Effect of Short-Term Phosphorus Supply on Rhizosphere Microbial Community of Tea Plants

Round 1

Reviewer 1 Report

The authors analyzed the bacterial and  fungal communities on tea plants using Illumina MiSeq sequencing of 16SrRNA and ITS2 regions to assess how short-term phosphorus supply influenced the microbial community changes as well as the impact on soil and host nutrient status. The author concluded that mediate P fertilizer level best favored microbial diversity and functioning. 

The manuscript is well written and the data analysis is appropriate. I would like to address some points which can make the manuscript stronger. 

(1) L96-97 How did authors treat with the experimental soil? The different microbial communities in the pots may be not simply influenced by P rate, but possibly due to the different communities at the beginning of this experiment. Did authors consider it?

(2) L102-107 what was the environmental condition of this pot experiment?  Outdoor or greenhouse? How about the light strength, day/night time, temperature, soil moisture maintaining etc. ? Please specify those in the main text.

(3) L270 I strongly suggest putting Figure S3 in the main text. 

(4) L355-356 please clarify the ‘abundance’ and ‘diversity’ change of Glomeromycota.    For Illumina, sequencing abundance change is not necessarily correlated to diversity change of identified OTUs.

Author Response

Reviewer #1

Comments and Suggestions for Authors

The authors analyzed the bacterial and  fungal communities on tea plants using Illumina MiSeq sequencing of 16SrRNA and ITS2 regions to assess how short-term phosphorus supply influenced the microbial community changes as well as the impact on soil and host nutrient status. The author concluded that mediate P fertilizer level best favored microbial diversity and functioning. 

The manuscript is well written and the data analysis is appropriate. I would like to address some points which can make the manuscript stronger. 

Re: Thanks for your valuable comments.

Q1: L96-97 How did authors treat with the experimental soil? The different microbial communities in the pots may be not simply influenced by P rate, but possibly due to the different communities at the beginning of this experiment. Did authors consider it?

Re: The soil used for tea planting has been taken from the uncultivated land in Shengzhou Experimental Station of Tea Research Institute (29.74° N, 120.82° E), affiliated with Chinese Academy of Agricultural Sciences (TRI-CAAS), Shaoxing, China. The soil was mixed intensively and the clones of the same tea variety were planted. All the pots were put in a net house with the same management. Thus, we assumed that the pots were unified before the treatment with P fertilizer. On the other hand, we set six to eight replicates for each treatment and the growth of tea plants was responsive to P rates. As we know, microbial communities of rhizosphere soil were largely affected by plants. Since the plants were responsive to P rates, we think the different microbial communities in pots were mainly infuenced by P rates and plant responses instead of other factors.

Q2: L102-107 what was the environmental condition of this pot experiment?  Outdoor or greenhouse? How about the light strength, day/night time, temperature, soil moisture maintaining etc. ? Please specify those in the main text.

Re: The pots with P treatments were placed in a net house equipped with automatic sprinklers and sun-shading net to main the soil moisture from 25 to 30 (W%). The experimental site was in a typical subtropical region with average annual rainfall of 1200 mm and average annual temperature of 12.6 °C. The annual sunshine duration was nearly 2000 hours and daily sky sruface photosynthetically active radiation (PAR) was 110.38. The environmental condition of this pot experiment has been revised according to the comments (lines 95-102).

Q3: L270 I strongly suggest putting Figure S3 in the main text. 

 Re: Figure S3 has been moved to main text as Figure 4 accordingly.

Q4: L355-356 please clarify the ‘abundance’ and ‘diversity’ change of Glomeromycota. For Illumina, sequencing abundance change is not necessarily correlated to diversity change of identified OTUs.

 Re: We thanked the reviewer for pointing out the ambiguous descriptions. In fact, we used FUNGuild database to identify the AMF species as described in M&M section, the identified different AMF species could be used to calculate the diversity. While the abundance was calculated from the OTU table. The richness and diversity of Glomeromycota were decreased in our study (please see figure S2 and S4).

Author Response File: Author Response.docx

Reviewer 2 Report

Yang et al. investigated the responses of tea rhizosphere microbiomes to different P input rates. The work is well conducted, and I will recommend it to be accepted for Agronomy before some comments were addressed. 

Authors should tell us more information in part of Material and methods, such as the management of the experiment. Moreover, I recommend that authors should show us the amount of P, rather than the amount of P2O5, and why did choose this P gradient.

Author Response

Reviewer #2

Comments and Suggestions for Authors

Yang et al. investigated the responses of tea rhizosphere microbiomes to different P input rates. The work is well conducted, and I will recommend it to be accepted for Agronomy before some comments were addressed. 

Authors should tell us more information in part of Material and methods, such as the management of the experiment. Moreover, I recommend that authors should show us the amount of P, rather than the amount of P₂O₅, and why did choose this P gradient.

Re: Thanks for your suggestion. The pots with tea plants were placed in a net house equipped with automatic sprinklers and sun-shading net to main the soil moisture from 25 to 30 (W%). The experimental site was in a typical subtropical region with average annual rainfall of 1200 mm and average annual temperature of 12.6 °C. Regarding to the amount of P input, moderate-P (200 mg kg^-1) was calculated based on the recommended amount of P fertilizer application for medium-yield tea gardens in Zhejiang Province and high-P was close to the highest amount of P fertilizer applicated by local farm household. The unit of P fertilizer has been changed into the amount of P rather than P₂O₅ in the new submitted version, i.e. moderate-P, 87.3 mg P kg^-1 soil and high-P, 436.5 mg P kg^-1 soil. The text in M&M section has been revised accordingly (lines 108-111).

Author Response File: Author Response.docx

Reviewer 3 Report

please see attached file

Comments for author File: Comments.pdf

Author Response

Reviewer #3

1. Abstract

Q1. In summary, moderate P input favours the rhizosphere microbial diversity and functions. Therefore, we suggest that moderate P input should be 34 recommended in practical tea production.

➔ In summary, moderate P input favours the rhizosphere microbial diversity and functions in the short-term pot experiment. Therefore, we suggest that moderate P input should be recommended in practical tea production and further field test is required.

Re: Thanks for your valuable suggestion. The text in abstract has been revised in the new submitted version accordingly (lines 34-36).

2.1. Field site and experiment design

Q1. “granite-weathered red soil”

➔ Please describe the soil textural classes (sand/silt/clay or their ratio) for wild and experiment soil. This may be related to maintaining the concentration of P level in the soil.

Re: The soil used in our experiment was taken from local experimental site with the textural classes of 1.49%-30.00%-68.51% (sand-silt-clay). The text has been revised accordingly (lines 99-100).

2.2. Tea tree and soil sampling and preparation

Q1. line 177-179: It is said that rhizosphere soil was separated, and it seems that rhizosphere and rhizoplane are not clearly distinguished from each other in the pre-treatment method described.

Re: The rhizosphere compartment is composed of about 1 mm of soil, tightly attached to the root surface and not easily shaken from the root (Lakshmanan et al., 2017). However, the rhizoplane compartment microbiome is derived from a set of microorganisms on the root surface that cannot be removed by buffer washing but can be removed by ultrasound (Edwards et al., 2015). In this paper, we separated roots according to the method of PBS washing to obtain the rhizosphere soil.

Q2. It is necessary to define ‘rhizosphere’ in root associated soil (how far from the root is rhizosphere? How to separate from rhizoplane?)

Re: The rhizosphere compartment is composed of about 1 mm of soil, tightly attached to the root surface and not easily shaken from the root, while the rhizoplane compartment microbiome is derived from the suite of microbes on the root surface that cannot be removed by washing in buffer but is removed by sonication. In this study, we used washing buffer to seperate rhizosphere soil from the root attached with soil. According to the comment, we added the defination of rhizosphere in the text (lines 125-127).

2.3. Soil chemical properties and plant elemental analyses

Q1. ICP-AES ->Inductively coupled plasma atomic emission spectroscopy (ICP-AES)

Re: The full name instead of the abbreviation has been used in the new version (lines 135-136).

Q2. Please indicate the model number of the ICP

Re: The detailed information of Inductively coupled plasma atomic emission spectroscopy (ICP-AES, iCAP 6000 SERIES) has been revised accordingly (lines 135-136).

Q3. Please write how linearity (for accurate measurement) was derived. Did you use reference materials (standard P solution) before measuring samples?

Re: In the analysis, the standard curve of measured elements was established before analyzing the samples. The concentration range of the standard curve was set from 0 ppm to 100 ppm, then the instrument performed automatic peak finding. Thereafter, we opened the standardization icon, run the standard solution in turn and checked whether the spectral peak was interfered. After getting the standard curve, the element concentration of sample solution was determined according to the standard spectral line.

Q4. Please write the R² value for measuring P conc. using ICP.

Re: The correlation coefficient of the standard curve was 0.9998

Q5. Write a reference to the soil measurement method using ICP.

Re: The corresponding reference (Hamalovâ et al., 2020) has been inserted accordingly (line 139).

4.2. Effects of P input rate on rhizosphere microbiome of tea plant

Q1. Treatment of high concentrations of phosphoric acid appears to have reduced fungal diversity. Discuss the reason for the particular increase in chytridiomycota in the P1 treatment group, and the reason for the particular increase in Mortierellomycota in the P2 treatment group in relation to the properties of the phyllum and the symbiotic action with plant-fungs.

Re: Thanks for the reviewer’s valuable suggestions. We do agree with the reviewer;s opinion and have explained the particular increase in chytridiomycota in the P1 treatment group, and the reason for the particular increase in Mortierellomycota in the P2 treatment group in relation to the properties of the phylum and the symbiotic action with plant-fungs. Please see lines 366-377.

Q2. “Furthermore, the decrease of AM fungal species after P input in our study indicated that P fertilizer further decreased the fungal functions (Fig. S4).”

Just because high concentration of P decreases the diversity of fungi cannot necessarily be interpreted as decrease the function of fungi associated with plants.

Reason 1: The decrease in the diversity of fungi in general soil can be interpreted negatively, but the selective dominance of a particular phylum or genera in plant root or rhizoplane is not appropriate to interpret only as a negative phenomenon.

Reason 2: Various genera belongings to the Mortierellomycota are known to have symbiotic relation with their host plants, overcoming environmental stress caused by high concentrations of salts.

Therefore, consider in depth the discussion of detailed identification of fungi through the ITS gene in the high-concentration P treatment group.

Re: We thanked the reviewer for pointing out our mistake in the manuscript. We have revised the inappropriate description and added the potential functions of Mortierellomycota to further the depth of discussion. Please see lines 396-400.

References

Lakshmanan, V., P. Ray and K. D. Craven. Rhizosphere sampling protocols for microbiome (16S/18S/ITS rRNA) library preparation and enrichment for the isolation of drought tolerance-promoting microbes. Methods Mol Biol. 2017, 1631, 349-362.

Edwards, J., C. Johnson, C. Santos-Medellín, E. Lurie, N. K. Podishetty, S. Bhatnagar, J. A. Eisen and V. Sundaresan. Structure, variation, and assembly of the root-associated microbiomes of rice. Proc Natl Acad Sci U S A. 2015, 112(8), E911-920.

Hamalovâ, M., J. Hodslavská, P. Janos and V. Kanický. Determination of phosphorus, potassium, and magnesium in fertilizers by inductively coupled plasma-atomic emission spectroscopy and comparison with other techniques. J AOAC Int. 2020, 80(6), 1151-1155.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors have addressed all the review suggestion and made clear modification in the text.  Thus I recommend to accept in present form.

Reviewer 3 Report

The parts that need to be corrected and verified have been met