Review Reports

Round 1

Reviewer 1 Report

The work entitled Insights into Adaptation of Geotrichum citri-aurrantii in Highly Acid Environment demonstrates the adaptability of Geotrichum citri-aurantii in the environments where it proliferates, especially in very acidic environments.

The presented study delves into various aspects of pH changes both in vitro and during infection, detailing morphological changes and transcriptomic and expression studies for specific genes. Despite all this work, there are some aspects to consider, which I will list below.

 

1. Authors should follow the journal's instructions and list citations with numbers and in order of appearance.
2. Check for grammatical errors throughout the entire text.
3. The wounds created for artificial infection are actually cuts, not a single point of damage from a wound. It is difficult to measure the damage when the wound is so large.
4. What is the reason for using those pH values ​​2.1, 3.2, 4.03 and 4.99?
5. Why do you use PDB adjusted to pH 2.2 and 3.0 for the microcopy assay, and IM medium at the same pHs for the transcriptomic studies?
6. Figure 1A is not clear and the lines are indistinguishable; perhaps a bar graph would be clearer. I am also unable to discern the infection on day 1 in Satsuma Mandarin.
7. In section 3.2 of the results, the terms up-regulated and down-regulated are mentioned. These terms do not seem correct to me since we are not observing expression but rather a decrease or increase in pH.
8. When you say the results showed that citri-aurantii alkalized the environment when the pH was below 3.02 and acidified it when the pH was above 3.02, it is not quite correct to speak of alkalization when the resulting pH is acidic.
9. In Figure 5, CM should be CW for cell wall.
10. In Figure 6, section C, it is not possible to read.
11. The protein network interactions shown in Figure 7A are unintelligible. I would remove that part and focus only on the smaller interrelationships. I also don't understand what it contributes to the work.
12. Since a large amount of information is shown in Figure 9, panels B and C may have maintained the same colors for the same genes, making it easier to follow and compare.
13. No clear explanation is given other than the differences in medium because when it grows in PDB in the lag phase the pH drops and then stabilizes, but when it grows in IM it drops and maintains that drop without recovering the initial pH.
14. It would be advisable to summarize some aspects of the discussion, which is too extensive, and some sections describe aspects that are already known in excessive detail.

Author Response

Comments 1: Authors should follow the journal's instructions and list citations with numbers and in order of appearance.

Response 1: Thank you for your comments. Revisions have been made as requested.

Comments 2: Check for grammatical errors throughout the entire text.

Response 2: Sorry for the language problem, we had improved the language using a language editing service.

Comments 3: The wounds created for artificial infection are actually cuts, not a single point of damage from a wound. It is difficult to measure the damage when the wound is so large.

Response 3: We appreciate your meticulous comments.

  The approach of creating cross-shaped wounds on fruit surfaces and subsequently performing inoculation has been documented in numerous publications, as evidenced by the method descriptions and symptomatic fruit photographs cited in these reference below:

    Louw, J.P.; Korsten, L. Pathogenicity and host susceptibility of Penicillium spp. on citrus. Plant Dis. 2015, 19, 21-30.  

    Wang, Z.; Meng, K.; Shen, X.; Li, L.; Chen, X.; Tan, X.; Tao, N. Xyloglucan-specific endo-β-1,4-glucanase (PdXEG1) gene is important for the growth, development and virulence of Penicillium digitatum. Postharvest Biol. Tec. 2024, 208, 112673.

    Trujillo-Salazar, J.A.; Mora-López, M.; López-Restrepo, D.A.; Pineda-Montoya, J.E.; Ramírez-Olier, J.P.; Botero-Botero, L.R. Antagonistic yeasts isolated from bioferments of mountain microorganisms (MM): an alternative for the control of fruit anthracnose caused by Colletotrichum tamarillo. J. Agric. Environ. Int. 2025, 119, 25-40.

In fact, to amplify the pH variation in the infected areas of the fruit, we created slightly larger cross-shaped wounds on the peels compared to those typically used in routine inoculation. This wound pattern facilitates the absorption of the spore suspension, enabling rapid colonization by the pathogenic fungi, with the intersection of the cross designated as the disease center. The lesion diameter can be determined based on the phenotypes of softening and collapse of the host tissue. The description of the experimental procedures has also been refined according to these references (lines 81-83).

Comments 4: What is the reason for using those pH values 2.1, 3.2, 4.03 and 4.99?

Response 4: Thank you for your comments. The pH range of 2.1 to 4.99 was selected to simulate the potential pH values found in various citrus varieties. This reason has been supplemented in Section 2.2 (line 89).

Comments 5: Why do you use PDB adjusted to pH 2.2 and 3.0 for the microcopy assay, and IM medium at the same pHs for the transcriptomic studies?

Response 5: Sorry for our mistake. Actually, the PDB medium was used for the transcriptome experiment because the IM medium is nutritionally deficient, and the mycelial biomass obtained after 24 h of cultivation does not meet the testing requirements. The experimental methods in this section have been revised in line 122.

Comments 6: Figure 1A is not clear and the lines are indistinguishable; perhaps a bar graph would be clearer. I am also unable to discern the infection on day 1 in Satsuma Mandarin.

Response 6: Thank you for your constructive comments. To present the infection on day 1 more clearly, Figure 1A has been revised to a bar graph format.

Comments 7: In section 3.2 of the results, the terms up-regulated and down-regulated are mentioned. These terms do not seem correct to me since we are not observing expression but rather a decrease or increase in pH.

Response 7: Thank you for your positive and constructive comments. Revisions have been made in lines 165-168.

Comments 8: When you say the results showed that citri-aurantii alkalized the environment when the pH was below 3.02 and acidified it when the pH was above 3.02, it is not quite correct to speak of alkalization when the resulting pH is acidic.

Response 8: Thank you for these positive and constructive comments. Revisions have been made in lines 167-168.

Comments 9: In Figure 5, CM should be CW for cell wall.

Response 9: Sorry for our mistake. Revisions have been made in Figure 5.

Comments 10: In Figure 6, section C, it is not possible to read.

Response 10: Thank you for your comments. Figure 6C and its corresponding analysis of results have been revised.

Comments 11: The protein network interactions shown in Figure 7A are unintelligible. I would remove that part and focus only on the smaller interrelationships. I also don't understand what it contributes to the work.

Response 11: Thank you for your comments. Figure 7A has been deleted.

The six sub-networks identified via PPI analysis indicated that nitrogen metabolism, carbon metabolism, cell wall synthesis and remodeling, as well as nucleotide and amino acid biosynthesis in G. citri-aurantii underwent significant alterations under acidic conditions. The key genes involved in these pathways were screened for subsequent qRT-PCR analysis.

To emphasize the significance of the PPI analysis, we have revised the result analysis in lines 265-269.

Comments 12: Since a large amount of information is shown in Figure 9, panels B and C may have maintained the same colors for the same genes, making it easier to follow and compare.

Response 12: Thank you for your constructive comments. We maintained the same colors for the identical genes in panels B and C of Figure 9.

Comments 13: No clear explanation is given other than the differences in medium because when it grows in PDB in the lag phase the pH drops and then stabilizes, but when it grows in IM it drops and maintains that drop without recovering the initial pH.

Response 13: Thank you for your comments.

It is reported that external carbon affected fungal metabolism and thereby regulating the dynamic pH changes in their microenvironment (Bi et al., 2016). High glucose in PDB medium boosted mycelium growth and acid production, resulting in a rapid decrease in the pH value of the medium, while reduced glucose led to ammonia accumulation and pH rebound (Vylkova, 2017). However, under nutrient-limiting conditions, fungi recycle substances via autophagy, maintaining acid production (Kaur et al., 2019). This may explain the differences in the pH regulation of G. citri-aurantii in the two media. The aforementioned content has been supplemented in lines 339-346.

References:

Bi, F.C.; Barad, S.; Ment, D.; Luria, N.; Dubey, A.; Casado, V.; Glam, N.; Mínguez, J.D.; Espeso, E.A.; Fluhr, R.; et al. Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi. Mol. plant pathol. 2016, 17, 1178-1195.

Vylkova, S. Environmental pH modulation by pathogenic fungi as a strategy to conquer the host. PLoS pathog. 2017, 13, e1006149.

Kaur, B.; Punekar, N.S. Autophagy is important to the acidogenic metabolism of Aspergillus niger. PLoS One 2019, 14, e0223895.

Comments 14: It would be advisable to summarize some aspects of the discussion, which is too extensive, and some sections describe aspects that are already known in excessive detail.

Response 14: Thank you for your constructive comments. The Discussion section has been streamlined.

Reviewer 2 Report

This is an article that determines the morphological changes,  spore germination and gene expression profiles of G. citri-aurantii under highly acidic conditions.

I have some questions and comments.

1. In line 86, it is necessary to include references that expand upon or detail the information about any method.
2. In line 99, it is necessary to include references that expand upon or detail the information about any method.
3. In Figure 5C and H, there is an error in the letter CM; it should be CW.
4. What is the objective and hypothesis of the work?
5. The presentation of Figure 1C should be modified because it does not clearly show the results.

 

This is an article that determines the morphological changes,  spore germination and gene expression profiles of G. citri-aurantii under highly acidic conditions.

I have some questions and comments.

1. In line 86, it is necessary to include references that expand upon or detail the information about any method.
2. In line 99, it is necessary to include references that expand upon or detail the information about any method.
3. In Figure 5C and H, there is an error in the letter CM; it should be CW.
4. What is the objective and hypothesis of the work?
5. The presentation of Figure 1C should be modified because it does not clearly show the results.

 

Author Response

Comments 1: In line 86, it is necessary to include references that expand upon or detail the information about any method.

Response 1: Thank you for your constructive comments. The reference was added in line 92.

Comments 2: In line 99, it is necessary to include references that expand upon or detail the information about any method.

Response 2: Thank you for your constructive comments. The reference was added in line 107.

Comments 3: In Figure 5C and H, there is an error in the letter CM; it should be CW.

Response 3: Sorry for our mistake. Revisions have been made in Figure 5.

Comments 4: What is the objective and hypothesis of the work?

Response 4: Thank you for your constructive comments. The objectives and hypotheses of this work are as follows:

During years of substantial rainfall, citrus sour rot frequently results in significant fruit losses. None of the fungicides currently approved for use are capable of effectively managing this disease. Considering that citrus cultivars with varying acidity levels, including high-acidity varieties such as lemons, are all vulnerable to G. citri-aurantii, this study aims to elucidate the acidophilic and acid-tolerant characteristics of the pathogen. The pathogen fortifies the physical structure of its cell wall to prevent acid influx while simultaneously enhancing signal transduction and optimizing carbon metabolic pathways, as well as energy metabolism processes, thereby facilitating adaptation to acidic conditions. Additionally, the Pal/Rim pH signaling pathway may contribute to regulating environmental pH in response to the pathogen. These findings offer potential targets for the development of new fungicides.

The above content has been summarized in Conclusion (lines 411-421).

Comments 5: The presentation of Figure 1C should be modified because it does not clearly show the results.

Response 5: Thank you for your constructive comments. To present the results more clearly, Figure 1C has been revised to a bar graph.

Round 2

Reviewer 1 Report

The authors have reviewed the guidelines point by point and have followed the suggested changes, significantly improving the quality of the manuscript. The manuscript is accepted for publication in its current form.

There is no minor detail that I want to highlight.      

Reviewer 2 Report

The authors made all the corrections to the manuscript.

The authors made all the corrections to the manuscript.