Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic of the article is relevant to the interests of Insects. These studies are necessary to contribute to the improvement of scientific knowledge in the area of study. However, the article must be reviewed.

Comments to authors:

Line 79: Please, add geographic coordinates of the study sites.

Line 91: Morphological macroscopic characterization of fungal species is incomplete. The complete morphological characterization of isolates should be provided. It is recommended to provide data on the growth of the fungi of interest in at least two different culture media. Please provide sufficient details macroscopically (description of colony appearance, color, growth diameter, etc.) in different culture medium.

Microscopic characterization is an essential requirement for the identification of entomopathogenic fungi. In this study, the authors document the discovery of new species of entomopathogenic fungi infecting B. tabaci; therefore, it is necessary for them to provide a complete description of the microscopic morphological characters of taxonomic interest. For example, the dimensions of the conidia and phialides, the shape of the conidiophores, and other structures of taxonomic importance in entomopathogenic fungi are missing. It is not possible to determine the taxonomic genus based on the color or diameter of the colonies.

Line 125: the section on molecular identification is incomplete. The molecular marker ITS is not enough to distinguish fungal species. No details are provided about the PCR protocol used in the experiments either. Please, provide it. ¿Were the sequences submitted to the GenBank database? Please, indicate accession numbers.

Line 144: Please provide details on the breeding procedure for B. tabaci.

Line 156: ¿How many replicates were there? The authors provide the number of repetitions but do not mention how many replicates were conducted for each treatment.

Line 158: Please replace “dilution” by “spore suspension”.

Line 167: The authors note that mortality was recorded over five consecutive days. However, according to the results, mortality did not reach 100% during that period. In light of this, the question is: on what basis did the authors decide to monitor mortality for only five days?

Line 176: ¿What is the number of replicates and repetitions?

Line 222: The microscopic descriptions for each of the isolates are missing.

Comments on the Quality of English Language

English need to be improved.

Author Response

Reviewer 1 Round 1

Comments to authors:

1. Line 79: Please, add geographic coordinates of the study sites.

Response: Thank you very much for the observation. The study region corresponds to a polygon with the following geographical coordinates: 18° 50' and 19° 15' N, 95° 50' and 96° 25' W

2. Line 91: Morphological macroscopic characterization of fungal species is incomplete. The complete morphological characterization of isolates should be provided. It is recommended to provide data on the growth of the fungi of interest in at least two different culture media. Please provide sufficient details macroscopically (description of colony appearance, color, growth diameter, etc.) in different culture medium.

Response: We appreciate the remark. Although morphological characterization is a classic criterion in the identification of entomopathogenic fungi, the main objective of this study was to evaluate the biological efficacy of native strains for whitefly control, rather than to perform an exhaustive taxonomic characterization. Therefore, the macroscopic characterization was performed only in SDA medium as a preliminary evaluation at the genus level as mentioned in the manuscript, while the identification at the species level of the strains was carried out using molecular tools, which offer greater precision and reduce the variability associated with different culture media. We recognize that multi-media characterization and microscopic analysis can complement the description; however, these aspects exceed the scope of this work

3. Microscopic characterization is an essential requirement for the identification of entomopathogenic fungi. In this study, the authors document the discovery of new species of entomopathogenic fungi infecting  tabaci; therefore, it is necessary for them to provide a complete description of the microscopic morphological characters of taxonomic interest. For example, the dimensions of the conidia and phialides, the shape of the conidiophores, and other structures of taxonomic importance in entomopathogenic fungi are missing. It is not possible to determine the taxonomic genus based on the color or diameter of the colonies.

Response: We appreciate the remark. However, we wish to clarify that this study does not report the discovery of new species of entomopathogenic fungi that infect B. tabaci, which are already described in the literature. In line with the focus of the work, aimed at evaluating the biological efficacy of native strains, the taxonomic identification of the isolates was carried out using molecular tools, which offer greater precision than morphological characters. Since no new species were discovered, we consider that this requested level of detail is beyond the scope of the present work.

4. Line 125: the section on molecular identification is incomplete. The molecular marker ITS is not enough to distinguish fungal species. No details are provided about the PCR protocol used in the experiments either. Please, provide it.

Response: Thank you very much for the observation. We agree that the ITS marker may have limited resolution for distinguishing closely related fungal species in certain taxonomic groups. However, the ITS region is widely accepted as the universal DNA barcode for fungi. It is commonly used for preliminary identification, particularly in studies focused on ecological characterization and isolate screening. In our study, ITS sequencing was used as an initial approach to identify the fungal isolates, which were then selected after pathogenicity evaluation. In line 135 acknowledges and mentions that it is an initial approach to species assignment. The PCR conditions were added in lines 136 to 139.

5. ¿Were the sequences submitted to the GenBank database? Please, indicate accession numbers.

Response: Thank you very much for the observation. The sequences were uploaded to NCBI, with the ID SUB16150138, this clarification was left on line 152.

6. Line 144: Please provide details on the breeding procedure for  tabaci.

Response: Thank you very much for the observation. Whitefly (B. tabaci) breeding was established with adults collected in serrano pepper plantations in the Sotavento region, Veracruz, where entomopathogenic fungi had never been applied. The insects were kept in entomological cages (40 × 40 × 40 cm) covered with anti-aphid mesh, which contained serrano pepper plants as a food source, under greenhouse conditions. Environmental conditions were controlled at a temperature of 28–32 °C, relative humidity of 60–80 % and a photoperiod of 12:12 h (light:dark). After five generations, the adult whiteflies were used for bioassays. Lines 154-160.

7. Line 156: ¿How many replicates were there? The authors provide the number of repetitions but do not mention how many replicates were conducted for each treatment.

Response: Three replications were performed in each repetition. Line 180

8. Line 158: Please replace “dilution” by “spore suspension”.

Response: Change made. Line 176

9. Line 167: The authors note that mortality was recorded over five consecutive days. However, according to the results, mortality did not reach 100% during that period. In light of this, the question is: on what basis did the authors decide to monitor mortality for only five days?

Response: We appreciate the observation. The five-day period was defined based on previous trials that showed high mortality in that time. Therefore, five days were defined to capture the most active phase of infection and the differences in virulence between strains. Our results confirm its relevance, since in that interval some strains reached 100% mortality (PV02 to 4 DDA; SV01 and MV03 to 5 DDA) Lines 315 and 332. Extending the evaluation could introduce bias due to natural mortality (19% in the control) or other factors not attributable to the treatments.

10. Line 176 ¿What is the number of replicates and repetitions?

Response: three repetitions and three replications per repetition were performed. Line 195.

11. Line 222: The microscopic descriptions for each of the isolates are missing.

Response: Detailed microscopic descriptions of each isolate were not included in this study, since the fungi evaluation does not correspond to new species, but to taxa previously reported and described in the literature. The identification of the isolates was carried out at the genus level based on their general morphological characteristics and the identification at the species level was carried out using molecular tools, which offer greater precision

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewed paper presents interesting and valuable research on the potential use of native strains of entomopathogenic fungi in controlling the whitefly B. tabaci.

Overall, the paper is well written, and the results are documented. However, I have some comments before publishing it.

Detailed comments:

1. Line 63 - "Paecelomyces" is used, but it should be "Paecilomyces"; 2. In the Materials and Methods section, the authors write: "The dead flies were removed from the cages and cultured on SDA medium to confirm their infection by the applied fungus." The question is, wouldn't it have been better to place the dead individuals in wet chambers to allow for fungal growth and sporulation? The next question is, were the dead individuals surface sterilized? Could bacteria have been present on the insects' bodies, which could have interfered with fungal growth on SDA?; 3. In my opinion, Fig. 3 and Fig. 4 are unnecessary and add nothing new.4. There is some discrepancy in the numbering of references. In the discussion, the authors cite references 58, 59, 60, and 61, yet the list of references ends at reference 57?

Author Response

Reviewer 2_Round 1

 

1.Line 63 - "Paecelomyces" is used, but it should be "Paecilomyces"

 

Response: Change made

 

2. In the Materials and Methods section, the authors write: "The dead flies were removed from the cages and cultured on SDA medium to confirm their infection by the applied fungus." The question is, wouldn't it have been better to place the dead individuals in wet chambers to allow for fungal growth and sporulation?

 

Response: We agree that the use of wet chambers is a widely used technique to promote the sporulation of entomopathogenic fungi and visually confirm the infection. However, in our study we opted for direct seeding in the SDA culture medium, this technique was chosen with the purpose of confirming more precisely the presence of the applied fungus through its growth in a selective medium, which allows its identification, in addition to reducing the ambiguity associated with the superficial sporulation that can be observed in wet chambers. Although the use of wet chambers favors sporulation and allows visual confirmation of the infection, it can also increase the risk of contamination or make it difficult to differentiate between the inoculated fungus and other saprophytic microorganisms. Therefore, we consider that direct isolation in culture medium offers a more robust verification of the fungi.

 

3.The next question is, were the dead individuals surface sterilized?

Could bacteria have been present on the insects' bodies, which could have interfered with fungal growth on SDA?;

 

Response: Flies were disinfected in sodium hypochlorite (0.5% of the active product) for 5 minutes and rinsed three times with sterile distilled water. Lines 95-96.

In no case were bacteria observed in the flies that were handled in the experiment, since when the flies were seeded in the culture medium it would have been observed, so this possibility is ruled out.

 

4. In my opinion, Fig. 3 and Fig. 4 are unnecessary and add nothing new.

 

Response: We appreciate your observation. Although Figures 3 and 4 may seem redundant, we consider that they provide relevant information, since they illustrate the microscopic characteristics briefly described in the text for the isolates of Cordyceps spp. and Beauveria spp., which facilitates their interpretation and supports their morphological identification. They were also included to complement the understanding of the results, by providing visual evidence that is not fully appreciated in the text.

 

5. There is some discrepancy in the numbering of references. In the discussion, the authors cite references 58, 59, 60, and 61, yet the list of references ends at reference 57?

 

Response: the references indicated were added. Line 608-617.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Many thanks to the authors for addressing the comments.

Please accept my apologies; I intended to refer to new isolates of entomopathogenic fungi, not new species.

The authors state that the aim of the study is to isolate, morphologically and molecularly characterize, and evaluate the pathogenicity and virulence of native entomopathogenic fungal strains from the Sotavento region of Veracruz against adult Bemisia tabaci under laboratory conditions (Lines 72–75).

I understand that the primary objective of the study is not to provide an exhaustive taxonomic characterization. However, since the authors indicate that the objective includes the morphological and molecular characterization of entomopathogenic fungi, I believe that a more comprehensive characterization should be provided for these new fungal isolates. Based on the data presented, it is not possible to identify the isolates at the species level. The information on germination rates, colony diameter, and colony color is insufficient to support identification beyond the genus level.

In addition, molecular characterization does not replace microscopic characterization, but rather complements it. Furthermore, the ITS marker may not provide sufficient resolution, resulting in identification only at the species complex level.

For accurate species-level identification, particularly in studies involving strain characterization, it is recommended to complement ITS analysis with secondary markers, especially protein-coding genes such as TEF1-α (Translation Elongation Factor 1-alpha), RPB1/RPB2 (RNA polymerase II subunits), and β-tubulin (TUB2).

 

Comments on the Quality of English Language

English need to be improved.

Author Response

Comment 1: The English could be improved to more clearly express the research.

Response: Thank you for your remarks. The suggestions were incorporated, and the changes are indicated in the document.

Comment 2: Figures and tables must be improved.

Response: The suggestions were taken into account. Table 1, line 126; figure 2, line 231; figure 3, line 240.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

Lines 108-111: I do not understand how the researchers were able to observe and determine the color of the spores while they were being stained. ¿Could the authors please clarify this point? The color of the spores is not a taxonomically significant characteristic for the identification of fungi within the phylum Ascomycota; however, spore dimensions—which the authors do not provide—are of taxonomic importance.

I would like to reiterate that the materials and methods used to describe and characterize the fungal strains are insufficient and incomplete, and therefore do not provide adequate evidence to confirm the identity of the fungi. 

Taxonomic identification at the genus level cannot be reliably established based solely on germination rates, colony color, or colony diameter. I believe the authors should revise the taxonomically informative characters commonly used for the identification of insect-pathogenic fungi. This aspect is crucial to the manuscript and therefore should be supported by rigorous and comprehensive data.

The identification of entomopathogenic fungi is based primarily on the morphological study of their reproductive structures. Therefore, the authors should provide a measurement of the fungal structures of taxonomic importance, including conidia, phialides, and conidiophores.

Line 135: Please remove the period after the parenthesis.

Line 144: Could you please provide the list of sequences and the sequence information (host, GenBank number, strain number or designation, fungal species, etc.) that you used for the comparisons? ¿Could you please clarify whether the sequences used for the comparisons correspond to the “type” strains of each fungal species? 
It is very important to validate the obtained data against reliable reference data. Given the abundance of information available in databases, the sequences selected for comparison must be chosen with great care.

Author Response

Comments 1. Lines 108-111: I do not understand how the researchers were able to observe and determine the color of the spores while they were being stained. ¿Could the authors please clarify this point? The color of the spores is not a taxonomically significant characteristic for the identification of fungi within the phylum Ascomycota; however, spore dimensions—which the authors do not provide—are of taxonomic importance.

I would like to reiterate that the materials and methods used to describe and characterize the fungal strains are insufficient and incomplete, and therefore do not provide adequate evidence to confirm the identity of the fungi.

Taxonomic identification at the genus level cannot be reliably established based solely on germination rates, colony color, or colony diameter. I believe the authors should revise the taxonomically informative characters commonly used for the identification of insect-pathogenic fungi. This aspect is crucial to the manuscript and therefore should be supported by rigorous and comprehensive data.

The identification of entomopathogenic fungi is based primarily on the morphological study of their reproductive structures. Therefore, the authors should provide a measurement of the fungal structures of taxonomic importance, including conidia, phialides, and conidiophores.

Response: We appreciate the reviewer's observation. For microscopic characterization, fungal tissue preparations were stained with lactophenol blue and mounted on unstained, transparent Scotch tape to observe mycelial type, spore color, and spore shape. These characteristics were compared with taxonomic keys. Lines 108-111.

We agree that the dimensions of spores and other reproductive structures are important taxonomic traits for taxonomic identification. However, the objective of this study was not to provide an exhaustive taxonomic description or to delimit new species, but to identify isolates of previously reported entomopathogenic fungi. The identification of the isolates was based mainly on molecular analysis of the ITS region, complemented by general morphological characteristics and comparison with descriptions in the specialized literature. Since the dimensions of the spores and other reproductive structures were not recorded during the development of this study, such information is not available for incorporation into the manuscript.

 

Comments 2. Line 135: Please remove the period after the parenthesis.

Response: The observation was made. Line 142.

 

Comments 3. Line 144: Could you please provide the list of sequences and the sequence information (host, GenBank number, strain number or designation, fungal species, etc.) that you used for the comparisons? ¿Could you please clarify whether the sequences used for the comparisons correspond to the “type” strains of each fungal species?

It is very important to validate the obtained data against reliable reference data. Given the abundance of information available in databases, the sequences selected for comparison must be chosen with great care.

Response: We thank the reviewer for this valuable suggestion. To improve the transparency and reproducibility of the phylogenetic analysis, we have added information regarding the reference ITS sequences used for phylogenetic comparisons. Specifically, the Materials and Methods section (Lines 153–158) has been revised to describe the selection of reference sequences from GenBank and to indicate that detailed information, including GenBank accession numbers, taxonomic identification, and ITS sequences, is provided in Supplementary File S1.

Additionally, the legend of Figure 5 has been modified to indicate that the complete information for the reference sequences used in the phylogenetic reconstruction is available in the supplementary materials. Lines 313-314.

Author Response File: Author Response.pdf