Identification of Botrytis cinerea as a Walnut Fruit Rot Pathogen, and Its Biocontrol by Trichoderma

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

The authors investigated the fungal pathogens associated with walnut (Juglans regia L.) fruit rot, identifying several species and highlighting Botrytis cinerea sensu lato as a newly recognized pathogen. They characterized the morphology, temperature-dependent growth, and pathogenicity of multiple B. cinerea strains across different fruit developmental stages. The authors presented a solid study as a previously unreported and significant pathogen of walnut fruit, with effective biocontrol potential demonstrated by Trichoderma strains. However, before the manuscript can be considered for acceptance, I have some major concerns regarding the experimental design and clarity of the study that need to be addressed.

Dear Authors: The following comments need to be addressed:

Major Comments

3.1. Assessment of symptoms of immature walnuts

It is unclear whether these symptoms were observed in the field, after sample collection, or during your pathogenicity test. Please clarify the context in which these symptoms were recorded and describe your results more clearly.

I am curious about the pathogenicity tests, sample collection, and fungal isolation. Figure 1 shows only lesions, while Figure 2 shows a bunch of mycelium, but Figures 9 and 10 show lesions, necrosis. You mention inoculating walnut fruits for 21 days (L 138) in dark conditions, but it’s unclear why mycelium is not visible, especially after such a long incubation. According to Koch’s postulates, re-isolation and confirmation of the pathogen are essential, particularly if only lesions and decay are visible without mycelial growth. It's also possible that fruit can decay in darkness under high humidity, even without active fungal growth.

In Figure 3, the Authors provided various Botrytis isolates grown on PDA after 10 days. However, in Figure 4,  only provided images of spores, conidia, and hyphae for a single Botrytis species. If the colony morphology differs among isolates, it would be beneficial to also include representative microscopic images (spores, conidia, and hyphae) for the other isolates. This would provide better clarity and help highlight morphological differences among the different Botrytis Isolates.

In Table 1, the authors listed different fungal genera, but in Table 2, ITS sequencing was performed for only 8 Botrytis cinerea isolates. Could you clarify the selection criteria for these specific isolates? Were they chosen based on distinct morphological characteristics, geographic origin (e.g., different orchard collections), or exposure to different fungicide treatments? Please provide a clear explanation of the experimental design and rationale behind the selection of these isolates.

Lines 220–226: In the BLAST analysis, you mentioned the sequence similarity between Botrytis cinerea type strain and other known strains. To enhance clarity, I suggest including the BLAST percentage identity and positivity values for these strains in Table 3.

Similarly, in Figure 5, in the phylogenetic tree authors identified strains (J5008–J5012) cluster together with B. cinerea 05.10 and other known strains in the same clades, indicating close genetic relatedness and likely placement within the B. cinerea sensu lato complex. The grouping of these strains within the Botrytis cinerea sensu lato clade appears reasonable; however, the current tree lacks low bootstrap support (e.g., 55) and lacks clarity of sub-clades/Branches or sequence similarity values. Please also show the similarity among strains (J5008–J5012) and B. cinerea 05.10 and other known strains, especially for J5012 and B. cinerea 05.10, and strains/isolates next to their.  Moreover, although Sclerotinia sclerotiorum shows 99% similarity between the two species, the phylogenetic tree branches do not clearly support this relationship. To strengthen the phylogenetic analysis and support the taxonomic placement, I  recommend reconstructing the tree using IQ-TREE (http://iqtree.cibiv.univie.ac.at/).

Line 131-133 previously studied four Botrytis cinerea (J5011/1, J5013/1, J5017, J5021) isolates were used as pathogens based on their different morphological characters.

Why did the author select these isolates? First, according to Figure 3 (Colony morphology), all eight isolates appeared different. Moreover, the selection of isolates should consider both morphological and genotypic differences. As shown in Figure 5, the phylogenetic analysis indicated that J5011/1 is most closely related to J5013/1, and similarly, J5017 is closely related to J5021.

Table 4: The standard error is very high, especially for  J5013/1 and J5021. This raises the possibility of contamination in your isolates or mixed culture, as such high SE values are not common in pure cultures.

Conclusion: L382-383- Trichoderma strains  TR04 and TR05 show promising potential in controlling B. cinerea infections. If the authors are concluding controlling B. cinerea infections, it suggests that these strains may be applied to reduce the pathogenicity of B. cinerea on walnut. Did you consider applying TR04 and TR05 in a pathogenicity assay to test their effectiveness against B. cinerea? This would provide more promising evidence to support their potential use in future disease management strategies for walnut orchards.

Minor Comments

Comma (,) was missing in references 10 and 6, it should be (10, 6, 13), L 50.

Similarly, in L144, (3232) please check through the manuscript.

Table 4: In Colony diameter (mean + SE) should be dot (.), not a comma (,)

In Figure 4, the statistical letters are missing on the left side figure, and the right side of the figure statistical letters are merging with figure.

Figure 8: a) Above the graph title no need to write Husk Scale, as it is already presented on the Y-axis. b) The statistical lettering is merging with the bar graph. Please check carefully in all figures and ensure consistency in distance among the letters and the bar graph.

Author Response

Comments 1: 3.1. Assessment of symptoms of immature walnuts

It is unclear whether these symptoms were observed in the field, after sample collection, or during your pathogenicity test. Please clarify the context in which these symptoms were recorded and describe your results more clearly.

Response 1: The following sentence is now added for clarification. “In the orchard, fruits with dark lesions were collected from trees both directly and after shaking, ensuring only freshly fallen ones were gathered.”

 

Comments 2: I am curious about the pathogenicity tests, sample collection, and fungal isolation. Figure 1 shows only lesions, while Figure 2 shows a bunch of mycelium, but Figures 9 and 10 show lesions, necrosis. You mention inoculating walnut fruits for 21 days (L 138) in dark conditions, but it’s unclear why mycelium is not visible, especially after such a long incubation. According to Koch’s postulates, re-isolation and confirmation of the pathogen are essential, particularly if only lesions and decay are visible without mycelial growth. It's also possible that fruit can decay in darkness under high humidity, even without active fungal growth.

Response 2: Figure 1 displays representative samples from the orchard, exhibiting dark lesions of varying severity. Figure 2 shows the samples after Overnight Freezing-Incubation (ONFIT). This method – described in the Materials and Methods (section 2.2, ONFIT method) – was used to counteract the inhibitory effect of fungistatic plant chemicals, thereby enhancing mycelial development. We have clarified the Legend for Figure 2, which now reads as ‘Fungal mycelia developed after using the ONFIT process’.

ONFIT was not employed after the inoculation of green fruits. Therefore, lesions and necrosis have developed as shown by Figures 9, but mycelial formation was inhibited by plant fungistatic chemicals.

 

Comments 3: In Figure 3, the Authors provided various Botrytis isolates grown on PDA after 10 days. However, in Figure 4, only provided images of spores, conidia, and hyphae for a single Botrytis species. If the colony morphology differs among isolates, it would be beneficial to also include representative microscopic images (spores, conidia, and hyphae) for the other isolates. This would provide better clarity and help highlight morphological differences among the different Botrytis Isolates.

Response 3: The colony morphology of B. cinerea can be diverse (Garfinkel et al 2019, Duèkena et al 2024), unlike microscopic characteristics such as spore size (Fekete et al 2011, doi:10.1016/j.micres.2011.10.006). Therefore, members of the B. cinerea species complex can only be identified through appropriate marker sequences (Garfinkel et al 2019). These info was added to the Discussion.

 

Comments 4: In Table 1, the authors listed different fungal genera, but in Table 2, ITS sequencing was performed for only 8 Botrytis cinerea isolates. Could you clarify the selection criteria for these specific isolates? Were they chosen based on distinct morphological characteristics, geographic origin (e.g., different orchard collections), or exposure to different fungicide treatments? Please provide a clear explanation of the experimental design and rationale behind the selection of these isolates.

Response 4: Eight of 37 B. cinerea isolates with different colony morphology characteristics were selected for sequence-based identification. This information was added to the Results.

 

Comments 5: Lines 220–226: In the BLAST analysis, you mentioned the sequence similarity between Botrytis cinerea type strain and other known strains. To enhance clarity, I suggest including the BLAST percentage identity and positivity values for these strains in Table 3.

Response 5: The results were identical (100% similarity). This info is now added.

 

Comments 6: Similarly, in Figure 5, in the phylogenetic tree authors identified strains (J5008–J5012) cluster together with B. cinerea 05.10 and other known strains in the same clades, indicating close genetic relatedness and likely placement within the B. cinerea sensu lato complex. The grouping of these strains within the Botrytis cinerea sensu lato clade appears reasonable; however, the current tree lacks low bootstrap support (e.g., 55) and lacks clarity of sub-clades/Branches or sequence similarity values. Please also show the similarity among strains (J5008–J5012) and B. cinerea 05.10 and other known strains, especially for J5012 and B. cinerea 05.10, and strains/isolates next to their. Moreover, although Sclerotinia sclerotiorum shows 99% similarity between the two species, the phylogenetic tree branches do not clearly support this relationship. To strengthen the phylogenetic analysis and support the taxonomic placement, I  recommend reconstructing the tree using IQ-TREE http://iqtree.cibiv.univie.ac.at/).

Response 6: Phylogenetic analyses of Botrytis species frequently shows weak bootstrap support (Garfinkel et al 2019, Lukasko and Hausbeck, 2024). This info has been added to the Discussion.

 

Comments 7: Line 131-133 previously studied four Botrytis cinerea (J5011/1, J5013/1, J5017, J5021) isolates were used as pathogens based on their different morphological characters.

Why did the author select these isolates? First, according to Figure 3 (Colony morphology), all eight isolates appeared different. Moreover, the selection of isolates should consider both morphological and genotypic differences. As shown in Figure 5, the phylogenetic analysis indicated that J5011/1 is most closely related to J5013/1, and similarly, J5017 is closely related to J5021.

Response 7: Given that all isolates possessed identical ITS sequences, we tested for growth variations in response to changing inoculation temperature among strains displaying diverse colony morphologies. This approach seeks to elucidate the adaptive diversity of the pathogenic B. cinerea population in response to distinct environmental conditions within a given orchard. This information was added to the Results.

 

Comments 8: Table 4: The standard error is very high, especially for J5013/1 and J5021. This raises the possibility of contamination in your isolates or mixed culture, as such high SE values are not common in pure cultures.

Response 8: Growth observed after heat stress (Table 4) suggests B. cinerea strains can enter into a reversible state of stress that allows recovery and morphological shifts under optimal conditions. However, as these changes involve post-translational events (Widiastuti et al 2011 doi:10.1016/j.pmpp.2011.04.002, Zhang et al.2023 doi: 10.1128/mbio.01077-23), their characteristics are inherently more variable than those based on DNA.

Comments 9: Conclusion: L382-383- Trichoderma strains TR04 and TR05 show promising potential in controlling B. cinerea infections. If the authors are concluding controlling B. cinerea infections, it suggests that these strains may be applied to reduce the pathogenicity of B. cinerea on walnut. Did you consider applying TR04 and TR05 in a pathogenicity assay to test their effectiveness against B. cinerea? This would provide more promising evidence to support their potential use in future disease management strategies for walnut orchards.

Response 9: Trichoderma TR04 and TR05 also exhibited a high in vitro biocontrol index against other walnut pathogens that cause fruit decay (Botryosphaeria dothidea and Diaporthe eres) (Kovács et al., 2021). This broad-spectrum activity highlights their potential for comprehensive management of various deteriorating fungal pathogens in walnut orchards. This information was added to the results.

 

Minor Comments 1: Comma (,) was missing in references 10 and 6, it should be (10, 6, 13), L 50.

Response MC1: Corrected

 

Minor Comments 2: Similarly, in L144, (3232) please check through the manuscript.

Response MC2: Corrected

 

Minor Comments 3: Table 4: In Colony diameter (mean + SE) should be dot (.), not a comma (,)

Response MC3: Corrected

 

Minor Comments 4: In Figure 4, the statistical letters are missing on the left side figure, and the right side of the figure statistical letters are merging with figure.

Response MC4: There were no statistical differences at Day 3 (left side of the figures), therefore letters were not added there. The position of the letters on the right side was corrected.

 

Minor Comments 5: Figure 8: a) Above the graph title no need to write Husk Scale, as it is already presented on the Y-axis. b) The statistical lettering is merging with the bar graph. Please check carefully in all figures and ensure consistency in distance among the letters and the bar graph.

Response MC5: Figure was corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

1. In figure 7, you have applied the heat shock of 30 degrees. Have you ever tried other higher temperatures with shorter duration time? Would this heat shock affect the pathogenicity or symptom development of Botrytis in walnut?
2. You have tested and proved the growth inhibition triggered by TR4 and TR5 in vitro, have you ever tested it in vivo?
3. In figure 7 and figure 8(b), the significance letter "b" is obscured by or overlaps with the error bar, which may affect clarity.
4. The replication numbers for each statistical analysis were not provided. Please include the number of biological and/or technical replicates used in each experiment.

Author Response

Comments 1: In figure 7, you have applied the heat shock of 30 degrees. Have you ever tried other higher temperatures with shorter duration time? Would this heat shock affect the pathogenicity or symptom development of Botrytis in walnut?

Response 1: B. cinerea thrives at 18–22°C, but high temperatures (≥32°C) inhibit mycelial growth and spore germination. Our findings, consistent with thermotolerance, suggest certain B. cinerea strains might enter into a reversible stress-state, that facilitates recovery and morphological shifts once optimal conditions are re-established. It's worth noting that brief heat shocks (e.g., 50°C for 20 seconds) are reported to trigger plant resistance by activating peroxidase genes and priming defenses without direct fungal inhibition (Widiastuti et al 2011, Zhang et al.2023). While being out of the scope of this current study, this information was added to the Discussion.

 

Comments 2: You have tested and proved the growth inhibition triggered by TR4 and TR5 in vitro, have you ever tested it in vivo?

Response 2: Neither in planta or field trials have been evaluated yet.

 

Comments 3: In figure 7 and figure 8(b), the significance letter "b" is obscured by or overlaps with the error bar, which may affect clarity.

Response 3: Figure was corrected.

 

Comments 4: The replication numbers for each statistical analysis were not provided. Please include the number of biological and/or technical replicates used in each experiment.

Response 4: Five replicates were used in each experiment, and this was indicated in the relevant Materials and Methods chapters.

Reviewer 3 Report

Comments and Suggestions for Authors

This work identified the pathogen of walnut fruit rot disease and explored the growth behavior and pathogenicity of pathogens. The results will deepen the understanding of walnut fruit diseases. My concerns are as follows:

Title

According to the Introduction provided by the authors, there have been reports of Botrytis cinerea as a pathogenic pathogen in walnut fruit. Therefore, the current title cannot accurately summarize the research content

Line 2, “Botrytis cinerea sensu lato” suggests changing to “Botrytis cinerea isolates”.

Abstract

Lines 26-27, It is better to use range of inhibition percentage for four Botrytis cinerea isolates,

Materials and Methods

Line 83, How to disinfect?

Line 97, please add the primer sequence of ITS1 and ITS4.

Lines 86-88, Generally, single spore isolation should be conducted.

Lines 123-124, How many plates were used at each temperature?

Line 131, how to distinguish growth stages BBCH75 and 79. Please provide citation.

Lines 134-139, How many fruits were used for inoculation? How many replicates?

Line 144, Please remove duplicate references.

Lines 146-147, Please explain the sources of the two Trichoderma species or provide references.

Results

Figure 9 and 10, Please check the lesion data for control. Compared to Figure 10, the control lesion in Figure 9 appears to be larger, indicating possible contamination by pathogenic pathogens.

Line 311, Suggest providing representative pictures of the dual culture.

Figure 13, there is no valid information in the figure. Please mark the hyphae of Botrytis cinerea and Trichoderma in the figure, respectively.

Discussion

Generally, pathogen isolation should be carried out in the early stages of the disease, and infectious and non-infectious diseases often occur simultaneously in the later stages. In addition to Botrytis cinerea, the work also isolated Penicillium sp. and Aspergillus sp. Suggest discussing whether these two fungi can cause walnut fruit diseases.

Author Response

Comments 1: This work identified the pathogen of walnut fruit rot disease and explored the growth behavior and pathogenicity of pathogens. The results will deepen the understanding of walnut fruit diseases. My concerns are as follows:

Title

According to the Introduction provided by the authors, there have been reports of Botrytis cinerea as a pathogenic pathogen in walnut fruit. Therefore, the current title cannot accurately summarize the research content

Line 2, “Botrytis cinerea sensu lato” suggests changing to “Botrytis cinerea isolates”.

Response 1: Changed as suggested.

 

Comments 2: Abstract

Lines 26-27, It is better to use range of inhibition percentage for four Botrytis cinerea isolates,

Response 2: Agreed; corrected.

 

Comments 3: Materials and Methods

Line 83, How to disinfect?

Response 3: The description was moved from the previous chapter: ‘Samples were surface-disinfected using the method described by Kovács et al. (2014) to eliminate surface contaminants.’ Further details are now added: ‘A 10% chlorogen-sesquihydrate (Neomagnol; Parma Produkt Ltd., Budapest, Hungary) and 0.1% Tween20 (Merck KGaA, Darmstadt, Germany) solution was employed for one-minute disinfection of the fruit surfaces, followed by two washes in sterile distilled water.’

 

Comments 4: Line 97, please add the primer sequence of ITS1 and ITS4.

Response 4: The primer sequence of ITS1 and ITS4 were added.

 

Comments 5: Lines 86-88, Generally, single spore isolation should be conducted.

Response 5: A more detailed description for isolation was added: ‘To isolate individual fungal strains, hyphal tips were carefully extracted from fungal colonies growing on agar plates. Each tip was then transferred to a fresh potato dextrose agar (PDA, Scharlau, Spain) medium. This process was repeated multiple times to ensure the purity of the fungal cultures (Thi Minh Le  et al 2019).’

 

Comments 6: Lines 123-124, How many plates were used at each temperature?

Response 6: For each isolate, five agar plates were used. This information is now added.

 

Comments 7: Line 131, how to distinguish growth stages BBCH75 and 79. Please provide citation.

Response 7: Citation (Robin at al., 2024) was added.

 

Comments 8: Lines 134-139, How many fruits were used for inoculation? How many replicates?

Response 7: The experiment was conducted with five replicates, and five control walnuts were inoculated with sterile PDA disks. This info is now added to the paragraph.

 

Comments 9: Line 144, Please remove duplicate references.

Response 9: Removed

 

Comments 10: Results

Figure 9 and 10, Please check the lesion data for control. Compared to Figure 10, the control lesion in Figure 9 appears to be larger, indicating possible contamination by pathogenic pathogens.

Figure 9 green walnut stages were at BBCH 75 stage, when  ????…..

Response 10: Thank you for identifying the contradiction in the figures – it was our mistake. We have now modified the images, added the appropriate controls, and revised the figure legends.

 

Comments 11: Line 311, Suggest providing representative pictures of the dual culture.

Response 11:  Pictures of confrontation test showing the antagonistic activity of Trichoderma TR04 and TR05 strains against Botrytis cinerea were added.

 

Comments 12: Figure 13, there is no valid information in the figure. Please mark the hyphae of Botrytis cinerea and Trichoderma in the figure, respectively.

Response 12: Arrows indicate the thin hypha of Trichoderma, and its coiling around the thicker hypha of Botrytis. This information was added to the figure caption.

 

Comments 13: Discussion

Generally, pathogen isolation should be carried out in the early stages of the disease, and infectious and non-infectious diseases often occur simultaneously in the later stages. In addition to Botrytis cinerea, the work also isolated Penicillium sp. and Aspergillus sp. Suggest discussing whether these two fungi can cause walnut fruit diseases.

Response 13: Alternaria, Penicillium, and other fungi (but not Botrytis) have already been detected in symptomatic and asymptomatic walnut kernels in Hungary using the ONFIT method. This information was added to the discussion.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have significantly improved the manuscript; however, some questions remain to be addressed.

Dear authors, I am thankful to you for providing your response and improving the manuscript; however, the remaining comments need to be addressed.

L68, ensuring only freshly fallen ones were gathered. 

Change to: Only freshly fallen fruits were collected

L 96-97. The references (29, 12) have some problems; please correct them.

Table 1: Your heading is: Number of infected fruits, but in L 203, isolation frequency was shown, then at the same time, L204, you cited Figure 2. I can’t see any Botrytis, Aspergillus, and Penicillium isolation frequencies in Figure 2. Please check carefully and cite the correct Table and figure in their correct place. Also clear the mentioned numbers in Table 1 or infected fruites or number of isolates?

L212, why / in 2 and b (Figure 2/b)

Line 236. Eight of 37 B. cinerea isolates, but it is unclear from Table 1 whether these numbers refer to infected fruits or isolates?

L247 B05.10 and T4, and another strain, I didn,t observe T4 in Phylogenetic, and I think it should be T7, as in your figure 5.

L249 Phylogenetic analysis placed our strains within the  B. cinerea sensu lato clade

 This conflicts with your earlier statement in Line 245 that the strains have significant (100%) homology to known B. cinerea sequences, then how can you say your strains are sensu lato? The term sensu lato refers to a broader group, including related genera or species.  If possible, you can partition (Label) the sensu lato clade and  B. cinerea/B. cinerea or Botrytis sensu stricto in the figure for better understanding.

L250: Remove the dot in parentheses with Figure 5

Please adjust distances of your statistical letter and error bar at constant distance in each figure. Check Figure 8b and others. I mentioned also regard this in previous revision .

L259-260 Given that all isolates possessed identical ITS sequences, we propose to test for variations in temperature growth among strains displaying diverse colony morphologies.  If ITS sequences were similar, then why did you rely on ITS? Why didn't you consider other nuclear protein-coding genes or multi-locus sequence typing (MLST i.e., HSP60, B-tubulin, G3PDH, etc) to differentiate between Isolates? (https://doi.org/10.1094/PHYTO-06-15-0143-R)

(doi: 10.1016/j.crmicr.2021.100049).

Figure 12. There is a mismatch in isolates naming as in Figure TR4 and TR5, but in the text, you used TR04 and TR05, please make consistency in the text (Results section 3.5, Discussion and Conclusion) and figure legends.

Figure 13. If you want to provide the antagonistic activity of Trichoderma strains, then why only one isolate, J5021, presented? I suggest you can select the 8-day growth inhibition and show all four isolates for better consistency with Figure 12.

 L387, please correct the duplication of references [42,44,44].

Author Response

Comments 1: Authors have significantly improved the manuscript; however, some questions remain to be addressed.

Dear authors, I am thankful to you for providing your response and improving the manuscript; however, the remaining comments need to be addressed.

Response 1: We appreciate your feedback and will thoroughly address the remaining questions.

 

Comments 2: L68, ensuring only freshly fallen ones were gathered. 

Change to: Only freshly fallen fruits were collected 

Response 2: Changed.

 

Comments 3: L 96-97. The references (29, 12) have some problems; please correct them. 

Response 3: Corrected.

 

Comments 4: Table 1: Your heading is: Number of infected fruits, but in L 203, isolation frequency was shown, then at the same time, L204, you cited Figure 2. I can’t see any Botrytis, Aspergillus, and Penicillium isolation frequencies in Figure 2. Please check carefully and cite the correct Table and figure in their correct place. Also clear the mentioned numbers in Table 1 or infected fruites or number of isolates?

Response 4: We have modified the sentence to clarify that the numbers reflect infected fruits with different fungal genera, and changed the citation to Table 2.

 

Comments 5: L212, why / in 2 and b (Figure 2/b) 

Response 5: Corrected to (Figure 2b) 

 

Comments 6: Please adjust distances of your statistical letter and error bar at constant distance in each figure. Check Figure 8b and others. I mentioned also regard this in previous revision. 

Response 6: Corrected

 

Comments 7: L259-260 Given that all isolates possessed identical ITS sequences, we propose to test for variations in temperature growth among strains displaying diverse colony morphologies.  If ITS sequences were similar, then why did you rely on ITS? Why didn't you consider other nuclear protein-coding genes or multi-locus sequence typing (MLST i.e., HSP60, B-tubulin, G3PDH, etc) to differentiate between Isolates? (https://doi.org/10.1094/PHYTO-06-15-0143-R)

(doi: 10.1016/j.crmicr.2021.100049).

Response 7: ITS sequencing alone cannot resolve intraspecific diversity or distinguish between closely related Botrytis species or lineages, as ITS regions are often highly conserved within the genus. ITS sequencing was chosen for the initial phase because it is a rapid, cost-effective, and widely accepted method for identification and reporting new host-pathogen associations. The immediate goal was to establish the presence of B. cinerea on green walnut and to provide foundational data for the scientific record, with the understanding that more detailed molecular analyses would follow.

As we wrote in the paper: ‘While ITS-based analysis offers preliminary identification, definitive species delimitation and confirmation necessitate a polyphasic approach, involving the sequencing and analysis of additional genetic loci, particularly because bootstrap support is commonly weak in Botrytis phylogenetic trees.’

 

Comments 8: Figure 12. There is a mismatch in isolates naming as in Figure TR4 and TR5, but in the text, you used TR04 and TR05, please make consistency in the text (Results section 3.5, Discussion and Conclusion) and figure legends. 

Response 8: Figure corrected.

 

Comments 9: Figure 13. If you want to provide the antagonistic activity of Trichoderma strains, then why only one isolate, J5021, presented? I suggest you can select the 8-day growth inhibition and show all four isolates for better consistency with Figure 12. 

Response 9: Figure 12 was changed to show the growth inhibition of all four isolates at Day 8.

 

Comments 10: L387, please correct the duplication of references [42,44,44].

Response 10: Corrected.  

Reviewer 2 Report

Comments and Suggestions for Authors

I have no major concerns and recommend it for publication.

Author Response

Comment: I have no major concerns and recommend it for publication.

Reply: Thank you.

Reviewer 3 Report

Comments and Suggestions for Authors

All the letters marked on the bar chart need to be thoroughly checked. Please note that identical letters indicate insignificant differences. For example, C and J5013/3 in Figure 11, as well as Figures 12C and TR5, seem to reach a significant level.

Figure 14 is still unclear and cannot distinguish the hyphae of the two fungi. Suggest replacing the image or removing it.

Author Response

Comments 1: All the letters marked on the bar chart need to be thoroughly checked. Please note that identical letters indicate insignificant differences. For example, C and J5013/3 in Figure 11, as well as Figures 12C and TR5, seem to reach a significant level.

Reply: Since the assumptions of parametric tests (such as normality and homogeneity of variance) were not met, we conducted pairwise comparisons using the non-parametric Mann–Whitney U test. While visual inspection of the error bars suggests apparent differences between groups (most of the p values are very close to the 0.05 threshold), we now include the exact p-values of these comparisons to support validity of our claims.

Fig 11.:

C vs. J513/3 p=0.058

C vs. J5017 p=0.053

Fig. 12.:

J513/3 Day 8: C vs. TR05 p=0.076

J513/3 Day 10: C vs. TR05 p=0.066

J5017 Day 8: C vs. TR05 p=0.054

J5017 Day 10: C vs. TR05 p=0.072

J5011 Day 8: C vs. TR05 p=0.076

J5011 Day 10: C vs. TR05 p=0.066

J5021 Day 8: C vs. TR05 p=0.098

 

The threshold values (p < 0.05) were added to the Figure Legends.

 

Comments 2: Figure 14 is still unclear and cannot distinguish the hyphae of the two fungi. Suggest replacing the image or removing it. 

Reply: We could not improve the resolution, thus the figure is now removed.