacking… including

Lines 263 to 265.. It says: “On common fruit mulberry in Chongqing，29 isolates S. sclerotiorum exhibiting 93.1% high-level resistance to thiophanate-methyl was documented [18]” However., that reference is not correct, the authors do an study of the endophitic fungus in 4 varieties of mulberry, but not with fungigicides. Please correct the reference 18: .( [18] W.F. Xu, F. Wang, R.L. Wang, Y. Sui, Z.Y. Zhou, J. Xie, Z.H. Xiang. Seasonal characterization of the endophytic fungal microbiome of mulberry (Morus spp.) cultivars resistant and susceptible to sclerotiniosi.)

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Overall, the english language is correct, however there are some phrases that need reviewing,

The attached file highlights some errors in English grammar (in pink)

Author Response

Comments:

Response: An introduction to the two fungicides has been added to the second paragraph of the introduction section.

“Thiophanate-methyl belongs to the benzimidazole (BZI) class of fungicides (a subgroup of the methyl benzimidazole carbamates, MBCs) and acts by binding to β-tubulin sub-units, interfering with microtubule assembly and disrupting essential cellular processes such as mitosis, cell division and intracellular transport. Resistance to thiophan-ate-methyl is predominantly mediated by point mutations in the tub2 gene encoding β-tubulin, which alter the fungicide-binding domain and confer cross-resistance among other BZI fungicides.”

“Boscalid, a carboxamide fungicide, exerts its biocidal effect by inhibiting the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial respiratory chain, thereby disrupting energy metabolism and halting fungal growth and reproduction. The single-site mode of action of boscalid renders it highly prone to resistance devel-opment, which primarily arises from point mutations in the SdhB, SdhC or SdhD subu-nits of the SDH complex; these mutations reduce the binding affinity between the fun-gicide and its target site, leading to ineffective control in the field.”

.Figure legend figure 3 is incomplete: Frequency of What? It should say Figure 3. Frequency of sensitivity and of resistance to thiophanate-methyl and boscalid in both species…

Response: The figure 3 legends have been revised to “Frequency of sensitivity and resistance to thiophanate-methyl and boscalid in S. sclerotiorum and S. shiraiana”.

Lane 199 explain the concept of negative Cross resistance.

Response: An explanation of negative cross-resistance has been added to line 199. “when populations of harmful organisms such as pathogens and insect pests acquire re-sistance to one class of pesticides, they instead exhibit enhanced sensitivity to another class of pesticides”

. Figure legend figure 4 is not clear. It says “Figure 4. Growth performance of methyl thiophanate-methyl resistant isolate SS-4 (A) and sensitive isolate SS-2 (B) on thiophanate-methyl and diethofencarb plates. It should say “Figure 4. Growth performance of methyl thiophanate-methyl resistant isolate SS-4 (A) and sensitive isolate SS-2 (B) on thiophanate-methyl (upper panel) and diethofencarb plates (bottom panel). Also Indicate what is CK.

Response: The figure 4 legends have been revised to “Growth performance of methyl thiophanate-resistant isolate SS-4 (A) and sensitive isolate SS-2 (B) on thiophanate-methyl(upper panel) and diethofencarb(bottom panel) plates, CK: blank control containing solvent. ”

Figure 5. Indicate which sections correspond to A, B or C (is not written in the figure).

Response: Letters A, B, and C have been labeled in the upper-left corner of each panel in Figure 5.

Lane 221 to227. Background information is missing. There are several reports indicating that resistance to Thiophanate-methyl is linked to point mutations in the beta?-tubulin gene. Please include some of these references. This can explain why did the authors decided to do this assay, this was done Based on previously published research, Then cite it.

Response: We have added the point that the resistance of fungi to thiophanate-methyl is associated with the point mutation of the β-tubulin gene in the second paragraph of the introduction, with relevant literatures cited.

“Resistance to thiophan-ate-methyl is predominantly mediated by point mutations in the tub2 gene encoding β-tubulin, which alter the fungicide-binding domain and confer cross-resistance among other BZI fungicides.”

Response: We have added the relevant background information on the binding mechanism starting from the fourth line of the third paragraph in the Discussion section, and summarized the binding results obtained in this study. And citations to relevant references have been added.

“The affinity and binding specificity between fungicides and their target proteins can largely reflect the sensitivity of target organisms to the agents. By investigating the binding modes of fungicides to target proteins, the fungicidal mechanisms and the resistance mechanisms of pathogens can be elucidated. In previous studies, the binding modes of MBC fungicides to the 198th amino acid residue of the β2-tub gene have been extensively reported. In this study, we investigated whether there is a synergistic relationship between the newly identified V349I mutation and the E198A mutation. The results showed that the binding energy between thiophanate-methyl and the V349I+E198A double mutant was significantly lower than that between thiophanate-methyl and the E198A single mutant.”

Lane 260 it says “However, this group (B1, group 1) of fungicides”…please explain about the groups of fungicides, context and background information is lacking… including

Response: We have added the background introduction of this group in the introduction section, and revised the expression of this paragraph in the discussion section.

However, the MBC group (B1, Group 1) of fungicides, including thiophanate-methyl, thiophanate, benomyl, carbendazim, thiabendazole and fuberidazole, has been classified as a high-risk category for resistance development by the Fungicide Resistance Action Committee (FRAC) (www.frac.info)

Response: This data is sourced from a master's thesis of Chongqing Three Gorges University in China. Since the data has not been officially published, we have deleted this piece of data and its citation in the present paper. Thank you for your corrections.

Reviewer 2 Report

Comments and Suggestions for Authors

I have read the manuscript entitled '' Resistance of the mulberry fruit sclerotiniosis pathogens to thi-2 ophanate-methyl and bosalid'' (Manuscript ID: horticulturae-4009106). This is a solid study that examines the antifungal activity of two biocides against autochthonous phytopathogenic fungal isolates. The most interesting is the fact that the authors analyzed the resistance mechanism of the biocides which gives fundamental approach to their research. However, the manuscript requires major changes before publication.

General comments

The manuscript requires English checking, including punctuation and placement of spaces.

Specific comments

Abstract

Line 15 – replace fruit mulberry gardens with these gardens to avoid repetition

Lines 22-23 – please remove the repeating (n=12)

Introduction

Please supplement the Introduction with additional data about the biocides used in this study, introduce the facts about their chemical classification and description. For example: Boscalid is a broad spectrum fungicide compound which is a biphenyl amide derived inhibitor of succinate dehydrogenase…

Line 46 – please transfer the words from the brackets into the sentence to improve the style.

Material and methods

Line 73 – replace add water to a volume of 1000 mL with water up to 1000 mL

Line 118 - what exactly is DPS software? Provide further data about this software package and the full name for the abbreviation.

Lines 122-128 – Cross resistance methodology is vaguely described, please proved a more detailed and clear description.

Lines 130-135 – The authors did not state what was the incubation time for this assay.

Results

Line 161 – The colonies do not present themselves, please rephrase

Figure 1 – shows multiple sclerotia and multiple conidia under C and D, not a single one

Figure 2 – The phylogenetic tree should be rooted with a non Sclerotinia species and the quality of the figure should be imporved.

Figure 3 is not mentioned in the text

Discussion

Please explain what is the function of beta tubulin gene in the resistance metabolism and/or why the authors chose to examine this gene. Give appropriate references.

According to all the given comments, I suggest a major revision before acceptance to the Horticulturae.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English language should be improved.

Author Response

I have read the manuscript entitled '' Resistance of the mulberry fruit sclerotiniosis pathogens to thiophanate-methyl and boscalid'' (Manuscript ID: horticulturae-4009106). This is a solid study that examines the antifungal activity of two biocides against autochthonous phytopathogenic fungal isolates. The most interesting is the fact that the authors analyzed the resistance mechanism of the biocides which gives fundamental approach to their research. However, the manuscript requires major changes before publication.

General comments

The manuscript requires English checking, including punctuation and placement of spaces.

Response: Thank you for your corrections. We have carefully proofread the entire manuscript, including punctuation marks and spacing. Please refer to the tracked changes in the document for details.

Specific comments

Abstract

Line 15 – replace fruit mulberry gardens with these gardens to avoid repetition

Lines 22-23 – please remove the repeating (n=12)

Response: Line 15 –The content has been revised. Lines 22-23 –The relevant content has been deleted.

Introduction

Response: An introduction to the two fungicides has been added to the second paragraph of the introduction section.

“Boscalid, a carboxamide fungicide, exerts its biocidal effect by inhibiting the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial respiratory chain, thereby disrupting energy metabolism and halting fungal growth and reproduction. The single-site mode of action of boscalid renders it highly prone to resistance development, which primarily arises from point mutations in the SdhB, SdhC or SdhD subu-nits of the SDH complex; these mutations reduce the binding affinity between the fungicide and its target site, leading to ineffective control in the field.”

Line 46 – please transfer the words from the brackets into the sentence to improve the style.

Response: The wording of the full sentence has been optimized for clarity and accuracy.

Commonly referred to as “white fruit disease” by growers, it primarily affects female flowers, green fruits, as well as new shoots and tender buds of early-maturing mulberry trees

Material and methods

Line 73 – replace add water to a volume of 1000 mL with water up to 1000 mL

Response: The content has been revised.

Line 118 - what exactly is DPS software? Provide further data about this software package and the full name for the abbreviation.

Response: Revised to Data Processing System (DPS). DPS is a domestic Chinese statistical analysis software with independent intellectual property rights.

Lines 122-128 – Cross resistance methodology is vaguely described, please proved a more detailed and clear description.

Response: We have provided a more comprehensive description of this paragraph, primarily supplementing the specific operational steps involved in post-cultivation, measurement, and statistical analysis.

“The mycelial growth rate method was used to determine the susceptibility of the 12 isolates of S. sclerotiorum to three fungicides, diethofencarb, boscalid and procymidone. The adopted serials of gradient concentration were diethofencarb ( 0.5, 1, 2, 4, 8 μg/mL), 0.125, 0.25, 0.5, 1, 2, 4 μg/mL(boscalid), and 0.5, 1, 2, 4, 8 μg/mL(procymidone) with three replications per treatment. Only solvent-amended PDA plates were served as controls. After 3-day of incubation at 25°C in darkness, colony diameters were measured along two perpendicular axes. Mycelial growth inhibition rates were calculated, with toxicity regression equations and EC50 values derived using DPS software. To analyze whether there was cross-resistance between the two fungicides, Spearman rank correlation coefficient was assessed. when P < 0.05 and ρ > 0.6, it indicated that there was strong positive interaction resistance between them”

Lines 130-135 – The authors did not state what was the incubation time for this assay.

Response: The incubation time has been added to the manuscript.

“cultured in the dark for 3 days”

Results

Line 161 – The colonies do not present themselves, please rephrase

Response: We have revised the morphological description of the fungal colonies

“Colonies had a yellowish-white felt-like surface lacking aerial hyphae, with irregular margins. The colony reverse was dark brown. A small, creamy-white conidial mass appeared at the colony center, while granular sclerotia protuberances were visible at the margin”

Figure 1 – shows multiple sclerotia and multiple conidia under C and D, not a single one

Response: Sclerotium and conidium in the figure legends have been revised to sclerotia and conidia, respectively

Figure 2 – The phylogenetic tree should be rooted with a non Sclerotinia species and the quality of the figure should be improved.

Response: We have improved the quality of the images.

Figure 3 is not mentioned in the text

Response: We have newly added a mention of Figure 3 in Section 3.3.

Discussion

Please explain what is the function of beta tubulin gene in the resistance metabolism and/or why the authors chose to examine this gene. Give appropriate references.

Response: We have provided the relevant background description in the Introduction

According to all the given comments, I suggest a major revision before acceptance to the Horticulturae.