Colletotrichum capsici-Induced Disease Development in Postharvest Pepper Associated with Cell Wall Metabolism and Phenylpropanoid Metabolism

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The study “Colletotrichum capsici-induced disease development in postharvest pepper associated with cell wall metabolism and phenylpropane metabolism” presents a valuable and well-structured contribution to the understanding of postharvest disease mechanisms and plant defense responses in pepper fruit.

I have carefully read through the manuscript and provided several suggestions aimed at improving clarity and reproducibility. All suggestions have been entered in the manuscript as comments and can be tracked directly in the document itself. The comments have been inserted directly into the manuscript file using the comment function.

I hope that these suggestions will be helpful in refining your manuscript, and I commend you for your efforts in advancing this area of research.

Comments for author File: Comments.pdf

Author Response

Comments 1: line 70: Please insert the full Latin name, Diaporthe citri, since this is the first time this organism is mentioned in the manuscript.

Response 1: Done.
Comments 2: line 142: Please specify the brand and model of the centrifuge used.

Response 2: Done.
Comments 3: line 147: Please specify the type of apparatus used for cutting the sample sections (e.g., microtome).

Response 3: The cutting instrument we used is stainless steel blade. And we have noted in the manuscript.
Comments 4: line 152: Please specify the type and model of the microscope used for analyzing the pepper sections, as well as the magnification applied during observation.

Response 4: Done.

Comments 5: line 156: Again, please specify the type and model of the centrifuge used.

Response 5: Done.

Comments 6: line 160: Please cite the relevant reference for the anthrone method used.

Response 6: Done.

Comments 7: line 168: It is not usual to begin a sentence with a numeral. Please revise the sentence so that it begins with text instead.

Response 7: Done.
Comments 8: line 169: Again, the same comment applies regarding the type and manufacturer of the centrifuge.

Response 8: Done.

Comments 9: line 171: Please, provide the complete centrifugation details, including the rotational speed (rpm or ×g) and the duration (in minutes).

Response 9: Done.

Comments 10: line 173: Kindly provide the complete centrifugation details, including the rotational speed (rpm or ×g) and the duration (in minutes)

Response 10: Done. The centrifugation mentioned in the Line 173, in fact, was referred in Line 171.

Comments 11: line 188: Again, the same comment applies regarding the type and manufacturer of the centrifuge.

Response 11: Done.

Comments 12: line 234: Make sure the species name are italicized

Response 12: Done.

Comments 13: line 249: Please provide more detailed information regarding the images obtained using the scanning electron microscope, including the magnification settings applied during imaging.

Response 13: We have reloaded the clearer picture with a scale label and magnification in the manuscript.

Comments 14: line 268: please add a note explaining what the asterisks represent in terms of statistical significance (e.g., P < 0.05, P < 0.01).

Response 14: Done.

Comments 15: line 282: Please add a note explaining what the asterisks represent in terms of statistical significance (e.g., P < 0.05, P < 0.01).

Response 15: Done.

Comments 16: line 300: Please include the magnification and the specific method used for staining the histological sections. Additionally, the scale bars in the images are difficult to see. Kindly improve their visibility and ensure they are presented in an appropriate and standardized format.

Response 16: We have reloaded the clearer picture with a scale label and magnification in the manuscript.
Comments 17: Tline 303: Please correct the spelling.

Response 17: Done.

Comments 18: line 308: Please correct the spelling.

Response 18: Done.

Comments 19: line 312: Please add a note explaining what the asterisks represent in terms of statistical significance (e.g., P < 0.05, P < 0.01).

Response 19: Done.

Comments 20: line 340: For better clarity, I suggest writing out the full names of all enzymes analyzed, along with their corresponding abbreviations.

Response 20: Done. We supplemented the full names of the enzymes along with their corresponding abbreviations in the part of the Figure name.

Comments 21: line 341: Please add a note explaining what the asterisks represent in terms of statistical significance (e.g., P < 0.05, P < 0.01).

Response 21: Done.

Reviewer 2 Report

Comments and Suggestions for Authors

Please see the attachment.

Comments for author File: Comments.pdf

Author Response

Comments 1: In the specialist literature, C. gloeosporioides and C. acutatum are often considered to be the more common and aggressive agents of anthracnose in peppers on a global scale. The claim that C. capsici is the "main" pathogen may be true locally (e.g., in China), but it is too generalized.

Response 1: Thank you for your suggestion. We have rewrote this sentence in the text.

Comments 2: Some enzymes involved in lignin biosynthesis, such as LAC and CAD, can indeed be activated as a defensive reaction. However, the statement that the pathogen itself "accelerates lignin deposition" suggests that it is the pathogen that initiates the defense response, which is imprecise—it is the host (pepper) that triggers these pathways as an immune mechanism.

Response 2: Thank you for your insightful comment. We agree with your observation that the defensive response, including lignin deposition, is initiated by the host (pepper) and not directly by the pathogen itself. Rather, the pathogen indirectly stimulates this response by triggering the host’s defensive system. In the revised abstract, we have clarified that C. capsici infection acts as a stimulus that induces lignin deposition in the host tissue, rather than actively "accelerating" the process itself.

Comments 3: Not every increase in phenols and flavonoids means effective defense. Sometimes these are secondary effects of oxidative stress, rather than a deliberate defense of the plant. This would require confirmation that this actually translates into a reduction in the development of the pathogen.

Response 3: Thank you for raising this important point. We fully agree that not all increases in phenols and flavonoids compounds necessarily reflect effective defence, as such changes may also result from oxidative stress or general metabolic disturbance during pathogen infection. In our study, the early-stage accumulation of total phenolics and flavonoids was accompanied by enhanced activity of key enzymes in the phenylpropanoid pathway (e.g., PAL, C4H, 4CL), as well as the lignin biosynthesis-related enzymes CAD and LAC, suggesting a coordinated activation of host defense responses.

However, we acknowledge that this biochemical evidence alone does not directly confirm functional resistance or pathogen suppression. Future studies involving pathogen quantification, histochemical localization, and possibly gene expression profiling (e.g., of PR proteins or lignin pathway genes) would be necessary to determine whether the observed metabolic changes translate into effective restriction of C. capsici development. We have added a sentence to the abstract to clarify this limitation and suggest this as an important direction for further research.

Comments 4: There is no distinction whether these enzymes are of plant origin (e.g. the ripening process) or fungal (pathogen). In fact, it is the fungal enzymes, not the plants, that are mainly responsible for the breakdown of the cell wall during infection.

Response 4: We appreciate the reviewer’s insightful observation. We agree that distinguishing the origin of cell wall-degrading enzymes—whether from the host plant (e.g., during ripening or stress response) or from the invading pathogen—is crucial for accurately interpreting the mechanism of cell wall disassembly during infection. In our study, the measured enzymatic activities (e.g., PG, PME, Cx, β-glucosidase) represent the total activity in the tissue. Based on the timing of infection, we speculated that in the early stage, enzyme activity is primarily host-derived, and the pathogen is still establishing itself. As the infection progresses and pathogen colonization intensifies, the contribution of pathogen-derived enzymes likely increases. We acknowledge this as a limitation of the current study, and further research is needed to accurately distinguish the source of these enzymes.

Comments 5: Cellulase (Cx) is not a pectolytic enzyme—it is an enzyme that breaks down cellulose, not pectin.

Response 5: We appreciate the reviewer’s correction regarding enzyme classification. You are right that cellulase (Cx) is not a pectinolytic enzyme but rather a cellulolytic enzyme that explicitly targets cellulose. In the revised manuscript, we have corrected this misclassification and now refer more broadly to “cell wall-degrading enzymes”, which include both pectinolytic enzymes (e.g., PG, PME) and cellulolytic enzymes (e.g., Cx).

Comments 6: An increase in soluble pectin and a decrease in fiber do not always directly cause a "deterioration"; rather, it is associated with a loss of firmness, but "deterioration" is too general a term and can be misleading.

Response 6: We thank the reviewer for pointing out the imprecise use of the term “deterioration”. We agree that this term is too broad and may be misleading in the context of the cited study, which primarily addressed changes in texture and firmness during postharvest storage. We intended to refer specifically to softening resulting from alterations in cell wall polysaccharides. In the revised manuscript, we have replaced “deterioration” with more precise terms, such as “textural softening” to reflect better the specific physiological changes discussed in the referenced study.

Comments 7: Alternaria alternata can infect a variety of tissues, but its mechanism of action depends on the plant species and environmental conditions — this statement is too general.

Response 7: Thank you for your constructive comment. We agree that the mechanism of Alternaria alternata infection varies depending on host species and environmental conditions. In our manuscript, we addressed this complexity by illustrating one specific aspect of its pathogenic mechanism—namely, the induction of cell wall-related enzyme activity in cantaloupe tissue.

Comments 8: Diaporthe citri (D. citri) is indeed a disease agent, but "phomopsis rot" is a common name, not a precise botanical term. In addition, attributing this disease only to the tips of the stems is a simplification.

Response 8: Thank you for this thoughtful comment. The phrase “Phomopsis stem-end rot caused by Diaporthe citri” was used in reference to the original literature (Chen et al., 2021. Cell wall modification and lignin biosynthesis involved in disease resistance against Diaporthe citri in harvested pummelo fruit elicited by carvacrol),

 where this terminology was employed. However, we agree that it is a common name and may not precisely describe the full range of symptoms or the site of infection.

To improve clarity and botanical accuracy, we have revised the sentence in our manuscript to describe the disease as “Diaporthe citri-induced rind softening and tissue degradation”, avoiding both the use of the common name and the implication that symptoms are limited to the stem-end.

Comments 9: Only PAL is classically recognized as a rate-limiting enzyme in this pathway. C4H and 4CL are important, but not always limiting.

Response 9: We thank the reviewer for this important correction. We agree that among these enzymes, PAL is classically recognized as the rate-limiting enzyme in the phenylpropanoid pathway, whereas C4H and 4CL are essential components but not necessarily rate-limiting under all conditions. In the revised manuscript, we have amended the sentence to reflect this distinction more accurately and avoid overgeneralization.

 Comments 10: C. capsici is a fungus (Colletotrichum capsici), not a bacterium – "bacteriostatic" agents are a misnomer here.

Response 10: We appreciate the reviewer’s correction. You are absolutely right—Colletotrichum capsici is a fungal pathogen, not a bacterium. The use of the term “bacteriostatic agents” in our original text was incorrect. In the revised manuscript, we have replaced “bacteriostatic agents” with the more appropriate term “antifungal agents” to accurately reflect the nature of the pathogen and the direction of potential control strategies.

Comments 11: The text lacks information about the exact number of biological repeats (replications) – it is not known whether "three copies" means three biological repeats or only technical ones. It should be specified whether each batch of 30 fruits was treated as a biological unit.

Response 11: We thank the reviewer for this important comment. The “three replicates” mentioned in the manuscript refer to three biological replicates, each consisting of 30 independently treated fruits. Each group of 30 fruits was treated and analyzed as a biological unit. We have revised the relevant sentence in the manuscript.

Comments 12: The description does not mention the use of a sputter coater – it only says "spraying with gold foil", which is not technically correct.

Response 12: We thank the reviewer for this accurate observation. We acknowledge that the phrase “spraying with gold film” is technically incorrect. The samples were, in fact, gold-coated using a sputter coater, as per standard SEM preparation procedures. We have revised the sentence in the manuscript accordingly to reflect this correct terminology.

Comments 13: The antron method is mainly used to determine the total sugar content, and not to clearly distinguish between cellulose and hemicellulose. Therefore, using this technique to label them separately can lead to inaccurate results.

Response 13: Thank you for this important comment. We acknowledge that the anthrone method is a colorimetric assay primarily used to estimate total carbohydrate content and may not accurately distinguish between cellulose and hemicellulose. In our study, we followed the protocol described by Wang et al. (Scientia Horticulturae 2021), which applied anthrone-based detection after specific fractionation steps to estimate cellulose and hemicellulose content separately. Additionally, similar approaches have been employed in other published studies, such as Li et al. (Biomolecules, 2019) and Wang et al. (Food Chemistry, 2015), which also applied anthrone-based to estimate cellulose and hemicellulose.

Comments 14: The description of "amount of carboxymethylcellulose to reducing sugar" is imprecise – it is not stated exactly what reducing sugar is measured (e.g. glucose).

Response 14: Thank you for your helpful comment. We agree that the original description of cellulase (Cx) activity was imprecise. In our assay, cellulase activity was evaluated by measuring the amount of reducing sugar released from carboxymethyl cellulose (CMC), using glucose as the standard in a DNS (3,5-dinitrosalicylic acid) colorimetric assay. One unit of enzyme activity was defined as the amount of enzyme required to release 1 mg of glucose equivalent per hour per gram of fresh weight (FW). We have revised the sentence in the Materials and Methods section to clearly reflect this definition.

Comments 15: It is not stated whether and which standard was used for calibration (e.g. p-NP-chitosided or laminarin) or incubation parameters (time, pH), which makes it impossible to reproduce the results.

Response 15: We thank the reviewer for pointing out this important omission. In order to reduce the replication ratio of the article, we have briefly described the experimental method. We are very sorry for the inconvenience caused to the readers. In our experiment, chitinase (CHI) activity was determined using colloidal chitin as the substrate. The reaction mixture contained 0.5 mL of enzyme extract and 0.5 mL of 10g /L colloidal chitin in 0.1 mol/L sodium acetate buffer (pH 5.2), incubated at 37°C for 1 hour. And then 0.1 mL 30 g/L desalted snailase was added. After incubation at 37 ℃ for 1 h, the amount of NAG released was measured colorimetrically at 585 nm.

For β-1,3-glucanase (β-1,3-Glu) activity, laminarin was used as the substrate, and reducing sugars were quantified using the DNS method, with glucose as the standard. The reaction system contained 20 μL of enzyme extract and 20 μL of 4 g/L laminarin in 0.1 mol/L sodium acetate buffer (pH 5.2), incubated at 37°C for 30 min.

We have now included these experimental details in the revised manuscript to ensure reproducibility of the enzyme assays.

Comments 16: Oversimplification – in fact, pepper fruits can show natural changes related to the ripening and aging process even under control conditions, regardless of the pathogen. A six-day total surface stability is unlikely without additional data on storage conditions.

Response 16: Thank you for this insightful comment. In our study, we focused on the growth of visible disease lesion near the wound site and initially overlooked the potential physiological changes associated with natural ripening and senescence in the absence of pathogen infection. Although no visible disease symptoms were observed in the control group during the first 6 days of storage, we acknowledge that subtle biochemical and physiological changes likely occurred.

We have revised the relevant description in the Results section to clarify that the "intact surface" refers only to the absence of visible lesions, and that natural aging processes may still be taking place. This distinction improves the accuracy of our observations.

Comments 17: Incorrect assignment of the species to a specific disease — Colletotrichum capsici is one of the possible culprits of anthracnose, but in peppers more typical are, for example, Colletotrichum acutatum or C. gloeosporioides. In the literature, C. capsici is mainly found in the context of other plants and regions.

Response 17: Thank you for this important observation. We agree with the reviewer that Colletotrichum acutatum and C. gloeosporioides are more frequently reported as the primary causal agents of anthracnose in pepper worldwide. While C. capsici has been reported to have a broad host range, it may not be the dominant species cases in specific regions, including parts of Asia. In our study, based on experimental results, we found that inoculation with C. capsici rapidly induced typical postharvest anthracnose symptoms in peppers, severely affecting visual appearance and storage quality. To avoid overgeneralization, we have revised the relevant statements.

Comments 18: Insufficient control of variables — the described hardness of fruit could have been disturbed not only by the pathogen, but also by storage conditions (temperature, humidity, mechanical damage), which were not taken into account here.

Response 18: We appreciate these helpful comments. We agree that multiple factors, including temperature, humidity, and mechanical injury, can influence postharvest fruit firmness. In our study, all fruits were stored under the same controlled environmental conditions (28 ± 1 °C, 95 ± 5% RH). Standardized mechanical wounding was applied to both the control and treatment groups to minimize variability. Therefore, the observed decline in firmness is primarily attributed to C. capsici infection under these consistent conditions.

Comments 19: The wording suggests that C. capsici has the fruit, which is incorrect. It should read: “offended fruit infected with C. capsici” or "in pepper fruits infected with C. capsici".

Response 19: We agree that the phrase “C. capsici fruits” was inappropriate. We have revised the wording to “C. capsici-treated fruits” throughout the manuscript to ensure scientific accuracy.

Comments 20: It is expected that immediately after inoculation (i.e. on day "0") there will be no significant changes — this is not substantively valuable information. Scientific analysis should focus on the differences at a later time.

Response 20: We thank the reviewers for these constructive comments. We agree that the absence of structural differences at day 0 is an expected result. However, we included the 0 d microstructural images as a baseline reference to illustrate a clear comparison with the progression of cellular damage observed in later stages.

 Comments 21: Not every C. capsici infection leads to permanent destruction of the fruit—much depends on the stage of disease development, storage conditions and the plant's immune response

Response 21: We agree that not every C. capsici infection results in the irreversible destruction of pepper fruit. The severity of tissue damage depends on multiple factors, including the infection stage, storage conditions, and the host’s immune response. Accordingly, we have softened the language in our manuscript to reflect that the damage observed in this study occurred under specific conditions and may not be universally applicable.

Comments 22: The statement that only on the first day the level of protopectin was lower in the infected sample is problematic. From a biological point of view, infection most often leads to the rapid breakdown of protopectin — not just on the first day. This statement suggests that infection inhibits decay, which is contrary to the general knowledge of the pathogenesis of necrotrophic fungi.

Response 22: Thank you for your insight suggestion. We acknowledge that our wrong previous expression may have led to confusion regarding the trend of protopectin content. In fact, the content of protopectin decreased throughout the storage period in both the control and the inoculated fruit. Only on the first day, the content of protopectin in the inoculated fruit was slightly higher than that in the control fruit. We have revised the manuscript to accurately reflect this trend and avoid any misinterpretation.

Comments 23: Changes in cellulose, hemicellulose and pectin content with lignin without logical justification. Lignin has a different structural function than these polysaccharides and its changes do not necessarily reflect the same degradation mechanisms.

Response 23: Thank you for this important comment. We agree that lignin and polysaccharides such as cellulose, hemicellulose, and pectin serve different structural and functional roles within the plant cell wall. Our intention was not to imply that they undergo degradation through the exact mechanisms but rather to highlight that C. capsici infection affects multiple components of the cell wall matrix and the changing trend between them.

Comments 24: Values for enzymatic activities (e.g., PG, Cx, β-Glu) are presented without reference to the calibration and standardization method—suggesting an accuracy that may not be there. For example, a value of 111.75 mg/h·g for PME appears unusually high and may indicate a unit error or no conversion.

Response 24: Thank you for your thoughtful comment. In order to reduce the replication ratio of the article, we have briefly described the experimental method. However, we operate strictly following the methods of the references. When determining PME, we defined the enzyme activity unit by the production amount of galacturonic acid. On the sixth day of storage, the PME activities in both the control group and the treatment group significantly increased, with the treatment group reaching 111.75 mg/h·g. It was speculated that PME might play a crucial role in the modification of cell wall components in peppers. 

Comments 25: The claim that enzyme activity dropped sharply by day 9, but at the same time continued to increase steadily in the control group for PG – is inconsistent. Since the infection leads to degradation, there should be no sharp drop in enzyme activity as early as day 9 if the infection continues to progress. 

Response 25: We appreciate this insightful observation. The observed sharp decline in enzyme activities (particularly PG and PME) by day 9 in the infected group may be attributed to several factors. First, extensive tissue maceration at late infection stages may lead to substrate depletion and enzyme inactivation. Second, cell wall disintegration may reach a point where measurable enzyme activity becomes reduced due to cellular collapse or dilution effects. In contrast, the control fruit retained structural integrity, allowing a more gradual increase in PG activity. Additionally, this trend is consistent with previous studies by Chen et al. (2018) and Li et al. (2019), who also observed a peak and subsequent decline in cell wall-degrading enzyme activity during the late stages of infection or senescence.

Comments 26: The statement that the lignin content in the infected group decreases after day 6 is problematic. Lignification is a process associated with the defense response and is usually increased in the presence of biotic stress, so the decrease may indicate tissue degradation rather than a defense mechanism effect — which has not been adequately accounted for.

Response 26: Thank you for your insightful comment. We agree that lignification is typically considered a hallmark of plant defense against biotic stress. In our study, the lignin content in the C. capsici-infected group increased during the early storage period but declined significantly after day 6. This decline may not indicate a suppression of the defense response, but rather reflect advanced tissue degradation caused by pathogen progression. At this stage, the loss of cellular integrity and enzyme inactivation could impair both the biosynthesis and retention of lignin.

Comments 27: The passage about staining showing more intense staining in the infected group also suggests that mechanical injury is the main cause of lignin synthesis. This blurs the line between infection and mechanical damage, even though the plants' defenses differ in these cases. 

Response 27: We thank the reviewer for this important observation. We agree that the mechanisms of lignin induction differ between mechanical injury and pathogen infection. In our experimental design, both control and C. capsici-treated fruits were subjected to standardized wounding to simulate natural infection sites. Therefore, the control group represents the response to mechanical injury alone. In contrast, the infected group reflects the combined effects of wounding and pathogen-induced stress.

Comments 28: The formulation that the decrease in flavonoid content in the infected group is "less pronounced", without a statistical indicator or unit of change, makes it difficult to assess the significance of this phenomenon. The use of a qualitative term in a place that requires quantitative interpretation is a research error

Response 28: We appreciate the reviewer’s comment. We acknowledge that the phrase “less pronounced”was a qualitative description and lacked statistical support.  In the revised manuscript, we have replaced this wording with quantitative data and, where applicable, included statistical comparisons (P-values) to indicate the differences in flavonoid content between the C. capsici-infected and control groups.

Comments 29: The notation that the pathogen "induces" an increase in the total content of phenols and flavonoids is a simplification. This increase can be due to a number of factors: oxidative stress, hormonal signals, local damage — not necessarily directly from the presence of C. capsici. A more cautious interpretation and reference to possible indirect mechanisms is advisable.

Response 29: Thank you for this insightful comment. We agree that the phrasing “the pathogen induces an increase in phenols and flavonoids” may be an oversimplification of the underlying biological processes. The accumulation of these compounds in response to C. capsici inoculation could result from a combination of factors, including oxidative stress, hormonal signaling, and localized tissue damage. These responses may not be caused directly by the pathogen itself but rather by the host’s perception of biotic stress. To reflect this complexity, we have revised the manuscript to use more cautious language and to acknowledge the possibility of indirect regulatory mechanisms.

Comments 30: Such a drastic decrease in enzymatic activity (PAL) in the short term (from 131 U/g to 17.7 U/g) is biologically unlikely without simultaneous indication of strong stress factors, e.g. necrosis or necrosis. There is no physiological justification for such a course.

Response 30: Thank you for your valuable comment. We agree that the sharp decline in PAL activity from 131 U/g to 17.7 U/g appears biologically dramatic. However, this result was consistently observed in all biological replicates and confirmed across independent experiments. The observed decline may be attributed to severe tissue degradation caused by C. capsici infection at later stages of the disease. As cellular integrity breaks down, enzymatic activity may be compromised due to enzyme inactivation, loss of substrate availability, or necrosis-associated metabolic suppression.

To address this concern, we have revised the manuscript to include a discussion of these possible mechanisms and to clarify that such a drastic decline likely reflects the combined impact of pathogen progression and loss of host viability rather than a simple regulatory downshift.

Comments 31: The M-shaped description of fluctuations is not a scientific way of presenting results – it is subjective, imprecise and not based on standardized statistical analysis. Describing enzymatic trends should be based on quantitative parameters (e.g. maximum and minimum values, significance of differences).

Response 31: We thank the reviewer for these important observations. We acknowledge that the use of the term “M-shaped” to describe enzymatic activity patterns is subjective and lacks quantitative clarity. In the revised manuscript, we have removed such descriptive language and instead reported the actual activity values, along with their corresponding time points and statistical differences where applicable. 

Comments 32: The statement of “significantly higher” enzyme activity (CHI) does not refer to a specific p-value or statistical test. There is no indication whether this difference is statistically significant or only biologically noticeable.

Response 32: We thank for the valuable comment. Regarding CHI activity, we have clarified that the differences observed between the control and C. capsici-treated groups in the mid-to-late stages were statistically significant (P < 0.05), as determined by one-way ANOVA followed by Duncan’s multiple range test. The revised text now includes these statistical details to support our interpretation.

Comments 33: Phenols in many species of vegetables and fruits usually decrease during storage as a result of oxidation and degradation. A positive correlation between time and phenolic content can be unusual or misinterpreted.

Response 33: Thank you for the valuable comment. We agree that in many fruit and vegetable species, total phenolic content tends to decline over time due to enzymatic oxidation or degradation. However, in our study, we observed a consistent increase in phenolic levels, particularly in the C. capsici-infected group. This may reflect a host-induced stress response to pathogen invasion, which transiently activates phenylpropanoid metabolism. In contrast, the total phenolic content in the control fruitsdid not increase continuously. As shown in Fig. 6A, there was a significant increase during the first three days, followed by a relatively stable phase.

Comments 34: The comparison of “resistance to disease” as inversely correlated with firmness has no physiological basis — usually fruits with greater firmness show greater mechanical and biochemical resistance to infections.

Response 34: We appreciate the reviewer’s insightful comment. We acknowledge that this was a writing error in the original text. The intended meaning is that fruit firmness is negatively correlated with the disease index; that is, fruits with lower firmness are generally more susceptible to pathogen infection due to weakened structural barriers. We have revised the text accordingly to reflect that firmness is positively associated with disease resistance. 

Comments 35: β-Glu (β-glucosidase) is not always clearly classified as an enzyme that degrades the cell wall in the context of fruit tissues — its main role may be related to chemical defense or metabolism of phenolic glycosides.

Response 35: We thank the reviewer for the insightful comment. We acknowledge that its role extends beyond cell wall degradation and may also involve chemical defense and the metabolism of phenolic glycosides. We have updated the text to reflect this broader functional context and removed β-Glu from the strict “cell wall degradation enzyme” category.

Comments 36: A decrease in the disease index should not be interpreted as a "negative PC2 axis" without showing the specific contribution of the variable. Causality can be mistakenly equated with correlation.

Response 36: Thank you for pointing this out. We fully agree that PCA is a statistical technique that reveals covariation patterns and does not imply causality. Our original statement suggesting that a lower disease index “corresponds” to the negative axis of PC2 may have inadvertently implied a cause-effect relationship.

We have revised the manuscript to clarify that the disease index exhibits a negative loading on PC2, reflecting its inverse covariation with other variables on the positive side of PC2, such as lignin content and defense enzyme activities. We now describe this relationship more cautiously using neutral language (e.g., “negatively associated with”), and we avoid interpreting the directionality of PCA axes as causal.

Comments 37: PCA does not assign unambiguous "biological functions" to its constituents—the assignment of PC2 to "immunity" is arbitrary. PCA shows covariability, not cause-and-effect relationships.

Response 37: Thank you for this insightful comment. We agree that PCA is a data-driven technique that reveals covariation patterns among variables, and it does not assign inherent biological functions to the principal components. Our earlier phrasing suggesting that PC2 is “defined as” a disease resistance component was imprecise and may have implied a level of mechanistic interpretation that PCA does not provide. We have revised the text to avoid assigning fixed biological labels to the components.

Comments 38: If the control group shows symptoms of disease, other sources of infection or errors in the sterility of the experiment should be considered. Otherwise, the results on the effects of C. capsici are difficult to substantiate.

Response 38: Thank you for pointing this out. We acknowledge that the appearance of disease-like symptoms in the control group on day 9 may raise questions about experimental sterility or potential background infection. To clarify, all fruits were handled under sterile conditions during inoculation and storage, and no deliberate inoculation was applied to the control group. We believe that the symptoms observed on the final day in the control group likely reflect natural senescence and possible opportunistic microbial colonization due to prolonged storage under high humidity. We have revised the manuscript to make this point more straightforward and to avoid misinterpretation of the control results as C. capsici-specific effects.

Comments 39: The phenomenon of the breakdown of the cell wall structure is a metaphorical simplification. In fact, it is a process of gradual enzymatic degradation and remodeling. This is not a sudden phenomenon, but a long-term change.

Response 39: We appreciate the reviewer’s observation. We agree that describing the process as a “collapse” of the cell wall structure may be metaphorical and oversimplified. In reality, cell wall degradation during pathogen infection and fruit senescence is a gradual and enzyme-mediated process involving polysaccharide remodeling and depolymerization. We have revised the wording in the manuscript to reflect this continuous nature of the process.

Comments 40: The description suggests that only these two components form the original wall. In fact, pectins also play an important role in the structure of the primary wall, which are crucial for its porosity, elasticity and interaction with external factors.

Response 40: We appreciate the reviewer’s observation and fully agree that pectins are integral components of the primary cell wall, alongside cellulose and hemicellulose. While our initial wording may have inadvertently suggested that only cellulose and hemicellulose are involved, we acknowledge that pectins play a crucial role in determining wall porosity, hydration, elasticity, and cell-cell adhesion. We have revised the relevant sentence in the manuscript to more accurately reflect the composition and structural function of the primary wall.

Comments 41: Monolignols do not "fill" the wall space – they are polymerized in situ, forming lignin as a result of an oxidative process. The wording is imprecise – lignin is not "added" like cement, but is formed from monomers at the site of their deposit.

Response 41: We thank the reviewer for this valuable clarification. We agree that the previous wording was imprecise. Lignin is not physically “filled into” the wall space; instead, it is synthesized through the oxidative polymerization of monolignols that are deposited into the cell wall matrix. This in situ polymerization reinforces the wall structure, particularly during secondary wall formation or under biotic stress. We have revised the relevant sentence to reflect better the biochemical process involved in lignification.

Comments 42: Lignification does indeed strengthen cell walls, but it also has side effects, such as reduced transport of water and nutrients in the affected tissue. This is not an unequivocally beneficial process—excessive lignification can have a negative effect on the physiology of the fruit.

Response 42: Thank you for this important observation. We agree that lignification, while acting as a defense mechanism that reinforces cell walls and limits pathogen spread, is not unequivocally beneficial. Excessive lignin deposition can negatively impact fruit physiology by impeding the transport of water and nutrients, reducing tissue elasticity, and potentially accelerating senescence. Nonetheless, given the scope of our study, we have focused on the defensive role of lignification against C. capsici infection. We acknowledge, however, that its physiological trade-offs warrant further study.

Comments 43: Quinine is not the name given to the oxidation products of phenols or flavonoids. Quinine is a specific compound (quinoline derivative), not a product of phenolic oxidation in plants. This is probably a terminological mistake – the author meant quinones, which can be toxic.

Response 43: We thank the reviewer for catching this important terminological error. We indeed intended to refer to quinones, not quinine. Quinones are oxidative products of phenolic compounds and flavonoids, often formed under the action of polyphenol oxidases, and are known for their antimicrobial properties. We have corrected this mistake in the revised manuscript. 

Comments 44: It is not reported that the activity of these enzymes may vary strongly depending on the location of the infection and that the increase in their activity may be due to other signalling mechanisms (e.g. H₂O₂, SA, JA, ET) and not only the presence of the pathogen.

Response 44: We appreciate the reviewer’s insightful comment. We agree that the activity of defense-related enzymes such as β-1,3-glucanase and chitinase can vary depending on the localization of infection (e.g., at the site of pathogen entry versus distal tissues). Additionally, their induction is often regulated not only by direct pathogen presence but also by complex signaling pathways involving endogenous molecules such as hydrogen peroxide (H₂O₂), salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). In our study, we focused on the overall enzymatic activity in the tissue surrounding the inoculation site; however, we acknowledge that a more detailed spatial and signaling analysis would be valuable for future investigations.

Comments 45: The organism C. capsici is not generally recognized as a bacterial pathogen, but as a fungus-like organism (it belongs to the genus Colletotrichum, which includes plant pathogens of a fungal nature). Therefore, attributing to it characteristics typical of pathogenic bacteria may be misleading.

Response 45: Thank you for pointing out this important taxonomic clarification. We fully agree that Colletotrichum capsici is a fungal pathogen, not a bacterial one. In the revised manuscript, we have corrected the wording accordingly to avoid potential misinterpretation.

Comments 46: Although the increase in the activity of enzymes such as PG (polygalacturonase) and PME (pectinomethylesterase) does indeed affect the degradation of the pectin structure, not all of these enzymes directly break down each of these components. For example, β-glucosidase is not the main enzyme that breaks down cellulose — cellulases (EG, CBH) are mainly responsible for this process. Such simplification can lead to a distorted picture of cell wall degradation pathways.

Response 46: Thank you for this important clarification. We agree that the specific roles of cell wall–modifying enzymes should be more accurately described. While PG and PME are directly involved in pectin degradation, β-glucosidase primarily participates in the hydrolysis of glycosidic bonds in oligosaccharides and phenolic glycosides rather than in cellulose degradation per se. The main enzymes responsible for cellulose breakdown are cellulases, including endoglucanases (EG) and cellobiohydrolases (CBH). We have revised the relevant sentence in the conclusion to reflect this distinction more precisely and to avoid oversimplification of the enzymatic pathways involved in cell wall disassembly.

Comments 47: Emphasizing that infection with the pathogen leads to "strengthening" of the cell wall structure contradicts the earlier paragraphs of the text, which spoke of the degradation of this structure. Such contradictory information within the same text may suggest a lack of a clear definition of the infection phase, or an incorrect understanding of the mechanisms of the plant's response.

Response 47: Thank you for this valuable observation. Without a clear temporal context, the coexistence of cell wall reinforcement and degradation may appear contradictory. Our data suggest a two-phase response. During the early stage of C. capsici infection, the pepper fruit initiates structural reinforcement mechanisms— such as increased lignin deposition and a transient elevation of cellulose and protopectin—to limit pathogen invasion. However, as the infection progresses, these defenses are overwhelmed, leading to enhanced activities of cell wall-degrading enzymes (PG, PME, Cx), which accelerate pectin and cellulose breakdown and ultimately weaken the structural integrity of the cell wall.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript horticulturae-3681653 has been meticulously prepared, thereby evidencing the authors' substantial expertise in the subject matter. Although I did not find significant factual errors, I do see minor inaccuracies. Below is a detailed list of identified issues. Their elimination will improve the quality of the paper.

1. The authors used terms “phenylpropane metabolic pathway” and “phenylpropane metabolism” in the title, key words and many places in the manuscript. I would also consider using the much more common term "phenylpropanoid metabolism", especially among the keywords.
2. The authors wrote: “For instance, during pathogen invasion, plants trigger the phenylpropane metabolic pathway, producing secondary metabolites such as phenolic compounds, flavonoids, and lignin to prevent the spread of pathogens[19]”. I would modify this sentence to: “For instance, during pathogen invasion, plants trigger the phenylpropane metabolic pathway, producing secondary metabolites such as phenolic compounds, including flavonoids, and lignin to prevent the spread of pathogens[19]” because flavonoids are also phenolic compounds.
3. When describing the methodology for determining lignin content, the authors wrote: “The final results were expressed as OD₂₈₀ nm/g”. It is not entirely clear in what units the lignin content was expressed. I understand that the authors measured absorbance at 280 nm. First, the abbreviation “nm” should be in subscript. Second, would not it be better to write that it is absorbance, not optical density, which is nowadays most often referred to the measurement of microbial counts, since light scattering is then measured? Perhaps “The final results were expressed in absorbance units, A_{280 nm}/g” would be more clear?
4. The D graph of Figure 3 is more narrow in comparison to the other graphs. Its width should be adjusted to match the others.
5. The graphs of Figure 4 are shifted to the left. Authors should pay attention to whether they will be properly aligned in the final version of the publication.
6. The authors should check whether the resolution of Fig. 8 is high enough. In the attached manuscript for review, its resolution is not satisfactory.
7. In Figure 9 (I like the humorous approach), in the phrases “total phenolic” and “flavonoid,” I would use the words in the plural.
8. The semicolon on line 12 is definitely too big.
9. The name of the enzyme (β-1,3-Glucanase) inside the sentence should be written starting with a lowercase letter (lines 23 and 174).
10. On line 51, the conjunction “and” should be written in a simple font.
11. On lines 114, 267, 300, and 340, the Latin name of the fungus is missing a space after
12. In methodology, I would include the noun “pericarp” or “fruit” (line 141) to the phrase “pepper tissue.”
13. On line 149, in the phrase, ”(…) stained with 1% (w/v) phloroglucinol ethanol…”, a word “in” is missing.
14. On line 219, there is capsici -treated instead of C. capsici-treated. The extra space should be removed.
15. On line 221, the Latin name of the fungus should be italized.
16. In most cases, the letter denoting the significance level (P) is correctly italicized. However, the authors forgot to italicize the P in line 234 (twice) and in line 274.
17. On line 265, the Latin name of the fungus is missing a space, while the space before the comma should be removed in the rest of the sentence.
18. On line 303, there is capsic instead of C. capsici.
19. On line 308, there is capsic-treated instead of C. capsici-treated.
20. In the phrase “(…) and protopectin - key components of the cell wall”, an en dash should be used instead of a hyphen (line 485).
21. When providing references in the text, it is essential to insert a space before the parenthesis containing the number. In the current version of the manuscript, the notation of references is heterogeneous.
22. In References, in the names of journals, all words (except prepositions and conjunctions) should be written starting with capital letters. Unfortunately, most of the names of journals are written incorrectly, e.g. Industrial crops and products, instead of In Industrial Crops and Products.
23. References would be more useful if each included a DOI number.

 

June 10, 2025

Author Response

Comments 1: The authors used terms “phenylpropane metabolic pathway” and “phenylpropane metabolism”in the title, key words and many places in the manuscript. I would also consider using the much more common term "phenylpropanoid metabolism", especially among the keywords.

Response 1: Thank you for your kind suggestion, we have replaced the “phenylpropane” with “phenylpropanoid” in the manuscript. 

Comments 2: The authors wrote: “For instance, during pathogen invasion, plants trigger the phenylpropane metabolic pathway, producing secondary metabolites such as phenolic compounds, flavonoids, and lignin to prevent the spread of pathogens[19]”. I would modify this sentence to: “For instance, during pathogen invasion, plants trigger the phenylpropane metabolic pathway, producing secondary metabolites such as phenolic compounds, including flavonoids, and lignin to prevent the spread of pathogens[19]” because flavonoids are also phenolic compounds.

Response 2: Thank you for your kind suggestion, we have rewrote this sentence.

Comments 3: When describing the methodology for determining lignin content, the authors wrote: “The final results were expressed as OD280 nm/g”. It is not entirely clear in what units the lignin content was expressed. I understand that the authors measured absorbance at 280 nm. First, the abbreviation “nm” should be in subscript. Second, would not it be better to write that it is absorbance, not optical density, which is nowadays most often referred to the measurement of microbial counts, since light scattering is then measured? Perhaps “The final results were expressed in absorbance units, A280 nm/g” would be more clear?

Response 3: Thank you for your kind suggestion. We collected the absorbance at 280 nm. Additionally, regarding the representation of lignin content, we have referred to some articles that also used OD₂₈₀/g for expression, such as Chen et al. (2022) J Agric Food Chem, “Lignin biosynthesis pathway and redox balance act synergistically in conferring resistance against Penicillium italicum infection in 7-Demethoxytylophorine-treated navel orange”.

Comments 4: The D graph of Figure 3 is more narrow in comparison to the other graphs. Its width should be adjusted to match the others.

Response 4: Thank you for your kind suggestion. We have reloaded the Figure 3 in the manuscript.

Comments 5: The graphs of Figure 4 are shifted to the left. Authors should pay attention to whether they will be properly aligned in the final version of the publication.

Response 5: Thank you for your kind suggestion. We have adjusted the format of  Figure 4 to ensure consistency with the other graphics.

Comments 6: The authors should check whether the resolution of Fig. 8 is high enough. In the attached manuscript for review, its resolution is not satisfactory.

Response 6: Thank you for your kind suggestion. We have reloaded the Figure 8 in the manuscript.

Comments 7: In Figure 9 (I like the humorous approach), in the phrases “total phenolic” and “flavonoid,” I would use the words in the plural.

Response 7: Thank you for your kind suggestion. As an indicator, it may also be acceptable to use the singular.

Comments 8: The semicolon on line 12 is definitely too big.

Response 8: We have made revision in the manuscript.

Comments 9: The name of the enzyme (β-1,3-Glucanase) inside the sentence should be written starting with a lowercase letter (lines 23 and 174).

Response 9: Thank you for your kind suggestion. We have rewrote in the manuscript.

Comments 10: On line 51, the conjunction “and” should be written in a simple font.

Response 10: Thank you for your kind suggestion. We have rewrote in the manuscript.

Comments 11: On lines 114, 267, 300, and 340, the Latin name of the fungus is missing a space after

Response 11: Thank you for your kind suggestion. We have rewrote in the manuscript.

Comments 12: In methodology, I would include the noun “pericarp” or “fruit” (line 141) to the phrase“pepper tissue.”

Response 12: Thank you for your kind suggestion. We have rewrote in the manuscript.

Comments 13: On line 149, in the phrase, ”(…) stained with 1% (w/v) phloroglucinol ethanol…”, a word“in”is missing.

Response 13: Done.

Comments 14: On line 219, there is capsici -treated instead of C. capsici-treated. The extra space should be removed.

Response 14: Done.

Comments 15: On line 221, the Latin name of the fungus should be italized.

Response 15: Done.

Comments 16: In most cases, the letter denoting the significance level (P) is correctly italicized. However, the authors forgot to italicize the P in line 234 (twice) and in line 274.

Response 16：Thank you for your kind suggestion. We have revised the manuscript.

Comments 17: On line 265, the Latin name of the fungus is missing a space, while the space before the comma should be removed in the rest of the sentence.

Response 17：Done.

Comments 18: On line 303, there is capsic instead of C. capsici.

Response 18: Done.

Comments 19: On line 308, there is capsic-treated instead of C. capsici-treated.

Response 19: Done.

Comments 20: In the phrase “(…) and protopectin - key components of the cell wall”, an en dash should be used instead of a hyphen (line 485).

Response 20: Done.

Comments 21: When providing references in the text, it is essential to insert a space before the parenthesis containing the number. In the current version of the manuscript, the notation of references is heterogeneous.

Response  21: Thank you for your kind suggestion. We have revised the manuscript.

Comments 22: In References, in the names of journals, all words (except prepositions and conjunctions) should be written starting with capital letters. Unfortunately, most of the names of journals are written incorrectly, e.g. Industrial crops and products, instead of In Industrial Crops and Products.

Response 22: Thank you for your kind suggestion. We have revised the manuscript.

Comments 23: References would be more useful if each included a DOI number.

Response 23: Thank you for your kind suggestion. We have revised the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Horticulturae-3681653: This manuscript presents a detailed and well-structured investigation into the mechanisms of postharvest disease development in bell pepper fruits induced by Colletotrichum capsici, focusing on changes in cell wall components and phenylpropanoid metabolism. The topic is timely and significant for postharvest pathology and horticultural science. The manuscript requires careful language editing throughout to correct grammatical errors and improve the overall clarity and flow of the text.

L25: "remained a high level" should be "remained at a high level".

L27: “resulting in” should be “resulted from.” The symptoms are not the cause of disease.

L42: "easily perishable" is grammatically correct but sounds awkward; consider "highly perishable".

L108: “District”

L118: Is this “2 mm pulp tissue” a mixture of pepper skin and flesh or just flesh?

L192: "contentwere" spacing error: should be "content were".

L209: Confirm whether n = 3 for biological replicates, and how many technical replicates were there.

L235: “The same as bellow” typo: should be "below".

L343–358: The correlation analysis is insightful. Strengthen the interpretation by explaining potential causal links, not just statistical associations.

L379: “accelated” should be “accelerated”.

L428–431: The discussion about cell wall collapse is good, but clearer linkage between lignin, pectin changes, and fruit firmness is needed in discussion.

L421–426: Sentence is too long. Please consider splitting it and reducing repetition of “cell wall”.

Conclusion: Please add 2–3 sentences on practical applications, such as how monitoring lignin/pectin could serve as a disease progression marker or if this suggests any potential treatments.

Comments on the Quality of English Language

The manuscript requires careful language editing throughout to correct grammatical errors and improve the overall clarity and flow of the text.

Author Response

Comments 1: L25: "remained a high level" should be "remained at a high level".

Response 1: Thank you for your kind suggestion. We have checked and revised the whole manuscript.

Comments 2: L27: “resulting in” should be “resulted from.” The symptoms are not the cause of disease.

Response 2: Thank you for your kind suggestion. We have checked and revised the whole manuscript.

Comments 3: L42: "easily perishable" is grammatically correct but sounds awkward; consider "highly perishable".

Response 3: Thank you for your kind suggestion. We have revised the manuscript.

Comments 4: L108: “District”

Response 4: We have revised the manuscript. And we are sorry here for our careless work.

Comments 5: L118: Is this “2 mm pulp tissue” a mixture of pepper skin and flesh or just flesh?

Response 5: Due to the thin skin of pepper, we have to collect mixture samples includ both skin and flesh. In the manuscript, our expression was inappropriate, and we revised it instead of ‘2 mm mixture tissue (pepper skin and flesh)’.

Comments 6: L192: "contentwere" spacing error: should be "content were".

Response 6: Done.

Comments 7: L209: Confirm whether n = 3 for biological replicates, and how many technical replicates were there.

Response 7: Usually, we set up three biological replicates during experiment design and three technical replicates for performing data statistics.

Comments 8: L235: “The same as bellow” typo: should be "below".

Response 8: Done.

Comments 9: L343–358: The correlation analysis is insightful. Strengthen the interpretation by explaining potential causal links, not just statistical associations.

Response 9: According to your suggestion, we re-analyzed this part of the text.

Comments 10: L379: “accelated” should be “accelerated”.

Response 10: Done.

Comments 11: L428–431: The discussion about cell wall collapse is good, but clearer linkage between lignin, pectin changes, and fruit firmness is needed in discussion.

Response 11: According to your suggestion, we supplemented the relevant content later.

Comments 12: L421–426: Sentence is too long. Please consider splitting it and reducing repetition of “cell wall”.

Response 12: According to your suggestion, we rewrote this sentence.

Comments 13: Conclusion: Please add 2–3 sentences on practical applications, such as how monitoring lignin/pectin could serve as a disease progression marker or if this suggests any potential treatments.

Response 13: Thank you for your suggestion. We have added relevant content in the Conclusion.