Application of AMF Alleviates Growth and Physiological Characteristics of Impatiens walleriana under Sub-Low Temperature
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments for author File: 
Comments.pdf
Minor editing of English language required.
Author Response
1、-The title is not sufficiently clear and therefore requires revision, avoid using effects of...
Response：
Title has been changed into ‘ Application of AMF alleviates Growth and Physiological Characteristics of Impatiens walleriana under Sub-Low Temperature ’.
In ABSTRAC section, shown in line 9
2、-In line 17, please revise the concept for tolerance and resistance.
Response:
It has been modified to tolerance, shown in line 26 of revised one.
3、-In line 13, please revise Author guideline for italic’s use.
Response:
Has been reworked according to the author's guidelines, shown in line 13 of revised one.
In INTRODUCTION section, shown in line 23
4、-Please revise Author guideline for cite use. In the text, reference numbers should be placed
in square brackets [ ].
Response:
[ ] have been re-added.
5、-In the introduction section, it is necessary to include a clear statement of the study's
objectives. The authors must provide a rationale for the selection of this research.
Response:
New in the INTRODUCTION section of the article is a description of the characteristics and advantages of the Impatiens walleriana, as well as the effects of cold stress on it.
6、-In line 19, please remove “et al.”
Response:
Already deleted, shown in line 67 of revised one.
7、-In line 33, the sentence should be rewritten to provide greater clarity
Response:
It has been rewritten to change the order of words, see the revised line 55-58 yellow highlight section.
8、-In line 33, please add the scientific names of plant species.
Response:
Already added, shown in line 54-55 of revised one.
9、-In line 48, please revise the concept for tolerance and resistance.
Response:
It has been modified to tolerance, shown in line 73 of revised one.
10、-I recommend the use of English Language Editing service.
Response:
Modified.
In MATERIALS AND METHODS section, shown in line 80
11、-Check the city and country to companies and products.
Response:
Already checked.
12、-Please add the characterization of Acaulospora morrowiae, Gigaspora gigantean and
Glomus clarum.
Response:
The bacterial agent used in this experiment is a mixed bacterial agent.There are a total of 14 kinds of microbial agents, of which the most important one is Glomus mosseae, which is screened out by previous experiments .
13、-In line 66-67, please specify the method employed for checking the number of propagules
in the AMF inoculum. It is important to inform multiplication data (host, substrate for
production, percentage of infectivity, inoculant multiplication time) and maintenance of the
mycorrhizal inoculum (storage conditions of the inoculum before use, considering the
medium and temperature).
Response:
It has been resived in the section 2.2.1. Hole Tray and Strain Preparation and In the section of 2.2.2 Seed Pretreatment, shown in line 86 and 104 of revised one.
Huai'an chaimihe agricultural technology company China was entrusted for microbial agent preparation. 2/3 of cultivate bowl was filled with sterilized sand, then spore liquid including Glomus mosseae was dropped in the sand. The ratio of bacterial liquid to sand in weight was 3%, spore liquid mixed with another 13 kinds of AMF dropped in the sand with 3% ratio of bacterial liquid to sand in weight. Host seeds(clover) was sowed in the sand after disinfect with 5% potassium permanganate soaking for 1h. And then transferred cultivate bowl to an artificial climate chamber for cultivation. Hogland nutrient solution was irrigated weekly. During harvesting, cut off the above ground stems and leaves of the host plant during harvesting, and place the pot in a room with a temperature of 25℃ and a humidity of 50% to dry. all the culture materials (including plant roots, hyphae, spores, and substrate) in the pot were collected and store them at room temperature at 25℃ for later treatments.
The component of the substrates were 60% peat, 20% vermiculite, and 20% perlite. The tray was disinfected with 75% alcohol before placing the substrates.
The propagation time of the inoculum were given as 50 days. For the infection rate, the determination of mycorrhizal infection rate was supplemented in the text, shown in the result and table S1.
14、-Why you have chosen to work with AMF inoculant containing Acaulospora morrowiae,
Gigaspora gigantean and Glomus clarum and not another AMF species? Please explain the
choice of these species.
Response:
From the previous studies, Glomus mosseae is good for abiotic stress mitigation in many kinds of plant. However, the infection rate of AMF is lower than other plants, we found that these microbial agent of mixed AMF have a good effect on the infection of Impatiens walleriana according to the pre-experiment screening done in the early stage. After inoculation, it was found that the low temperature resistance of Impatiens balsamina increased significantly, which provided a basis for the next low temperature resistance experiment of Impatiens walleriana.
15、-I recommend the use of English Language Editing service.
Response:
Modified.
In RESULTS section, shown in line 170
16、-The results section should be rewritten to provide greater clarity.
Response:
The results section has been rewritten.
17、-In line 131-132, replace “mycelium” with “intracellular hyphae”.
Response:
It has been modified to intracellular hyphae.
18、-In line 137, replace “vaccinated” with “inoculated”.
Response:
It has been modified to inoculated.
In DISCUSSION section,shown in line 314
19、-In line 253-254, please specify what kind of affects physiological and biochemical?, add
some comparisons.
Response:
Already modified, shown in line 336of revised one.
20、-In line 257, please specify what kind of tolerance to biotic and abiotic stresses.
Response:
Already modified.
21、-In line 265, please add brackets.
Response:
Already added.
22、-In line 283 and 309, please revise Author guideline for cite use. In the text, reference
numbers should be placed in square brackets [ ].
Response:
Already added.
23、-Discussion section must be divided into subheadings.
Response:
Already added.
In CONCLUSIONS section, shown in line 387
24、-Conclusions are too simply and vague.
Response:
Already modified, shown in line 388 of revised one.
25、-In references section please revise Authors guidelines.
Response:
Already modified.
In FIGURES
26、-There are too many figures, please consider some of them as supplementary material.
Response:
It's already in Figure S 1.
27、-In figure 1, please add scale bars length. This figure should be supplementary online
material. The photos provided are not clear and do not provide enough information that would
be important to understand the results. Arbuscules, which are key fungal structures, are not
seen or quantified to later extrapolate the rest of the results. The mere presence of vesicles
cannot justify the results obtained.
Response:
Already added.
28、-In figure 5, 6 and 7, please add scale bars length.
Response:
Already added, shown in figure 4,5 and 6 of revised one.
29、-Figure 8 must be improved, letter number is too small.
Response:
Already modified, shown in figure 7 of revised one.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsGeneral comments
The manuscript titled “Effects of AMF on the Growth and Physiological Characteristics of I. wallerana under Sub-Low Temperature” has been submitted to Horticulturae. In this study, authors have tested the efficacy of AMF in the enhancement of cold tolerance of I. wallerana plant. The physiological and biochemical characteristics of I. wallerana seedlings were evaluated.
They concluded that inoculation with AMF improve the growth and development of I. wallerana under Sub-Low Temperature, by increasing plant growth, stem diameter, Fv'/Fm' and Y(II), and by decreasing plasma membrane permeability, the accumulation level of O2, H2O2 and MDA. In my opinion, the results of the research are of interest to researchers. However, several paragraphs are insufficiently developed and need to be expanded to explore the ideas presented in greater depth.
I have listed some comments that help the authors to improve the manuscript.
Specific comments
Title: In the title, please give the full name of I. wallerana.
Abstract: please present the percentages of improvement and reduction to better promote and demonstrate the benefits of using mycorrhizal inoculum.
Keywords: Please put in alphabetical order. In addition, keywords should not repeat words found in the title of the manuscript.
Introduction:
L37: Please give the full name of the AMF, as this is its first appearance in the manuscript.
- If possible, add a brief overview of the plant studied, its interest, and some effects (symptoms) of cold stress in I. wallerana.
- It is important to add some publications of specific work on AMF, which has been used to alleviate abiotic stress in I. wallerana.
M&M
- Plant Material: Please provide more information on the two I. wallerana cultivars used in this study, particularly regarding the tolerance or sensitivity of the cultivars to cold stress.
L57: Please give more details on the preparation of the mycorrhizal inoculum for inoculating plants, giving the key stages.
L65: Please pay attention to the tense used in formulating the sentences in the manuscript. Using the past rather than the future tense is preferable, as the work has already been done.
L68: Please give more details on the pre-treatment of the seeds, are the seeds sterilised, how? are they pre-germinated, describe these steps.
L73: rhizobial root infection?
L82; L107: please clarify‘’ with 10 pots for each treatment and 3 replicates‘’ ‘’ five biological replicates”. what is the number of replicates 3 , 7 or 10 ?.
Results
The photos in Figure 1 are not visible and the magnification scale is not indicated. If possible, please replace them with clearer photos at a higher resolution, including an enlargement scale.
Discussion
-In the discussion section, the authors are asked to compare their results with other works that have used specific AMF, Acaulospora morrowiae, Gigaspora gigantean and Glomus clarum
Conclusion
L322: Please replace ‘vaccination’ with ‘innoculation’.
References
- Add the missing DOI and check all references citation in the text and list (volume number and pages).
Author Response
General comments
The manuscript titled “Effects of AMF on the Growth and Physiological Characteristics of I. wallerana under Sub-Low Temperature” has been submitted to Horticulturae. In this study, authors have tested the efficacy of AMF in the enhancement of cold tolerance of I. wallerana plant. The physiological and biochemical characteristics of I. wallerana seedlings were evaluated.
They concluded that inoculation with AMF improve the growth and development of I. wallerana under Sub-Low Temperature, by increasing plant growth, stem diameter, Fv'/Fm' and Y(II), and by decreasing plasma membrane permeability, the accumulation level of O2, H2O2 and MDA. In my opinion, the results of the research are of interest to researchers. However, several paragraphs are insufficiently developed and need to be expanded to explore the ideas presented in greater depth.
I have listed some comments that help the authors to improve the manuscript.
Specific comments
1、Title: In the title, please give the full name of I. wallerana.
Response:
Modified to Impatiens walleriana.
2、Abstract: please present the percentages of improvement and reduction to better promote and demonstrate the benefits of using mycorrhizal inoculum.
Response:
The section has been rewritten, shown in line 14-27 of revised one.
3、Keywords: Please put in alphabetical order. In addition, keywords should not repeat words found in the title of the manuscript.
Response:
The modification has been completed as required, shown in line 28 of revised one.
Introduction:
4、L37: Please give the full name of the AMF, as this is its first appearance in the manuscript.
Response:
Already modified, shown in line 59 of revised one.
5、- If possible, add a brief overview of the plant studied, its interest, and some effects (symptoms) of cold stress in I. wallerana.
Response:
Already modified, shown in line 32-39 of revised one.
6、- It is important to add some publications of specific work on AMF, which has been used to alleviate abiotic stress in I. wallerana.
Response:
A new literature has been added, shown in line 48 of revised one.
M&M
7、- Plant Material: Please provide more information on the two I. wallerana cultivars used in this study, particularly regarding the tolerance or sensitivity of the cultivars to cold stress.
Response:
We screened different colors of Impatiens walleriana. These two kinds of Impatiens walleriana have similar tolerance to sub-low temperature, shown in line 84 of revised one.
8、L57: Please give more details on the preparation of the mycorrhizal inoculum for inoculating plants, giving the key stages.
Response:
The bacterial agent used in this experiment is a mixed bacterial agent.There are a total of 14 kinds of microbial agents, of which the most important one is Glomus mosseae, which is screened out by previous experiments and has been added with specific references.The section 2.2.1. Hole Tray and Strain Preparation and the section of 2.2.2 Seed Pretreatment has been rewritten.
9、L65: Please pay attention to the tense used in formulating the sentences in the manuscript. Using the past rather than the future tense is preferable, as the work has already been done.
Response:
Has been modified.
10、L68: Please give more details on the pre-treatment of the seeds, are the seeds sterilised, how? are they pre-germinated, describe these steps.
Response:
We re-describe the process of soaking seeds in warm soup in this paper, shown in line 104-107 of revised one.
11、L73: rhizobial root infection?
Response:
The word " rhizobial root " is due to a previous mistranslation and has now been reworded to correct semantics, shown in line 110 of revised one.
12、L82; L107: please clarify‘’ with 10 pots for each treatment and 3 replicates‘’ ‘’ five biological replicates”. what is the number of replicates 3 , 7 or 10 ?.
Response:
It has been modified and the specific number of repetitions is given, shown in line 125 and 150 of revised one.
Results
13、The photos in Figure 1 are not visible and the magnification scale is not indicated. If possible, please replace them with clearer photos at a higher resolution, including an enlargement scale.
Response:
Already added a scale to the Figure and also put in Figure S1.
Discussion
14、-In the discussion section, the authors are asked to compare their results with other works that have used specific AMF, Acaulospora morrowiae, Gigaspora gigantean and Glomus clarum
Response:
In the early stage, it has been found that these bacteria play a role in cultivated crops. However, because Impatiens walleriana belongs to the family of Impatiens, its infection effect is not high, so the authors chose the mixed bacterial agent as the inoculant ( the mixed bacterial agent is widely used in production and get good outcomes). After inoculation, it was found that the low temperature tolerance of Impatiens walleriana increased significantly, which provided a basis for further research of Impatiens walleriana.
Conclusion
15、L322: Please replace ‘vaccination’ with ‘innoculation’.
Response:
Already modified.
References
16、- Add the missing DOI and check all references citation in the text and list (volume number and pages).
Response:
The inspection and supplement have been completed.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have responded to all questions and suggestions.
Please pay attention to the term used: in line 89, the authors used ‘bacteria’ instead of ‘fungi’.
Author Response
Comment 1 : Please pay attention to the term used: in line 89, the authors used ‘bacteria’ instead of ‘fungi’.
Response 1 : We replaced 'bacteria' with 'fungi' in lines 90 and 95.
Author Response File: 
Author Response.docx
