Impact of Aerated Irrigation Duration on the Growth of Greenhouse Grape Seedlings and Rhizosphere Soil Microorganisms
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript reports on an experiment where different times of aerated irrigation were applied on grapevine seedlings, and the effects on selected growth parameters of the plants and the microbial diversity of the soil were investigated.
The experimental aims are clear, the set-up is adequate, though some details in the material and methods section should be improved. The presentation of results may be improved, and the results do not always support the statements in the discussion.
The main problem I have found in the manuscript is related to the references cited, not always appropriate, not always corresponding between text and reference list, not always available on international databases. This is the main factor leading to the impossibility of recommending the manuscript for publication in its present form.
Following are more detailed comments on the manuscript:
Introduction:
L42 The definition of the role of soil is too simplified and the reference [5] doesn’t seem appropriate.
L48 – “crop roots become increasingly sensitive to hypoxic stress [7]” there is no relation between this sentence and the reference 7. Karki, H.; Bargali, K.; Bargali, S.S. Spatial and Temporal Trends in Soil N-mineralization Rates under the Agroforestry….
L47-48 and L53-55 present the same concept.
L61-62 I don’t find the relation between the sentence “The micro-bubbles possess a large surface area and long lifespan, making them effective for mitigating soil hypoxia [15]” and the reference 15. Su, N.H. Generalisation of Various Hydrological and Environmental Transport Models Using the Fokker–Planck Equation…
A citation such as Zhou, Y., Li, Y., Liu, X. et al. Synergistic improvement in spring maize yield and quality with micro/nanobubbles water oxygation. Sci Rep 9, 5226 (2019). https://doi.org/10.1038/s41598-019-41617-z would be more appropriate.
L64-65 and L68-70 present the same concept and reference.
Materials and methods:
L111 what do you mean by “both thick and thin two-year-old ‘Flame Seedless’ grape seedlings”?
L112 before it was stated that the experiment was carried out from April 2023. What happened between April and May 16? Were the plants acclimatized and then transplanted?
L147 I was not able to find the cited reference on online databases. Perhaps it is an article in Chinese. More details on the method used should be provided, together with a reference that may be easily consulted by readers.
L168 change refrigerator to freezer
L197 It should be specified that Duncan’s multiple comparisons has been used as a post-hoc test after a significant difference was found in ANOVA.
Results:
L229 How was root vigor estimated? It was not specified in the methods section. Here only the % variation between treatments and no absolute value is reported.
Root system activity is reported in the heading and table but not mentioned in the text.
L347 Stem diameter was not significantly affected by aeration treatment. However correlation between this parameter and Botryotrichum abundance is reported. Can you explain this fact and its physiological significance?
Discussion:
L377 The SPAD results do not show a clear cut increase in chlorophyll content with aeration, but a decrease in T1 and T3 in the first weeks. At the end of the experiment there are no significant differences between control and treatments.
L414-415 repeats the concept of L410-412
L489 data do not support the conclusion that T2 increases leaf SPAD values.
The discussion of possible physiological reasons for improvements in growth related to differences in microbiota is not exhaustive.
Also the complexity of interpreting the relationships between plant growth and microbes should be addressed.
Figures:
Figure 1 is very good, clearly showing the experimental set-up.
L133 rephrase the caption, the figure does not show the effect of different aerated irrigation but the setup of the experiment.
Figure 2: change the major tick tabs, it is sufficient to write 15 – 18 -21 – 24 – 27 and specify Days after aeration on the axis title.
L221 eliminate “the same below”. Repeat “different lowercase letters indicate significant differences among treatments (P < 0.05)” in each caption where it is necessary.
L232 specify dry biomass.
References:
Following are some examples of problems encountered with references.
Ref 4: not found on database; DOI sends to article in Chinese with different authors.
Ref 6: not found on database. Link leads to a message in Chinese
Ref 13: not found
Ref 14: article on google scholar, but DOI not found
Ref 16: not found on Google, the link leads to a Chinese website
Ref 47: not found on database; DOI sends to article in Chinese
Ref 52: not found on database
I may also suggest to integrate the discussion with some of the following:
Finkel 2017 Understanding and exploiting plant beneficial microbes. Curr. Opin. Plant Biol. 38, 155–163.
Darriaut et al. 2022 Soil composition and rootstock genotype drive the root associated microbial communities in young grapevines. Front Microbiol. 2022
Bamba et al. 2024 Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus. FEMS Microbiology Ecology, Volume 100, Issue 5, May 2024
Author Response
Introduction:
Comments 1: L42 The definition of the role of soil is too simplified and the reference [5] doesn’t seem appropriate.
Response 1:Thank you for your suggestion. We have added the definition of soil action in lines 44-47: Soil, as an important resource in agricultural production systems, can provide the necessary water and nutrients for crop growth and development, and soil conditions such as nutrient effectiveness, porosity, and aeration can significantly affect crop root structure[5], and I apologize for the oversight in reference [5] citation earlier, and we have replaced it with a more appropriate literature.
[5]Luo, Z.K.; Zhang, S.; Zhao, Z.G.; Minasny, B., Chang, J.F., Huang, J.Y.; Li, B.H.; Shi, Z.; Wang, M.M.; Wu, Y.S.; et al. Soil-smart cropping for climate-smart production. Geoderma 2024, 451,117061, https://doi.org/10.1016/j.geoderma.2024.117061.
Comments 2: L48 – “crop roots become increasingly sensitive to hypoxic stress [7]” there is no relation between this sentence and the reference 7. Karki, H.; Bargali, K.; Bargali, S.S. Spatial and Temporal Trends in Soil N-mineralization Rates under the Agroforestry….
Response 2: Thank you for your suggestion. We apologize for the previous mistake. And we have replaced reference 7 .
[7] Ma, J.W.; Rukh, G.; Ruan, Z.Q.; Xie, X.C.; Ye, Z.Q.; Liu, D. Effects of Hypoxia Stress on Growth, Root Respiration, and Metabolism of Phyllostachys praecox. Life 2022, 12, 808, https://doi.org/10.3390/life12060808.
Comments 3: L47-48 and L53-55 present the same concept.
Response 3: Thank you for your suggestion. We have deleted the repetition in lines 47-48 and 53-55 in the revised draft: which can adversely affect their growth and overall health.
Comments 4:L61-62 I don’t find the relation between the sentence “The micro-bubbles possess a large surface area and long lifespan, making them effective for mitigating soil hypoxia [15]” and the reference 15. Su, N.H. Generalisation of Various Hydrological and Environmental Transport Models Using the Fokker–Planck Equation…
A citation such as Zhou, Y., Li, Y., Liu, X. et al. Synergistic improvement in spring maize yield and quality with micro/nanobubbles water oxygation. Sci Rep 9, 5226 (2019). https://doi.org/10.1038/s41598-019-41617-z would be more appropriate.
Response 4: Thank you for your suggestion. We have replaced reference [15] with: Zhou, Y.P.; Li, Y.K.; Liu, X.J.; Wang, K.Y.; Muhammad, T. Synergistic improvement in spring maize yield and quality with micro/nanobubbles water oxygation. Scientific Reports 2019, 9, 1-10, https://www.sci-hub.ee/10.1038/s41598-019-41617-z.
Comments 5: L64-65 and L68-70 present the same concept and reference.
Response 5: Thank you for your suggestion. We have removed concepts and references in L68-70 that are duplicated in L64-65 in the revised manuscript.
Materials and methods:
Comments 6: L111 what do you mean by “both thick and thin two-year-old ‘Flame Seedless’ grape seedlings”?
Response 6: Thank you for your suggestion. I mean that “Uniformly grown two-year-old own-rooted ‘Flame Seedless’ grape seedlings ”
Comments 7: L112 before it was stated that the experiment was carried out from April 2023. What happened between April and May 16? Were the plants acclimatized and then transplanted?
Response 7: Thank you for your suggestion. Between April and May 16, 2023 we were preparing the aerated irrigation units needed for the test. And the grapevines were transplanted after acclimatization [We have added in the revised manuscript: (L114-115)Uniformly grown two-year-old own-rooted ‘Flame Seedless’ grape seedlings were planted in the center of each pot after acclimatization on May 16, 2023.]
Comments 8: L147 I was not able to find the cited reference on online databases. Perhaps it is an article in Chinese. More details on the method used should be provided, together with a reference that may be easily consulted by readers.
Response 8: Thank you for your suggestion. We have added more details of the methods used in the revised manuscript and rechecked the references[31].
(L150-161) Root activity was estimated using the modified triphenyl tetrazolium chloride (TTC) method following root removal [31]. The fresh roots were washed with dH2O thrice, blotted on filter paper, and then stored at 4 °C to be used same day. Standard curve was plotted based on the spectrophotometric absorption (λ= 485 nm) of different amounts of TTC (1 g·0.1 L-1) solution with Na2S2O4 and ethyl acetate. Roots were then sliced into one centimeter pieces and immersed in 10 mL of an equally mixed solution of TTC (0.4%) and phosphate buffer (0.1 mol·L−1, pH 7.0) and kept in the dark for 3 h at 37 °C. Subsequently, 2 mL of H2SO4 (1 mol·L−1) was added to stop the reaction. The immersed root tips were dried with filter paper and extracted with ethyl acetate. The extracted solution was transferred into a tube with ethyl acetate cleaning solution to a total volume of 10 mL, and absorbance was read at 485 nm. Root activity was calculated from the standard curve and expressed as TTC reduction intensity: ㎍·g-1·h-1.
We have modified reference [31] to read: Richter, A.K.; Frossard, E.; Brunner, I. Polyphenols in the woody roots of Norway spruce and European beech reduce TTC. Tree Physiol. 2007, 27, 155–160. https://www.sci-hub.ee/10.1093/treephys/27.1.155.
Comments 9: L168 change refrigerator to freezer
Response 9: Thank you for your suggestion. We have revised it in the revised manuscript.[(L183)and stored in a freezer at -80 ℃ for subsequent 16S and ITS high-throughput genome sequencing.]
Comments 10: L197 It should be specified that Duncan’s multiple comparisons has been used as a post-hoc test after a significant difference was found in ANOVA.
Response 10: Thank you for your suggestion. We have revised it in the revised manuscript. [(L213-213)Duncan’s multiple comparisons has been used as a post-hoc test after a significant difference was found in ANOVA(P < 0.05].
Results:
Comments 11: L229 How was root vigor estimated? It was not specified in the methods section. Here only the % variation between treatments and no absolute value is reported.
Root system activity is reported in the heading and table but not mentioned in the text.
Response 11: Thank you for your suggestion. We have supplemented the calculation of root activity in the Materials and Methods section of the revised manuscript. And we have added the absolute values of the differences in root vigor among treatments in the Results section.[(L243-247)Root activity under different treatments showed in descending order as T2 (0.0520 ㎍·g-1·h-1) > CK (0.0482 ㎍·g-1·h-1) > T1 (0.0458 ㎍·g-1·h-1) > T3 (0.0455 ㎍·g-1·h-1), with T2 treatment yielding the highest value, which was 7.88%, 13.8% and 13.5% higher than CK, T1 and T3 treatments, respectively.]
Comments 12: L347 Stem diameter was not significantly affected by aeration treatment. However correlation between this parameter and Botryotrichum abundance is reported. Can you explain this fact and its physiological significance?
Response 12: Thank you for your suggestion. This is because the role of microbial communities on plants is closely related to species as well as environmental conditions. And since plant root phenotypes are more sensitive to differences in soil microbial communities than stem and shoot phenotypes(Bamba et al., 2024), the results of this study showed that stem thickness was not significantly affected by aeration treatments and there was no significant correlation with Botryotrichum abundance.
(Bamba et al. 2024 Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus. FEMS Microbiology Ecology, Volume 100, Issue 5, May 2024)
Discussion:
Comments 13: L377 The SPAD results do not show a clear cut increase in chlorophyll content with aeration, but a decrease in T1 and T3 in the first weeks. At the end of the experiment there are no significant differences between control and treatments.
Response 13: Thank you for your suggestion. We apologize for the inaccurate description of the effect of aerated irrigation on leaf SPAD values of grapevine seedlings due to our error. And we have made changes to this section in the revised version of the manuscript as follows: (L398-400)The results of this study showed that T2 treatment effectively increased plant leaf SPAD values, and at 15 d after aerated irrigation, leaf SPAD values were significantly lower under T1 and T3 treatments compared to CK treatment.
Comments 14:L414-415 repeats the concept of L410-412
Response 14: Thank you for your suggestion. We have removed the duplication of L414-415 with L410-412 in the revised manuscript: Aeration into the soil significantly increases the abundance and diversity of bacterial communities in the soil.
Comments 15: L489 data do not support the conclusion that T2 increases leaf SPAD values.
Response 15: Thank you for your suggestion. We are very sorry for the incorrect description of the leaf SPAD values in the conclusion section due to our previous mistake. We have checked and corrected the description of the effect of aerated irrigation treatments on the SPAD values of grapevine leaves and made changes to the conclusions section. (L525-528:An aerated irrigation duration of 20-minute significantly increased plant height, above-ground and below-ground biomass, total root length, and root volume in greenhouse grapes compared to other treatments. Additionally, a duration of 10 minutes notably increased the number of root tips.)
Comments 16: The discussion of possible physiological reasons for improvements in growth related to differences in microbiota is not exhaustive.Also the complexity of interpreting the relationships between plant growth and microbes should be addressed.
Response 16: Thank you for your suggestion. We have added to the discussion of the physiological reasons why differences in microbial communities may lead to improved growth and the complexity of the relationship between plant growth and microorganisms in sections 4.3 and 4.4 of the revised manuscript ( L432-440, L478-480, L482-488, and L499-502 red words sections for details).
Figures:
Comments 17: Figure 1 is very good, clearly showing the experimental set-up
Response 17: Thank you very much for your affirmation of Figure 1.
Comments 18: L133 rephrase the caption, the figure does not show the effect of different aerated irrigation but the setup of the experiment.
Response 18: Thank you for your suggestion.We have changed the title of L133 to “Schematic diagram of experimental design” in the revised manuscript.
Comments 19: Figure 2: change the major tick tabs, it is sufficient to write 15-18 -21-24-27 and specify Days after aeration on the axis title.
Response 19: Thank you for your suggestion. We have modified the Figure 2 horizontal coordinate representation in the revised manuscript.
Comments 20: L221 eliminate “the same below”. Repeat “different lowercase letters indicate significant differences among treatments (P < 0.05)” in each caption where it is necessary.
Response 20: Thank you for your suggestion. We have already deleted “the same below” in the revised manuscript. and repeated “different lowercase letters indicate significant differences among treatments (P < 0.05)” in each heading.
Comments 21: L232 specify dry biomass.
Response 21: Thank you for your suggestion. We have specified dry biomass in the revised manuscript 3.1.2 section.
References:
Comments 22:Ref 4: not found on database; DOI sends to article in Chinese with different authors.
Response 22: Thank you for your suggestion. We have rechecked and corrected the DOI of reference [4] in the revised manuscript.
(https://link.oversea.cnki.net/doi/10.13855/j.cnki.lygs.2019.01.001.)
Comments 23:Ref 6: not found on database. Link leads to a message in Chinese
Response 23: Thank you for your suggestion. We have rechecked and corrected the DOI of reference [6] in the revised manuscript.
(https://doi.org/10.11766/trxb201607060270)
Comments 24: Ref 13: not found
Response 24: Thank you for your suggestion. We have rechecked and corrected the DOI of reference [13] in the revised manuscript.
(https://doi.org/10.3969/j.issn.1000-2561.2022.01.015)
Comments 25: Ref 14: article on google scholar, but DOI not foun-d
Response 25: Thank you for your suggestion. We have changed reference [14] in the revised manuscript:
Seridou, P.; Kalogerakis, N. Disinfection applications of ozone micro- and nanobubbles. Environmental Science: Nano 2021, 8, 3493-3510, https://doi.org/10.1039/D1EN00700A
Comments 26:Ref 16: not found on Google, the link leads to a Chinese website
Response 26: Thank you for your suggestion. We have rechecked and corrected the DOI of reference [16] in the revised manuscript.
(https://doi.org/10.7606/j.issn.1000-7601.2022.06.12)
Comments 27: Ref 47: not found on database; DOI sends to article in Chinese
Response 27: Thank you for your suggestion. Because of the additions to the discussion section, the reference order in the revised manuscript was changed from the original manuscript, with the reference [47] ordering changed to [50], and the DOIs of the corrected documents were checked and corrected.
(https://link.oversea.cnki.net/doi/10.13344/j.microbiol.china.220899)
Comments 28: Ref 52: not found on database
Response 28: Thank you for your suggestion. Because of the additions to the discussion section, the reference order in the revised manuscript was changed from the original manuscript, with the reference [52] ordering changed to [60], and the DOIs of the corrected documents were checked and corrected.
(https://doi.org/10.3969/j.issn.1000-6850.2007.07.101)
Comments 29:I may also suggest to integrate the discussion with some of the following:
- Finkel 2017 Understanding and exploiting plant beneficial microbes. Curr. Opin. Plant Biol. 38, 155–
- Darriaut et al. 2022 Soil composition and rootstock genotype drive the root associated microbial communities in young grapevines. Front Microbiol. 2022
- Bamba et al. 2024 Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus. FEMS Microbiology Ecology, Volume 100, Issue 5, May 2024
Response 29: Thank you for your suggestion.
- Reference “Finkel 2017 Understanding and exploiting plant beneficial microbes. Curr. Opin. Plant Biol. 38, 155–163” Sorted in the revised manuscript as [41]. We have added “Previous studies have shown that various agricultural practices can have a profound impact on microbial communities[41], and rhizosphere soil aeration significantly affects the abundance and diversity of soil microbial communities in the rhizosphere soil of crops [42-43].”
- Reference “Darriaut et al. 2022 Soil composition and rootstock genotype drive the root associated microbial communities in young grapevines. Front Microbiol. 2022” Sorted in the revised manuscript as [40]. We have added “Rhizosphere soil microorganisms are an important bioindicator indicator for assessing plant health and productivity[40].”
- Reference “Bamba et al. 2024 Synergistic effects of plant genotype and soil microbiome on growth in Lotus japonicus. FEMS Microbiology Ecology, Volume 100, Issue 5, May 2024 ” Sorted in the revised manuscript as [44]. We have added “And climatic conditions, soil type, aeration intensity, field management, and other environmental conditions can influence plant physiology and soil microbial activity, thereby affecting the structural composition of the rhizosphere microbial community[44].”
- Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsCongratulations for your research paper !
Comments for author File: Comments.pdf
Author Response
Dear Reviewer:
Thank you very much for taking the time to review our manuscript and for providing such detailed and constructive feedback. We greatly appreciate your recognition of this study and article. If you have any questions, feel free to contact me.
Yours sincerely,
Liang Yinchi
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
The work is very interesting, simple, and well-designed. The topic is relevant and the method innovative. Given that special attention is paid to the quality of the grapevine and the issue of irrigation, restrictions, etc., experiment with reduced, but optimal irrigation seems very useful, especially considering the advantages of the aeration method. Which can have quite important applications in the case of grapevines, as a perennial crop and, depending on the climate, can be grown on very heavy soils, with poor drainage and problems with aeration and microbiological activity. However, this research is in controlled conditions, but it seems like a good basis for further research and optimization. Perhaps you could briefly mention possible problems, for example, if there is a blockage, do air bubbles also help in the longevity of the irrigation system itself, etc. It would be interesting to conduct the results at a time when the grapevine is fruitful, for example, five years old and more. All in all, an interesting research and idea.
Please find attached the pdf of the paper with a few more comments.
Kind Regards,
Reviewer
Comments for author File: Comments.pdf
Author Response
Comments 1: how many plants, replicates per treatment?
were they own-rooted or on rootstock, if on rootstock, please indicate which one?
Response 1: Thank you for your suggestion. There were a total o-f three treatments in this experiment with nine plants per treatment and single replications. All grapevines used in this experiment wer-e own-rooted. It has been added to L91 and L114 in the revised manuscript.(L19: Our experiment focuses on two-year-old own-rooted ‘Flame Seedless’grape seedlings; L114: Uniformly grown and thickness two-year-old own-rooted ‘Flame Seedless’ grape seedlings were planted in the center of each pot after acclimatization on May 16, 2023 .)
Comments 2: very nice image!
here you can think of highlighting treatments with different colors, either plants are in different colors or pots, or CK, T1, T2 and T3 can be included in the image, above corresponding treatment row, (with a little symbolic clock visually indicating time for each treatment - optional)
Response 2: Thank you for your suggestion. We made changes to Fig. 1 in the revised manuscript: treatment names (CK, T1, T2, T3) were added above each row of plants.
Comments 3: L146-L147
Response 3: Thank you for your suggestion. We have added and refined the assay for root activity in section 2.2.2 of the revised manuscript Materials and Methods.
Root activity was estimated using the modified triphenyl tetrazolium chloride (TTC) method following root removal [31]. The fresh roots were washed with dH2O thrice, blotted on filter paper, and then stored at 4 °C to be used same day. Standard curve was plotted based on the spectrophotometric absorption (λ= 485 nm) of different amounts of TTC (1 g/0.1 L) solution with Na2S2O4 and ethyl acetate. Roots were then sliced into one centimeter pieces and immersed in 10 mL of an equally mixed solution of TTC (0.4%) and phosphate buffer (0.1 mol·L−1, pH 7.0) and kept in the dark for 3 h at 37 °C. Subsequently, 2 mL of H2SO4 (1 mol·L−1) was added to stop the reaction. The immersed root tips were dried with filter paper and extracted with ethyl acetate. The extracted solution was transferred into a tube with ethyl acetate cleaning solution to a total volume of 10 mL, and absorbance was read at 485 nm. Root activity was calculated from the standard curve and expressed as TTC reduction intensity: ㎍·g-1·h-1.
Comments 4: L155-L156
Response 4: Thank you for your suggestion. We have verified the root scanner and image analysis software models and have made changes in the revised manuscript.(The root systems were then scanned with a root scanner, and the scanned images were analyzed by WinRHIZO (Canada)).
Comments 5: maybe you can use the abbreviation dpa (days post aeration)? also the first time in the text where the full term is mentioned, state the abbreviation and continue using the abbreviation
Response 5: Thank you for your suggestion. We have modified the Figure 2 horizontal coordinate representation in the revised manuscript.
Comments 6: Are in the graphs indicated standard error or standard deviation?
Response 6: Thank you for your suggestion. The graphs in this manuscript show standard errors.
Comments 7: Perhaps because in a shorter period, shorter irrigation would encourage the plant to grow, so shorter intervals do not affect overall success as much, but they encourage the growth of young roots.
e.g. Under severe drought, the finer roots/root tips are the first to suffer and are similar to the leaves in their sensitivity to drought. (even though root in general is the most resistant part of grapevine under drought)
Response 7: Thank you for your suggestion. Thank you for your valuable feedback and insightful suggestions. I understand your point regarding the potential influence of shorter irrigation intervals on promoting the growth of younger roots within a shorter time frame.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Authors, the changes made have improved the quality of the manuscript. I am satisfied by the answers to my comments on the previous version.
