Mycorrhizal Fungi of Phalaenopsis japonica (Orchidaceae) and Their Role in Seed Germination and Seedling Development

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1、In general, the article is too verbose, especially the introduction and methods, which should be simplified greatly, and many contents unrelated to this study can be deleted. The introduction of Phalaenopsis japonica can be moved to the materials and methods. For example, the paragraph of lines 142-147 can be deleted.。

2、The three questions raised are not scientific, and it is suggested to reorganize the scientific questions according to the results

3、Again, in the "materials and methods" part, it should be simplified, some experimental steps described too detailed.

4. The results are not clearly presented, for example, how many fungal strains were isolated, and which fungi were molecularly identified, and what were the fungi?

5、Table 2, Why were these six fungal strains selected for the germination experiment? The experiment had only six replicates with too few runs

6. The color discrimination of the different growth stages in Figure 5 is not obvious enough, and this figure confused the reader and I suggested redrawing it to clearly express seed germination, protocorm formation and seedlings at different times

 Once again, the writing of the manuscript needs to be improved greatly, too much repeated sentences!

Comments on the Quality of English Language

 Once again, the writing of the manuscript needs to be improved greatly, too much repeated sentences!

Author Response

Rev#1

Thank you for the recent comments on our manuscript. We found the comments very helpful and have done our best to integrate them into the revised version of this manuscript.

1、In general, the article is too verbose, especially the introduction and methods, which should be simplified greatly, and many contents unrelated to this study can be deleted. The introduction of Phalaenopsis japonica can be moved to the materials and methods. For example, the paragraph of lines 142-147 can be deleted.

 

• As the reviewer suggested, we have simplified the introduction and methodology. Changes can be identified with track changes. We have removed some unrelated and repetitive points from the introduction and methodology and detailed methodological explanations were replaced with some citations.
• The introduction of Phalaenopsis japonica was moved to the first paragraph of the methodology section.
• The paragraphs of lines 142-147 were deleted.

 

2、The three questions raised are not scientific, and it is suggested to reorganize the scientific questions according to the results

 

• We have removed these questions and reorganized the last paragraph of the introduction according to the results.

 

3、Again, in the "materials and methods" part, it should be simplified, some experimental steps described too detailed.

 

• We have removed some unrelated points from the methodology, and detailed methodological explanations were replaced with some citations.

 

4. The results are not clearly presented, for example, how many fungal strains were isolated, and which fungi were molecularly identified, and what were the fungi?

 

• We have added the following sentences in the first paragraph of the results to explain about the fungal strains: “All fungal isolates belonged to the Ceratobasidiaceae family. Specifically, 13, 2, and 1 OTUs were identified as CE6, CE22, and CE12, respectively”.

 

5、Table 2, Why were these six fungal strains selected for the germination experiment? The experiment had only six replicates with too few runs.

 

• We have added the following sentences in the materials and methods section 2.7.1.: “All fungal isolate OTUs, except for TU27, were detected from adult japonica. Four Ceratobasidiaceae isolates were obtained from P. japonica roots. To assess the effect of Tulasnellaceae fungi, we utilized two Tulasnellaceae isolates, TU18 and TU27. Since isolating Tulasnellaceae fungi from P. japonica proved unsuccessful, we utilized TU18 isolates obtained from another epiphytic orchid.”

 

• In the seed germination experiment, one replicate comprised four discs, each containing approximately 20–40 seeds per plate, totaling 120–240 seeds per treatment. We have followed some previous references (Zhang et al. 2022. Diversity, 14, 1119; Zhang et al. 2022. Frontiers in Ecology and Evolution, 10, 994641; Rammitsu et al. 2023. Plant Species Biology, 38(6), pp.306-318). Therefore, we believe the number of replicates chosen for the experiment is sufficient.

 

6. The color discrimination of the different growth stages in Figure 5 is not obvious enough, and this figure confused the reader and I suggested redrawing it to clearly express seed germination, protocorm formation and seedlings at different times.

 

• We have modified Figure 5 considering the comments of both reviewers. We have used distinct colors for early development and seedlings establishment phases.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have provided some interesting findings, e.g., the different effects of one tulasnelloid fungus for seedling establishment and subsequent growth, which may be helpful to better understanding the mutualisms among epiphytic orchids and their symbiotic fungi.

Several suggestions are as following:

1.     The abstract (statements of lines 22-30) can be further modified. The phylogenetic analysis can be reconstructed with incorporating results infered from sanger and HTS sequencings, and accordingly, the results based on two different sequencings also need by integratively discussed, rather than in two separate parts (4.1 and 4.2). The OMF bottlenecks in seedling establishment might be prevalent for Orchidaceae and can be further discussed.

2.     Figures 2 and 4 are space consuming but not very informative, which need to be reconsidered (e.g., the height-width ratio of figure 4 can be adjusted, and the tulasnelloid and ceratobasidioid OTUs may be summarized as two columns).

3.     The germination stages 1-4 can be categrized as early germinataion or porotocorm formation, while stages 5-6 mean protocrom differentiation or seedling establishment, therefore it make sences to highlight the two periods. At least in Figure 5, the authors may use different colors but not a gradient orange to show the stages 4 and 5. Similarly, stages 2-3 might be merged given that there are little significant difference, according to the commonly used criteria for division (e.g., Arditti J. 1967. Factors affecting the germination of orchid seeds. The Botanical Review 33:1-97.)

4.     Try to use more informative subtitles in Discussion, rather than “Molecular identification…”, “Molecular identification…”, and “Symbiotic culture”, and move the satements about synergistic effects from conclusions to discussions.

Lines 27, 221 and 685:  “Ceratobasidiaceae were”: change to “fungi of Ceratobasidiaceae were” or “Ceratobasidiaceae was”

Line 64: “Phalaenopsis”: change to “P.”

Line 687: Qin et al. (2021), instead of 2023

Line 694: the OTU CE6

Comments on the Quality of English Language

N/A

Author Response

Rev#2

 

Thank you for the recent comments on our manuscript. We found the comments very helpful and have done our best to integrate them into the revised version of this manuscript.

The authors have provided some interesting findings, e.g., the different effects of one tulasnelloid fungus for seedling establishment and subsequent growth, which may be helpful to better understanding the mutualisms among epiphytic orchids and their symbiotic fungi. 

Several suggestions are as following:

 

1. The abstract (statements of lines 22-30) can be further modified. The phylogenetic analysis can be reconstructed with incorporating results infered from sanger and HTS sequencings, and accordingly, the results based on two different sequencings also need by integratively discussed, rather than in two separate parts (4.1 and 4.2). The OMF bottlenecks in seedling establishment might be prevalent for Orchidaceae and can be further discussed.

 

• We have added the following sentences in the abstracts: “Sanger sequencing found 9 OTUs, 4 CE, and 5 TU; HTS detected 22 OTUs, 4 CE, 16 TU, and 2 Serendipitaceae”. We have deleted one sentence (Changes can be identified with Track changes).
• Regarding the phylogenetic analysis, we generated the analysis using Sanger sequencing results that used the whole ITS region including ITS1, 5.8S, and ITS2, approximately 600bp in length. The HTS methods primarily focused on only the ITS2 region, approximately 250bp long. To ensure a more robust tree, we utilized the whole ITS sequences obtained from Sanger sequencing rather than ITS2.
• Discussion of the Sanger sequencing and HTS (4.1 and 4.2) were modified into one section.
• We have added the following sentences in the discussion section 4.2: “The primary inducers of seed germination and early development in P. japonicawere CE6, with CE22 and TU18 also significantly contributing to increased root and leaf numbers. Therefore, OMF bottlenecks in seed germination might be prevalent for P. japonica”.

 

2. Figures 2 and 4 are space consuming but not very informative, which need to be reconsidered (e.g., the height-width ratio of figure 4 can be adjusted, and the tulasnelloid and ceratobasidioid OTUs may be summarized as two columns).

 

• We have adjusted the height-width ratio in Figure 2.

 

• In Figure 4, we have modified the height-width ratio and OMF family arrangement. We have retained Figure 4 as a single column and showed labels to identify the relative abundances of Ceratobasidiaceae and Tulasnellaceae.

 

3. The germination stages 1-4 can be categrized as early germinataion or porotocorm formation, while stages 5-6 mean protocrom differentiation or seedling establishment, therefore it make sences to highlight the two periods. At least in Figure 5, the authors may use different colors but not a gradient orange to show the stages 4 and 5. Similarly, stages 2-3 might be merged given that there are little significant difference, according to the commonly used criteria for division (e.g., Arditti J. 1967. Factors affecting the germination of orchid seeds. The Botanical Review 33:1-97.) 

 

• We have used distinct colors for e for early germination and seedling establishment in Figure 5. We have also indicated early germination for Stages 1- 4 and seedling establishment as Stages 5 – 6.
• We have retained seed germination and protocorm development categorized as it is, because we followed our previous study (Zhang et al. 2022. Diversity, 14, 1119).

 

4. Try to use more informative subtitles in Discussion, rather than “Molecular identification…”, “Molecular identification…”, and “Symbiotic culture”, and move the satements about synergistic effects from conclusions to discussions.

• We have modified the subtitles as follows,

          4.1. Mycorrhizal fungal composition of Phalaenopsis japonica.

          4.2. Fungal effects on seed germination and seedling growth.

 

5. Lines 27, 221 and 685: “Ceratobasidiaceae were”: change to “fungi of Ceratobasidiaceae were” or “Ceratobasidiaceae was”

• Corrected .

 

6. Line 64: “Phalaenopsis”: change to “P.”

• We believe that we can retain it as it is.

 

7. Line 687: Qin et al. (2021), instead of 2023

• Corrected.

 

8. Line 694: the OTU CE6

• Corrected.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved accordingly, therefore I suggest to accept it for publication.