Arbuscular Mycorrhizal Fungi Improve Growth, Photosynthetic Activity, and Chlorophyll Fluorescence of Vitis vinifera L. cv. Ecolly under Drought Stress

Round 1

Reviewer 1 Report

In my opinion the manuscript  has improved satisfactorily and  can be published

Author Response

Thank you for reviewing the manuscript and giving the positive comments.

Reviewer 2 Report

 

GENERAL FINDINGS

The effects of arbuscular mycorrhizal fungi on grapevine physiology in interaction with water stress is of high interest.

The experiments were well designed and it became clear the effects of arbuscular mycorrhiza in improving vine growth and photosynthetic activity, but some more information on the water stress level of the vine might be appropriate to better discuss the results: the authors report the foliar water content, but in my opinion it is also necessary the foliar water potential to have an indication of the actual water stress level reached. So, my suggestion is to add this parameter, or in its absence indicate in the discussion that more specific parameters on the water stress indication might be appropriate in further studies.

Furthermore, it is irrelevant to underline some possible effects on the basis of non-statistically different data.

 

SPECIFIC REVISIONS

Page 5, lines 199-202: since the statistical differences were not significant this sentence is irrelevant “The relative water content in the leaves was numerically higher in mycorrhizal than non-mycorrhizal plants that were well-watered; however, the difference was not significant.”

Page 6 line 228: Tab. 1 should be Tab. 2

Page 6 line 225-238: it is appropriate to refer mainly to statistically different data as reported in table 2 and not to the percentage of variation even if not statistically significant.

Page 8 lines 263-267: it is important to give some indication on the level of water stress about the sensibility of GFS-3000 and FluorCam FC 800-C/1010 and not just “prolonged water stress”

Page 9 Figure 5: why is there no NPQ parameter as in Figure 4?

Author Response

Response to the reviewers

The effects of arbuscular mycorrhizal fungi on grapevine physiology in interaction with water stress is of high interest.

 

The experiments were well designed and it became clear the effects of arbuscular mycorrhiza in improving vine growth and photosynthetic activity, but some more information on the water stress level of the vine might be appropriate to better discuss the results: the authors report the foliar water content, but in my opinion it is also necessary the foliar water potential to have an indication of the actual water stress level reached. So, my suggestion is to add this parameter, or in its absence indicate in the discussion that more specific parameters on the water stress indication might be appropriate in further studies.

 

Furthermore, it is irrelevant to underline some possible effects on the basis of non-statistically different data.

 

Thank you for taking your time to review this manuscript. We really appreciate all your comments and suggestions. According to your advice, we amended the relevant part in the manuscript. The discussion relating to foliar water potential parameter has been added in Line 328-329.

 

1. Page 5, lines 199-202: since the statistical differences were not significant this sentence is irrelevant “The relative water content in the leaves was numerically higher in mycorrhizal than non-mycorrhizal plants that were well-watered; however, the difference was not significant.”

 

Reply: Thank you for your advice. We had revised in Line 199-200.

 

2. Page 6 line 228: Tab. 1 should be Tab. 2

 

Reply: Thank you for your reminder. We had revised in Line 227.

 

3. Page 6 line 225-238: it is appropriate to refer mainly to statistically different data as reported in table 2 and not to the percentage of variation even if not statistically significant.

 

Reply: Thank you for your advice. We had revised in Line 225-228.

 

4. Page 8 lines 263-267: it is important to give some indication on the level of water stress about the sensibility of GFS-3000 and FluorCam FC 800-C/1010 and not just “prolonged water stress”

 

Reply: Thank you for your advice. We had added the information in Line 262-265.

 

5. Page 9 Figure 5: why is there no NPQ parameter as in Figure 5?

 

Reply: The FluorCam FC 800-C/1010 system was provided by the large-scale instrument sharing platform. The technical staff taught and showed how to use the equipment. However, she did not know how to get the NPQ parameter. Other people used this system all only acquired for Fv/Fm, Fv/Fo and photos. So, there was no NPQ parameter in Figure 5.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript by Qiuhong Ye et al., entitled “Arbuscular mycorrhizal fungi improve growth, photosynthetic 2 activity and chlorophyll fluorescence of Vitis vinifera L. cv. 3 Ecolly under drought stress” addresses the answers of a host plant and an arbuscular mycorrhizal fungus to water stress. The topic is really interesting and adequate. The manuscript style is OK. Nevertheless, to have the paper suitable for publication in Agronomy, the authors should address several points:

- Line 40 the authors mention “abundant research” but give only one example and one reference. Please give more or add references of review papers that addressed the topic.

- Lines 90 and 91, and following occurrences in the paper: please use italic style for Latin names

- the authors must indicate the supplier of their inoculum and the method of preparation of the inoculum (pots, containers, in-vitro,...). This, to know the purity of the inoculum

- are the fungi in the inoculum are deposited in a reference collection such as the International Bank of Glomeromycota? This would be useful to compare the results obtained with those of other possible studies using one or more of those strains

- the experiment was conducted over 9 months but the authors state that "water stress treatments were stopped watering". This is not clear. Over such a long period the plants could not survive without watering. Please detail the protocol for the duration of the experiment

- furthermore, this non-watering also results in an increase in the temperature of the soil, so it is not probably just a question of water stress. Instead, I would have recommended reducing the amount of watering over a longer period

- Figure 1: how were standard error or deviation calculated? The look really équal for all conditions. In some other figures the standard error or deviation is really weak for 15 plants. But no number of analysed plants is indicated. Was it n=15 for all analysis and treatments?

- When we look at figure 2, apart from the differences in leaf size, it is not obvious to observe any difference between the treatments. The gradient from right to left observed on all leaves is surprising. Is this due to the lighting angle?

- the photographs shown in figure supplementary material 1 are not of very good quality and are difficult to analyze. On the photographs a to d we can see spores and I think like the authors that these roots have been mycorrhized. But I don't see any arbuscule. No scale is indicated on these photographs. It is sometimes difficult to orientate oneself because one cannot see the central cylinder. Also, on the photo f we can see fungal structures. Is it a pathogen? A mycorrhizal fungus? Impossible to say. Two hours in an autoclave is perhaps not enough to sterilize a soil. The authors could have presented a kinetic of mycorrhization which would have allowed to put their observations in comparison with the intensity of the mycorrhization. When was it set up? What other fungi are observed in f? What is photo a focused on; the spores look as big as the root?

 

Author Response

Response to the reviewers

The manuscript by Qiuhong Ye et al., entitled “Arbuscular mycorrhizal fungi improve growth, photosynthetic activity and chlorophyll fluorescence of Vitis vinifera L. cv. Ecolly under drought stress” addresses the answers of a host plant and an arbuscular mycorrhizal fungus to water stress. The topic is really interesting and adequate. The manuscript style is OK. Nevertheless, to have the paper suitable for publication in Agronomy, the authors should address several points:

Thank you for taking your time to review this manuscript. We really appreciate all your comments and suggestions. According to your advice, we amended the relevant part in the manuscript. All of your questions were answered one-by-one. Additionally, we revised the language by English editing service of MDPI.

1. - Line 40 the authors mention “abundant research” but give only one example and one reference. Please give more or add references of review papers that addressed the topic.

Reply: Sorry to make misunderstanding due to confused writing. We had changed the writing and also added more references in Line 43.

2. - Lines 90 and 91, and following occurrences in the paper: please use italic style for Latin names.

Reply: Thank you for your reminder. We had checked italic style for Latin names throughout the paper.

3. - the authors must indicate the supplier of their inoculum and the method of preparation of the inoculum (pots, containers, in-vitro,...). This, to know the purity of the inoculum.

Reply: We had added the information in Line 102-108 as requested (Line number will be normal when close “Track change” function).

4. - are the fungi in the inoculum are deposited in a reference collection such as the International Bank of Glomeromycota? This would be useful to compare the results obtained with those of other possible studies using one or more of those strains.

Reply: The fungi are deposited in INVAM (International culture collection of (vesicular) arbuscular mycorrhizal fungi). The link of INVAM (https://invam.wvu.edu/) is also listed on the website of International Bank for Glomeromycota.

5. - the experiment was conducted over 9 months but the authors state that "water stress treatments were stopped watering". This is not clear. Over such a long period the plants could not survive without watering. Please detail the protocol for the duration of the experiment.

Reply: We had revised the writing in Line 100-101 and 106-110 as requested. The grapevines were under the regular water management for five months after inoculation. Then started the water stress treatment.

6. - furthermore, this non-watering also results in an increase in the temperature of the soil, so it is not probably just a question of water stress. Instead, I would have recommended reducing the amount of watering over a longer period.

Reply: Thank you for your recommendation. We agree that your drought stress treatment would be better and will use in the future. As for this study, we referred Nikolaos Nikolaou et al. (2003) study on grapevine with AMF. They stopped watering to conduct the water stress treatment. We thought it’s precise and faster. Also, we did not realize the influence of temperature increase caused by non-watering on plants. Thank you for your advice.

7. - Figure 1: how were standard error or deviation calculated? The look really équal for all conditions. In some other figures the standard error or deviation is really weak for 15 plants. But no number of analyzed plants is indicated. Was it n=15 for all analysis and treatments?

Reply: The data were analyzed by Tukey’s test. The standard error from result of Tukey’s test was equal for all treatments. We chose the plants with similar vigor for measurements, not 15 plants for analysis. We had indicated the number of analyzed plants in legends.

8. - When we look at figure 2, apart from the differences in leaf size, it is not obvious to observe any difference between the treatments. The gradient from right to left observed on all leaves is surprising. Is this due to the lighting angle?

Reply: The green area was different in each leaf. However, it’s not very obvious to observe, especially when leaf size is different. The FluorCam FC 800-C/1010 system exported the data at the end and the photo was an assistant tool to show the result. For another question, we also thought the gradient from right to left on leaves was due to the lighting angle.

9. - the photographs shown in figure supplementary material 1 are not of very good quality and are difficult to analyze. On the photographs a to d we can see spores and I think like the authors that these roots have been mycorrhized. But I don't see any arbuscule. No scale is indicated on these photographs. It is sometimes difficult to orientate oneself because one cannot see the central cylinder. Also, on the photo f we can see fungal structures. Is it a pathogen? A mycorrhizal fungus? Impossible to say. Two hours in an autoclave is perhaps not enough to sterilize a soil. The authors could have presented a kinetic of mycorrhization which would have allowed to put their observations in comparison with the intensity of the mycorrhization. When was it set up? What other fungi are observed in f? What is photo a focused on; the spores look as big as the root?

Reply: We added more photographs. It could see arbuscule in new photos. The roots in photos were taken after five-month inoculation. We are not sure what it is in photo f as well. As your experience, two hours in the autoclave is perhaps not enough to sterilize a soil. However, this experiment focused on the effect of AMF on grapevines. We did not pay much attention to the mycorrhization. Thank you for your suggestion. We will consider this part if the experiment could be continued. As for photo a, we put it just because it found spores and looked clear.

 

Reference:

Nikolaou, N.; Angelopoulos, K.; KARAGIANNIDIS, N. Effects of Drought Stress on Mycorrhizal and Non-Mycorrhizal Cabernet Sauvignon Grapevine, Grafted onto Various Rootstocks. Experimental Agriculture 2003, 39, doi:10.1017/S001447970300125X.

Reviewer 2 Report

General comments

In general, this paper gives interesting results on the effect of mycorrhizal fungi (AMF) on the growth and some physiological parameters of own rooted grapevine (Ecolly cultivar) under water stress. The entire study and its results rest on the fact that the mycorrhizal fungi improve   the root and shoot growth, leaf chlorophyll content and photosynthetic parameters. Even similar results have been previously reported by others, the findings of this manuscript could be interesting if some failures had been avoided.

Materials and Methods

Ln.89:  The vine in most viticultural regions is usually cultivated grafted onto rootstocks resistant to phylloxera. Why the authors used own rooted plants? The use of own rooted vines of a local cultivar in the present experimentation is of limited interest to international grapevine growing.

The pot medium was : cinammon soil. Please use the scientific category of the soil.

Ln 96: Plant size or vigour is very low.  Shoot dry weight of 2-4 g is very low repr esenting a pruning weight of 5- 8 g and a shoot length of about 45cm with 10-12 nodes and very small leaves. I think that the length of the cuttings from which the experimental vines created   was about 35cm? It is impossible to use experimental vines in the same year of their creation.  Thus, the measurement conditions may have yielded non-useful results for grapevine growing. In my opinion the experiment should be done on plants at least two years old and planted in 10 L pots.

There are several unclear issues :

Ln 98: The authors may give more detailed information on the mycorrhizal inoculation:

-The commercial name and the company producing the mycorrhizal inoculant is not reported by authors.

-Ηow the experimental vines were inoculated and how the inoculum incorporated in pot medium and the rhizosphere (the authors state the beginning of experimentation at January 2021 and the inoculation at 25March2021).

-The inoculum was a troof of inoculated roots or a spore suspension?

 Ln 310:  It should be noted if there were visual symptoms of drought stress on leaf blades and which day after the application of water stress.

Ln 341:The authors reported that in stressed vines the fluorescence of chlorophyll is  more sensitive than gas exchange parameters. However, it is not supported on any reference. On the contrary, in my experience but also according to recent and earlier publications, net assimilation rate and stomatal conductance decreased earlier than chlorophyll fluorescence in stressed plants: 

-Nieva et al 1999. Gas exchange and chlorophyll fluorescence of C 3 and C4 saltmash species. PHOTOSYNTHETICA 36(3)397-406.

-Subrahmanyam (2008), Effects of chromium toxicity on leaf photosynthetic characteristics

Author Response

Response to the reviewers

In general, this paper gives interesting results on the effect of mycorrhizal fungi (AMF) on the growth and some physiological parameters of own rooted grapevine (Ecolly cultivar) under water stress. The entire study and its results rest on the fact that the mycorrhizal fungi improve the root and shoot growth, leaf chlorophyll content and photosynthetic parameters. Even similar results have been previously reported by others, the findings of this manuscript could be interesting if some failures had been avoided.

Thank you for taking your time to review this manuscript. We really appreciate all your comments and suggestions. According to your advice, we amended the relevant part in the manuscript. All of your questions were answered one-by-one. Also, we revised the language by the English editing service of MDPI.

1. Ln.89: The vine in most viticultural regions is usually cultivated grafted onto rootstocks resistant to phylloxera. Why the authors used own rooted plants? The use of own rooted vines of a local cultivar in the present experimentation is of limited interest to international grapevine growing.

Reply: Your suggestion is great. However, most vineyards use own-rooted vines in China because there is no phylloxera in China and own-rooted vines are cheaper than vines grafted onto rootstocks.

Thank you for reminding that local cultivar would attract limited interest to international grapevine growers. We will consider this in the future experiment.

2. The pot medium was: cinammon soil. Please use the scientific category of the soil.

Reply: The pot medium had changed to “Lou soil (Eum-Orthic Anthrosol in FAO soil taxonomy)” in Ln.93 as requested (The line number will be normal when close “Track change” function). “Lou soil” is Chinese name in scientific category of the soil in Chinese soil taxonomy.

3. Ln 96: Plant size or vigour is very low. Shoot dry weight of 2-4 g is very low representing a pruning weight of 5-8 g and a shoot length of about 45cm with 10-12 nodes and very small leaves. I think that the length of the cuttings from which the experimental vines created was about 35cm? It is impossible to use experimental vines in the same year of their creation. Thus, the measurement conditions may have yielded non-useful results for grapevine growing. In my opinion the experiment should be done on plants at least two years old and planted in 10 L pots.

Reply: Sorry for misuse the “cutting” in the paper and had changed to “two-year own rooted grapevines”. We used “Own rooted Vitis vinifera L. cv. Ecolly cuttings” and wanted to express that cutting was not grafted and had roots grown.

Your speculation of pruning weight is correct; however, the leaves were not small due to cultivar character. The plant size/vigour looked not very low because of big leaves.

We agree that it would be better to use 10L pots. However, we referred to Jia-Dong He et al. (2019), 2L pot was used in study on trifoliate orange with AMF; Zhongfeng Zhang et al. (2014), 1.5kg soil each pot in study on Cyclobalanopsis glauca with AMF; Dong Huang et al. (2020), 12cm*12cm pot in study on apple with AMF. So, we thought our pot size were appropriate.

There are several unclear issues:

4. Ln 98: The authors may give more detailed information on the mycorrhizal inoculation:

-The commercial name and the company producing the mycorrhizal inoculant is not reported by authors.

Reply: We had added the information as requested (Ln. 102).

5. -Ηow the experimental vines were inoculated and how the inoculum incorporated in pot medium and the rhizosphere (the authors state the beginning of experimentation at January 2021 and the inoculation at 25March2021).

Reply: We dug a small hole at the same position in the pot, then put the inoculum in the hole and covered by the soil dug out. Then watered the pots. The manuscript was also revised in Ln. 106-108.

The grapevines were stored in the sand and at dormancy period before the experiment. They were potted at the end of January 2021, but did not germinate soon. Then we inoculated at 25 March 2021.

6. -The inoculum was a troof of inoculated roots or a spore suspension?

Reply: The product was powder and can be used directly without making suspension. We watered the pot after inoculation.

7. Ln 310: It should be noted if there were visual symptoms of drought stress on leaf blades and which day after the application of water stress.

Reply: We had added some descriptions in Ln. 324 as requested.

There were several leaves became wilt in several pots at around 7 days after stopping watering.

8. Ln 341: The authors reported that in stressed vines the fluorescence of chlorophyll is more sensitive than gas exchange parameters. However, it is not supported on any reference. On the contrary, in my experience but also according to recent and earlier publications, net assimilation rate and stomatal conductance decreased earlier than chlorophyll fluorescence in stressed plants: 

-Nieva et al 1999. Gas exchange and chlorophyll fluorescence of C 3 and C4 saltmash species. PHOTOSYNTHETICA 36(3)397-406.

-Subrahmanyam (2008), Effects of chromium toxicity on leaf photosynthetic characteristics

Reply: We have added some discussions in Ln.357-365. Thank you for providing these references. We did not notice these papers and also lacked the experience in this aspect. We were thinking to share what we observed, so put the observation in the manuscript. As for contrary result, we proposed some reasons as following: 1. The sensitivity of machines is different. 2. The chlorophyll fluorescence was measured in the room with little disturbance; however, the gas exchange parameter was measured outdoor with more disturbance. 3. The variability between plants could be great or smaller. The variance of data from chlorophyll fluorescence was smaller than gas exchange parameters. These may be the reasons why we had the contrary results.

 

References:

Huang, D.; Ma, M.; Wang, Q.; Zhang, M.; Jing, G.; Li, C.; Ma, F. Arbuscular Mycorrhizal Fungi Enhanced Drought Resistance in Apple by Regulating Genes in the MAPK Pathway. Plant Physiology and Biochemistry 2020, 149, 245–255, doi:10.1016/j.plaphy.2020.02.020.

Zhang, Z.; Zhang, J.; Huang, Y. Effects of Arbuscular Mycorrhizal Fungi on the Drought Tolerance of Cyclobalanopsis Glauca Seedlings under Greenhouse Conditions. New Forests 2014, 45, 545–556, doi:10.1007/s11056-014-9417-9.

He, J.; Ying-Ning, Z.; Wu, Q.-S.; Kuca, K. Mycorrhizas Enhance Drought Tolerance of Trifoliate Orange by Enhancing Activities and Gene Expression of Antioxidant Enzymes. Scientia Horticulturae 2019, 262, 108745, doi:10.1016/j.scienta.2019.108745.

Round 2

Reviewer 1 Report

The authors have done a good job on their revised version. However, I have a problem with the authors' response to one of my comments on the previous version, namely "As your experience, two hours in the autoclave is perhaps not enough to sterilize a soil. However, this experiment focused on the effect of AMF on grapevines. We did not pay much attention to the mycorrhization. Thank you for your suggestion. We will consider this part if the experiment could be continued. As for photo a, we put it just because it found spores and looked clear.". Indeed, studying the impact of mycorrhizal fungi without studying the interaction with the plant is for me rather strange. But above all, the fact of having a non-sterile substrate as indicated by the figures of the non-mycorrhizal roots (presence of fungi) makes it difficult, if not impossible, to know if the effects observed are due in totality, partially or not at all to the mycorrhizal fungi, or to other microorganisms present in the substrate but not identified. These should at least be taken into account and discussed.

Author Response

Response to the reviewers

Thank you for taking your time to review the revised manuscript. We really appreciate your comments and suggestions.

First, we think we need to clarify that “We did not pay much attention to the mycorrhization…” is the response to your suggestion “present a kinetic of mycorrhization”. We did not present a kinetic of mycorrhization, so we feel like we did not pay much attention to the mycorrhization. We think this is a great suggestion, so we will consider in the next experiment. We read previous statement again and realize it might make people feel we do not care mycorrhization. Sorry if make this wrong feeling. We had added discussions in Line 302-305 as your advice.

Additionally, we would like to explain that we did not realize fungal structures in the photo because the mycorrhizal roots were full of fungi and can be easily seen; The non-mycorrhizal roots were basically clear, and it looked empty, especially compared to mycorrhizal roots with obvious stained fungi. We realized it was possible fungi as you reminded that “two hours in an autoclave is perhaps not enough to sterilize a soil”.

We thought sterilized soil had negative effect on plants due to absence of microorganisms. This opinion came from Kleinschmidt, G. (1972). So, we thought a few microorganisms left in the sterilized soil might have a little positive effect on plants. This did not influence the conclusion that soil inoculated with mycorrhiza had higher positive effect on plants.

If you have any questions, please contact us without hesitate.

Reference

Kleinschmidt, G. Stunting of Citrus Seedlings in Fumigated Nursery Soils Related to the Absence of Endomycorrhizae. Phytopathology 1972, 62, 1447.

Reviewer 2 Report

I believe that the publication has improved considerably in line with my suggestions and can be published in the journal agronomy. Α few more improvements to the expressions could still be made to the points I have highlighted

 

Comments for author File: Comments.pdf

Author Response

Response to the reviewers

Thank you for taking your time to review the revised manuscript. We really appreciate all your suggestions. The manuscript improved greatly with your comments and suggestions. According to your advice, we amended the relevant part in the manuscript.

Line 96, “from Shidan Bao” has corrected as “of Shidan Bao”.

Line 97, “C” has corrected as “℃”

Line 314-315. It was one report with the following sentence. We had revised the writing to avoid misunderstanding.

Line 339, “chlorophylls” has corrected as “chlorophylls”

Line 344 “metals” has corrected as “heavy metals”

Line 346 “although” has corrected as “though”

Line 369-373. We had revised the writing as requested.