Differential Effects of Arbuscular Mycorrhizal Fungi on Rooting and Physiology of ‘Summer Black’ Grape Cuttings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please find attached

Comments for author File: Comments.pdf

Author Response

Responses to Reviewer 1 Abstract: Comment 1: The abstract is clearly written but appears dense with numerical data, which could be streamlined for better readability. Author Response: Thank you, we have simplified the content as much as possible, as follows: Arbuscular mycorrhizal fungi (AMF) symbiosis has great potential in improving grape-vine performance and reducing external input dependency in viticulture. However, the precise, strain-specific impacts of different AMF species on ‘Summer Black’ grapevine cuttings across multiple physiological and morphological dimensions remain underexplored. To address this, we conducted a controlled greenhouse pot experiment, systematically evalu-ating four different AMF species (Diversispora versiformis, Diversispora spurca, Funneliformis mosseae, and Paraglomus occultum) on ‘Summer Black’ grapevine cuttings. All AMF treatments suc-cessfully established root colonization, with F. mosseae achieving the highest infection rate. In detail, F. mosseae notably enhanced total root length, root surface area, and volume, while D. versiformis specifically improved adventitious and secondary lateral root numbers. Phosphorus (P) uptake in both leaves and roots was significantly elevated across all AMF treatments, with F. mosseae leading to a 42% increase in leaf P content. Furthermore, AMF inoculation generally enhanced the activities of catalase, superoxide dismutase, and peroxidase, along with soluble protein and soluble sugar contents in leaves and roots. Photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were dramatically increased in AMF colonized cutting seedlings. whereas, P. occultum exhibited inhibitory effects on several growth metrics such as shoots length, leaf and root biomass, adventitious lateral root numbers, decreased the contents of Nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) in both leaves and roots. These findings conclusively demonstrate that AMF symbiosis via optimizing root morphology, enhancing nutrient acquisition, and boosting photosynthetic efficiency and stress resilience, thus providing valuable insights for developing targeted bio-fertilization strategies in sustainable viticulture. Comment 2: Briefly mention the experiment timeline. Author Response: Thank you for such a good suggestion. The entire experimental period from cutting to harvesting plants is 12 weeks. We have provided detailed explanations in the revised manuscript. Comment 3: The inhibitory effects of Paraglomus occultum need further elaboration. Author Response: Thank you. Based on your suggestions, we have made corresponding revisions in the revised draft, specifically as follows: Whereas, P. occultum exhibited inhibitory effects on several growth metrics such as shoots length, leaf and root biomass, adventitious lateral root numbers, decreased the contents of Nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) in both leaves and roots. Introduction Comment 4: The introduction is well-structured but could be improved by breaking down long paragraphs and simplifying complex sentences for easier understanding by a broader audience. Author Response: Thanks, based on your suggestion, we have revised the entire preface section in the revised manuscript as follows: Grape (Vitis vinifera L.), belonging to Vitaceae family, is globally recognized as one of the most widely cultivated and economically fruit crops. Its popularity is largely attributed to delicious fruits, which are rich in diverse nutrients, including organic acids, glucose, and various vitamins [‎1], as well as a wide range of beneficial phytochemicals [‎2,‎3]. These compounds contribute to the well-established health benefits of grapes, such as anti-inflammatory, cardioprotective, anti-cancer, and anti-diabetic properties [‎3]. The rapid expansion of the global grape industry in recent decades has highlighted the urgent need for sustainable agricultural practices, as current intensive cultivation often relies heavily on chemical fertilizers, leading to soil fertility degraded, ecosystem balance disrupted, etc. In this context, exploring biological alternatives for enhancing grape cultivation is of paramount for fostering ecological and sustainable development. Root system structure directly determines nutrient and water absorption efficiency, which profoundly influencing plant growth performance, stress resilience (e.g., drought and disease resistance), and ultimately, fruit quality [‎4]. Specific root components, such as root hairs, adventitious roots, and lateral roots, all play crucial roles. Especially lateral roots are particularly prominent in anchoring plant, expanding the absorptive surface, and storing nutrients. The dense network of root hairs is indispensable for the efficient uptake of less mobile nutrients, notably the easily fixed element phosphorus (P) [‎5]. Arbuscular mycorrhizal fungi (AMF) represent a ubiquitous and ecologically significant group of soil microorganisms that form mutualistic symbioses with the roots of the vast majority of terrestrial plants [‎6]. This ancient symbiosis is fundamental to ecosystem stability and sustainable productivity in agricultural systems [‎7]. Upon infecting host roots, they form characteristic structures such as intraradical hyphae, vesicles, and arbuscules, and extensive extraradical hyphal networks in the soil [‎8]. This network acts as an extension of host’s root system, significantly increasing water and nutrient uptake, thereby boosting plant growth and vigor [‎9]. AMF are particularly renowned for their capacity to improve plant acquisition of mineral nutrients, especially P, by mobilizing and transporting it from the soil, thus providing a robust nutritional foundation for plant development [‎10]. Beyond P, they also enhance the absorption and utilization of other essential nutrients, such as nitrogen, potassium, calcium, iron, copper, zinc, manganese, and boron. Furthermore, AMF inoculation could improve plant photosynthetic efficiency by influencing pigment content and enzyme activities [‎11]. Meanwhile, AMF contribute significantly to plant stress tolerance by enhancing osmotic regulation [‎12] and resistance to various abiotic stresses, e.g. drought [‎13], salinity [‎14], heavy metals [‎15], and temperature extremes [‎16]. They also play a role in improving soil structure [‎17] and conferring disease resistance [‎18]. It is well-known that AMF has positive effects grapevines, which including promoting growth, improving nutrient absorption, enhancing tolerance to abiotic stresses such as water deficit and high temperatures, as well as biotic stresses [‎19]. Notably, the effective-ness of AMF inoculation is highly variable and strain-specific, with different fungal species or isolates exhibiting distinct impacts on various plant species and genotypes. For in-stance, Meng et al. [‎20] demonstrated that inoculation with different AMF strains (Diversispora versiformis, Funneliformis mosseae, and Rhizophagus intraradices) significantly enhanced multiple growth parameters and root morphological traits in Tinospora sagittata plants. In grape cultivation, Luciani et al. [21] inoculated AMF on one-year-old 'Sangiovese' self-rooted seedlings and three-year-old 'Sangiovese' grafted seedlings (rootstock 420A), found that Glomus iranicum significantly increased total root volume and promoted root extension in grapevines. Similarly, inoculated with G. intraradices or G. mosseae enhanced new shoot growth in grapes after five weeks [22]. In addition, Fattahi et al. [23] demonstrated that inoculation with G. mosseae, G. intraradices, G. etunicatum, and G. verruciforme on 'Asgari' grapevines significantly increased chlorophyll content, relative leaf water content, and concentrations of P, K+, and Ca2+ in grape tissues. Under water-limited conditions, G. verruciforme and G. etunicatum provided the most significant protection to grape-vines. These variability highlights the importance of characterizing specific AMF strains for targeted applications. While existing research has identified the general benefits of AMF for grape growth and stress tolerance, there are still critical knowledge gaps regarding the specific impacts of AMF on rooting process and early growth of grape cuttings. Establishment of a robust root system is fundamental for the successful propagation and subsequent field performance of grapevines, but the exact effects of different AMF strains on the development of complex root architecture in cuttings remain underexplored. Vitis Vinifera ‘Summer Black’ is a European-American hybrid triploid variety developed in Japan through the crossbreeding of Vitis vinifera and Vitis labrusca. This variety is characterized by its dark purple to black, seedless berries, which are primarily utilized for fresh consumption and wine production. While ‘Summer Black’ grapes exhibit strong stress resistance and high yield, while their root systems tend to be shallower compared to those of other varieties. Hence, we hypothesized that AMF inoculation would significantly enhance the rooting success rate and early growth performance of ‘Summer Black’ grape cuttings. Therefore, the present study was aimed to evaluate the mycorrhizal colonization capabilities of each AMF strain in grape roots. Simultaneously quantify their impact on overall growth performance and detailed root system architecture of grape cuttings, as well as elucidate their impact on the physiological activities regulation of nutrient absorption and accumulation in grape tissues. Thus, highly efficient AMF strains with great practical application potential in sustainable viticulture could be identified. The findings will provide a crucial theoretical and practical foundation for optimizing AMF technology in grape production, ultimately contributing to establish more resilient and environmentally friendly cultivation systems. Material and Methods Comment 5: It would be better to specify pot size in liters rather than dimensions. Author Response: Thanks, in this study, the plastic pots (upper inner diameter: 20 cm, bottom inner diameter: 15 cm, height: 18 cm) were applied approximately 3L, we have already indicated it in the revised draft. Comment 6: Indicate how much growing media was added to each pot. Author Response: Thanks, we have made detailed revisions, the specific content as follows:The Vitis vinifera L. ‘Summer Black’ cuttings were planted into plastic pots (upper inner diameter: 20 cm, bottom inner diameter: 15 cm, height: 18 cm) with 2.5 Kg growth substance. For AMF treatment, 80 g inoculum with about 3,500 spores were applied, each inoculum consisted of colonized root segments, AMF spores, and river sand, propagated in maize trap cultures. The control (non-AMF) treatment consisted of 80 g of sterilized my-corrhizal inoculum (autoclaved at 0.11 MPa and 121 ◦C for 2 h) supplemented with2.5 mL of a filtrate (through a 25 µm flter) from the inoculum to maintain similar microbial communities, excluding the AMF strain. Comment 7: Clarify whether the cuttings were procured from a single mother plant; include cutting size (length and diameter). Author Response: Yes, to ensure consistent genetic background of the cuttings, all cuttings come from the same mother plant, each branch was approximately 30 cm long with the stem thickness of 1 cm. We have also provided a detailed description of this in the revised draft, please refer to the revised draft for details. Comment 8: Specify how long cuttings were kept under sand, as callusing might have started. Author Response: Thank you. We collected the mother branches in December of the previous year and stored them in sand for 8 weeks at 4-7 ° C, with stable humidity and relative humidity maintained at around 80%. Cutting will be carried out in February of the following year. Before cutting, we checked all the cuttings and found no signs of aging. We have provided a detailed description of this information in the revised manuscript. Comment 9: Indicate if pots were covered with transparent polyethylene sheets to prevent desiccation. Author Response: OK. In this experiment, the flowerpot we used was made of polyethylene film, which has anti drying properties, so there is no need to use an additional transparent polyethylene pot. Comment 10: Ensure consistent use of standardized units and formatting throughout. For example, enzyme activities (catalase, SOD, etc.) should include units. Author Response: Thank you for your valuable feedback. We have checked all the data to ensure that each data is using standard units. Please refer to the revised draft for details. Comment 11: Clarify sample size by stating the total number of plants or replicates. Author Response: Thanks. In this study, a single-factor completely randomized experimental design was employed comprising five treatments: inoculation with D. versiformis, D. spurca, F. mosseae, P. occultum, and a non-inoculated control (non-AMF). Each treatment consisted of six replicates, one grape cutting was planted per pot, with each pot representing an experimental unit, totaling 30 pots and 30 seedlings. We also provided a detailed description in the revised manuscript. Comment 12: Provide more details on environmental monitoring (e.g., light intensity, humidity measurements). Author Response: Thank you, based on your suggestion, we have added detailed environmental condition parameters as follows:: The specific environmental parameters maintained were: the photosynthetic photon flux density was 982 µmol/m2/s with a day/night temperature regime of 28°C/20°C, relative humidity of 65 ± 5%, and a 16-hour light/8-hour dark photoperiod with a photosynthetically active radiation intensity of approximately 400 µmol m-2·s-1 at plant canopy height, provided by natural sunlight supplemented with artificial grow lights. Comment 13: Include citations for all methods used, particularly for assays and sterilization procedures, with brief descriptions referencing previous studies. Author Response: Thank you for your feedback. In all of our studies, to ensure the influence of other microorganisms in the cultivation substrate, we used autoclaved sterilization at 121 ℃ and 0.1MP for 2 h (Meng et al., 2023). We have added relevant literature in the revised draft, please check. Meng, L.L.; Xu, F.Q.; Zhang, Z.Z.; Alqahtani, M.D.; Tashkandi, M.A.; Wu, Q.S. Arbuscular mycorrhizal fungi, especially Rhizophagus in-traradices as a biostimulant, improve plant growth and root columbin levels in Tinospora sagittata. Horticulturae, 2023, 9:1350. Results & Discussion: Comment 13:The section is generally well-written, but the following improvements are recommended: Include ± values (standard deviation or standard error) on all graphs/bars. Author Response: Thank you. All the bar charts and histograms in the revised manuscript now display± error bars to indicate variability; full details are provided in the figure legends. Comment 14: Clearly label units on all figures and graphs. Author Response: Thanks. We carefully checked and redrawn the graph to ensure that all data were clearly expressed in units. Comment 15: Remove lines 409–411 as they appear redundant or off-topic. Author Response: Thank you. This sentence is indeed irrelevant to the topic and caused our negligence. We have removed it from the revised manuscript. In addition, we also do the needful editing according to the editor’s comments and author guidelines. All changes are marked in highlighted. Please see the revised manuscript for details.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The review of the manuscript entitled „Differential effects of arbuscular mycorrhizal fungi on growth, root architecture, and physiological responses of ‘Xiahei’ grape  cuttings”

 

The authors of the submitted manuscript evaluated root development, plant growth, physiological activity - photosynthetic parameters, stress resiliance measured as the concentration of antioxidant enzymes and nutrient acquisition in ‘Xiahei’ grape cuttings, treated with four species of arbuscular mycorrhizal fungi (AMF). The use of two species of AMF: Diversispora versiformis and Funneliformis mosseae led to the optimization of the assessed traits. The presented study is a valuable contribution, enriching the knowledge on the possibilities of using AMF in grapevine cultivation and indicated the diversified response of grapevines, to inoculation of the AMF species used. The research topic is justified and necessary for the sustainable development of grapevine cultivation.

In the introduction, the authors describe the economic and dietary importance of grapevines, the benefits resulting from the use of AMF and the objectives of the study that fit well into the knowledge gaps. The research methodology, equipment used, statistical analysis and presentation of results are correct and typical for the described research topic.

The authors conducted research on the ‘Xiahei’ (Vitis vinifera L.)- a table variety grown in China. The manuscript should contain a very short description of this variety - origin, use of fruit, growth strength and productivity of the vines. A short description of the conditions and technology of cultivation is also missing. The research was conducted on own-root plants. Such plants will be probably planted in future field studies and in vineyards, where AMF inoculation will be implemented. In most important growing regions in the world, grapevines are grafted onto rootstocks. The variety, the type of cuttings used, including rootstocks, are of significant importance for the uptake and acquisition of nutrients by the grapevine and the mineral composition of plants, as well as the colonization of roots by AMF. Therefore, it is worth explaining the specificity and technology of local grapevine cultivation, to which the research was dedicated. Brief information about the variety and cultivation conditions (a few sentences) will inform readers about the realities of grapevine cultivation in China.

If the authors have data on the concentration of nutrients and organic matter in the used potting substrate, it is advisable to include them in the description of the research methodology. These parameters could be important for the uptake of macronutrients and the effectiveness of AMF. In this type of research, it would be interesting to use soil from the cultivation area in parallel to or instead of the standard substrate. The manuscript is interesting and valuable, and the results provide a basis for continuing research in field conditions.

In connection with future research, the role of fertilization and biostimulants for the species composition of AMF and the level of colonization in grapevine roots can be mentioned in the discussion. In this context, the following articles can be cited:

Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.;  Frąc, M.; Sitarek, M.; Przybył, M. Occurrence of arbuscular mycorrhizal fungi in the roots of two grapevine cultivars in response to bioproducts. S. Afr. J. Enol. Vitic. 2019, 40(1), 1-4.  https://doi.org/10.21548/40-1-3115.

Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.;  Frąc, M.; Sitarek, M.; Przybył, M.; Górnik, K. Diversity of arbuscular mycorrhizal fungi in the rhizosphere of Solaris and Regent grapevine plants treated with bioproducts. S. Afr. J. Enol. Vitic. 2020, 41(1), 83-89. https://doi.org/10.21548/41-1-3725.

 

In my opinion, after taking into account the above comments, the manuscript may be the subject of further editorial work.

Author Response

Responses to Reviewer 2

Comment 1: The authors of the submitted manuscript evaluated root development, plant growth, physiological activity - photosynthetic parameters, stress resiliance measured as the concentration of antioxidant enzymes and nutrient acquisition in ‘Xiahei’ grape cuttings, treated with four species of arbuscular mycorrhizal fungi (AMF). The use of two species of AMF: Diversispora versiformis and Funneliformis mosseae led to the optimization of the assessed traits. The presented study is a valuable contribution, enriching the knowledge on the possibilities of using AMF in grapevine cultivation and indicated the diversified response of grapevines, to inoculation of the AMF species used. The research topic is justified and necessary for the sustainable development of grapevine cultivation.

Author Response: Thank you for your affirmation.

 

Comment 2: In the introduction, the authors describe the economic and dietary importance of grapevines, the benefits resulting from the use of AMF and the objectives of the study that fit well into the knowledge gaps. The research methodology, equipment used, statistical analysis and presentation of results are correct and typical for the described research topic.

Author Response: Thank you for your compliment.

 

Comment 3: The authors conducted research on the ‘Xiahei’ (Vitis vinifera L.)- a table variety grown in China. The manuscript should contain a very short description of this variety - origin, use of fruit, growth strength and productivity of the vines. A short description of the conditions and technology of cultivation is also missing. The research was conducted on own-root plants. Such plants will be probably planted in future field studies and in vineyards, where AMF inoculation will be implemented. In most important growing regions in the world, grapevines are grafted onto rootstocks. The variety, the type of cuttings used, including rootstocks, are of significant importance for the uptake and acquisition of nutrients by the grapevine and the mineral composition of plants, as well as the colonization of roots by AMF. Therefore, it is worth explaining the specificity and technology of local grapevine cultivation, to which the research was dedicated. Brief information about the variety and cultivation conditions (a few sentences) will inform readers about the realities of grapevine cultivation in China.

Author Response: Thank you for your valuable suggestion. We have made detailed revisions in the “Introduction”, please check. The specific content is as follows:

Vitis Vinifera ‘Summer Black’ is a European-American hybrid triploid variety developed in Japan through the crossbreeding of Vitis vinifera and Vitis labrusca. This variety is characterized by its dark purple to black, seedless berries, which are primarily utilized for fresh consumption and wine production. While ‘Summer Black’ grapes exhibit strong stress resistance and high yield, while their root systems tend to be shallower compared to those of other varieties.

 

Comment 4: If the authors have data on the concentration of nutrients and organic matter in the used potting substrate, it is advisable to include them in the description of the research methodology. These parameters could be important for the uptake of macronutrients and the effectiveness of AMF. In this type of research, it would be interesting to use soil from the cultivation area in parallel to or instead of the standard substrate. The manuscript is interesting and valuable, and the results provide a basis for continuing research in field conditions.

Author Response: Thank you for your suggestion. We have made detailed revisions in the revised draft, as follows: The potting substrate consisted of a 1:1 (v/v) mixture of garden soil and sand. The soil had a pH of 6.1, and organic carbon (C), available N, Oslen-phosphorus (P), and available potassium (K) were 9.7 mg/kg, 11.8 mg/kg, 15.3 mg/kg, and 21.5 mg/kg, respectively.

 

Comment 5: In connection with future research, the role of fertilization and biostimulants for the species composition of AMF and the level of colonization in grapevine roots can be mentioned in the discussion. In this context, the following articles can be cited:

Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.; Frąc, M.; Sitarek, M.; Przybył, M. Occurrence of arbuscular mycorrhizal fungi in the roots of two grapevine cultivars in response to bioproducts. S. Afr. J. Enol. Vitic. 2019, 40(1), 1-4.  https://doi.org/10.21548/40-1-3115.

Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.; Frąc, M.; Sitarek, M.; Przybył, M.; Górnik, K. Diversity of arbuscular mycorrhizal fungi in the rhizosphere of Solaris and Regent grapevine plants treated with bioproducts. S. Afr. J. Enol. Vitic. 2020, 41(1), 83-89. https://doi.org/10.21548/41-1-3725.

Author Response: Thank you for such a good suggestion. We have made detailed revisions based on your suggestions, and the specific contents are as follows:

In addition, in this study, we observed different responses of AMF inoculation to various mineral elements, which may be related to the nutritional level of the cultivation substrate itself, as additional NPK fertilization may inhibit AMF infection and thus affect nutrient absorption [43-44].

we observed differential responses of AMF inoculation to various mineral elements, potentially due to the interplay between N, P, K fertilization and AMF colonization, especially the content of N, P, K. Previous field research has demonstrated that some bio stimulants such as Ausuma, Bioilsa, and BF-Ecomix can enhance the abundance and species richness of mycorrhizal fungi in the grapevine rhizosphere [43-44].

 

In addition, we also do the needful editing according to the editor’s comments and author guidelines. All changes are marked in highlighted. Please see the revised manuscript for details.

 

 

 

 

 

 

 

 

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides a detailed study evaluating the influence of four AMF strains on the growth, root architecture, nutrient uptake, and physiological traits of Vitis vinifera ‘Xiahei’ cuttings. The scope of the study is scientifically sound and the research question is clearly presented. However, I find the manuscript overburdened with detailed comparisons and numerical data that obscure the broader patterns and conclusions. For a more accessible and impactful contribution, the authors should streamline the results and sharpen their discussion on practical implications, particularly for viticultural applications. The authors have done a thorough job collecting a breadth of parameters, but the manuscript must benefit from tighter focus, improved clarity, and more critical synthesis of the findings. Below, I provide specific comments to support a major revision.
Title & Abstract
The title reflects the manuscript content well, though could be slightly shortened for clarity (e.g., omit “physiological responses” or rephrase to “on rooting and physiology of ‘Xiahei’ grape cuttings”).
The abstract is overly dense with statistics. Simplify by focusing on the most important findings and general trends rather than exact percentages.
Introduction
The introduction is well-structured and adequately references relevant literature. However:
L42-57: Consider tightening the background on the significance of grapevine cultivation and AMF to avoid redundancy.
L94-108: The knowledge gap and hypotheses are clearly presented but should more directly identify why understanding strain specificity matters for viticulture practitioners.
Materials and Methods
Generally clear, but several methodological details are missing or need clarification for reproducibility:
L124–131: Indicate the exact propagation date of the cuttings and clarify how uniformity was ensured.
L133–134: Provide a more precise description of substrate sterilization protocol, e.g., confirm whether a single or repeated autoclaving cycle was used.
L142–147: It is unclear if the inoculation layer was directly in contact with cuttings or if any separation barrier was present. Also, were non-AMF pots inoculated with a sterilized inoculum of the same matrix?
L165–170: Please clarify whether the LI-6400 chamber settings (e.g., CO₂ concentration, PAR) were standardized across measurements.
L188–198: Confirm how root sampling locations were randomized or standardized, as root colonization may vary spatially.
Results
The main weakness of the results section is excessive detail and statistical granularity, which makes it hard to extract key findings.
3.1 Mycorrhizal Colonization
L225–247: The description of colonization success is clear. However, linking this immediately to downstream performance (e.g., colonization vs. root growth) would add value.
3.2–3.3 Growth and Root Traits
Tables 1–2 & Figures 3–5: Consider summarizing the most impactful traits in bar graphs for visual clarity (e.g., shoot length, root volume, adventitious root number).
Suggest consolidating data presentation:
Present only significantly different traits.
Avoid reporting decimal-level precision when biologically irrelevant.
Consider using principal component analysis (PCA) or heatmaps to show the differentiation among AMF treatments.
3.4 Mineral Uptake
While comprehensive, the presentation is too complex for a general reader. Group nutrients into categories (e.g., macro vs. micro) and summarize trends.
It’s unclear whether the authors assessed the nutrient content per gram DW or per organ — this should be clarified.
3.5–3.7 Physiological Data
Figures 6–7 and Table 4: Improve clarity by presenting treatment comparisons for each physiological trait side-by-side.
L387–406: Leaf temperature (Tl) changes are minor and their interpretation is weak. Either remove or explain the relevance more clearly.
Discussion
The discussion is long and repeats results extensively. It needs to be refocused to:
Synthesize patterns rather than restate numbers.
Clearly identify which AMF strains are agronomically promising.
Explicitly contrast positive vs. inhibitory effects (e.g., F. mosseae vs. P. occultum).
L449–466: Discussion on nutrient uptake should connect more with field relevance (e.g., do these effects persist in soil vs. sterile media?).

L467–499: Antioxidant and sugar/protein results are well contextualized but would benefit from reference to field stress mitigation, particularly under water or salt stress.
Conclusion
A strong summary of findings, but could be condensed.
Suggest concluding with a clearer recommendation: e.g., “F. mosseae and D. versiformis are recommended candidates for AMF-based inoculation in grapevine propagation.”

Minor Issues and Language
Scientific names (e.g., Vitis vinifera, Funneliformis mosseae) must be italicized consistently throughout.
Acronyms (e.g., AMF) should be defined only once.
Tables should use superscript letters for significance for readability.
There are minor English issues that require copyediting, including overly long sentences and passive constructions.

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

Responses to Reviewer 3

 

Title & Abstract

Comment 1: The title reflects the manuscript content well, though could be slightly shortened for clarity (e.g., omit “physiological responses” or rephrase to “on rooting and physiology of ‘Summer Black’ grape cuttings”).
Author Response: We sincerely appreciate your valuable suggestions and have implemented all recommended revisions accordingly. Specifically, as follows: Differential effects of arbuscular mycorrhizal fungi on rooting and physiology of ‘Summer Black’ grape cuttings

 

Comment 2: The abstract is overly dense with statistics. Simplify by focusing on the most important findings and general trends rather than exact percentages.

Author Response: Thank you, we have simplified the content as much as possible, as follows: Arbuscular mycorrhizal fungi (AMF) symbiosis has great potential in improving grape-vine performance and reducing external input dependency in viticulture. However, the precise, strain-specific impacts of different AMF species on ‘Summer Black’  grapevine cuttings across multiple physiological and morphological dimensions remain underexplored. To address this, we conducted a controlled greenhouse pot experiment, systematically evaluating four different AMF species (Diversispora versiformis, Diversispora spurca, Funneliformis mosseae, and Paraglomus occultum) on ‘Summer Black’  grapevine cuttings. All AMF treatments successfully established root colonization, with F. mosseae achieving the highest infection rate. In detail, F. mosseae notably enhanced total root length, root surface area, and volume, while D. versiformis specifically improved adventitious and secondary lateral root numbers. Phosphorus (P) uptake in both leaves and roots was significantly elevated across all AMF treatments, with F. mosseae leading to a 42% increase in leaf P content. Furthermore, AMF inoculation generally enhanced the activities of catalase, superoxide dismutase, and peroxidase, along with soluble protein and soluble sugar contents in leaves and roots. Photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were dramatically increased in AMF colonized cutting seedlings. whereas, P. occultum exhibited inhibitory effects on several growth metrics such as shoots length, leaf and root biomass, adventitious lateral root numbers, decreased the contents of Nitrogen (N), potassium (K), magnesium (Mg) and iron (Fe) in both leaves and roots. These findings conclusively demonstrate that AMF symbiosis via optimizing root morphology, enhancing nutrient acquisition, and boosting photosynthetic efficiency and stress resilience, thus providing valuable insights for developing targeted bio-fertilization strategies in sustainable viticulture.

 

Introduction

The introduction is well-structured and adequately references relevant literature. However:
Comment 3: L42-57: Consider tightening the background on the significance of grapevine cultivation and AMF to avoid redundancy.
Author Response: Thanks so much. Based on your suggestion, we have made modifications to this part of the manuscript as follows: Grape (Vitis vinifera L.), belonging to Vitaceae family, is globally recognized as one of the most widely cultivated and economically fruit crops. Its popularity is largely attributed to delicious fruits, which are rich in diverse nutrients, including organic acids, glucose, and various vitamins [‎1], as well as a wide range of beneficial phytochemicals [‎2,‎3]. These compounds contribute to the well-established health benefits of grapes, such as anti-inflammatory, cardioprotective, anti-cancer, and anti-diabetic properties [‎3]. The rapid expansion of the global grape industry in recent decades has highlighted the urgent need for sustainable agricultural practices, as current intensive cultivation often relies heavily on chemical fertilizers, leading to soil fertility degraded, ecosystem balance disrupted, etc. In this context, exploring biological alternatives for enhancing grape cultivation is of paramount for fostering ecological and sustainable development.

 

Comment 4: L94-108: The knowledge gap and hypotheses are clearly presented but should more directly identify why understanding strain specificity matters for viticulture practitioners.
Materials and Methods

Author Response: Thanks so much. Based on your suggestion, we have made modifications to this part of the manuscript as follows: It is well-known that AMF has positive effects grapevines, which including promoting growth, improving nutrient absorption, enhancing tolerance to abiotic stresses such as water deficit and high temperatures, as well as biotic stresses [‎19]. Notably, the effective-ness of AMF inoculation is highly variable and strain-specific, with different fungal species or isolates exhibiting distinct impacts on various plant species and genotypes. For in-stance, Meng et al. [‎20] demonstrated that inoculation with different AMF strains (Diversispora versiformis, Funneliformis mosseae, and Rhizophagus intraradices) significantly enhanced multiple growth parameters and root morphological traits in Tinospora sagittata plants. In grape cultivation, Luciani et al. [21] inoculated AMF on one-year-old 'Sangiovese' self-rooted seedlings and three-year-old 'Sangiovese' grafted seedlings (rootstock 420A), found that Glomus iranicum significantly increased total root volume and promoted root extension in grapevines. Similarly, inoculated with G. intraradices or G. mosseae enhanced new shoot growth in grapes after five weeks [22]. In addition, Fattahi et al. [23] demonstrated that inoculation with G. mosseae, G. intraradices, G. etunicatum, and G. verruciforme on 'Asgari' grapevines significantly increased chlorophyll content, relative leaf water content, and concentrations of P, K⁺, and Ca²⁺ in grape tissues. Under water-limited conditions, G. verruciforme and G. etunicatum provided the most significant protection to grapevines. These variability highlights the importance of characterizing specific AMF strains for targeted applications.

While existing research has identified the general benefits of AMF for grape growth and stress tolerance, there are still critical knowledge gaps regarding the specific impacts of AMF on rooting process and early growth of grape cuttings. Establishment of a robust root system is fundamental for the successful propagation and subsequent field performance of grapevines, but the exact effects of different AMF strains on the development of complex root architecture in cuttings remain underexplored.

Vitis Vinifera ‘Summer Black’ is a European-American hybrid triploid variety developed in Japan through the crossbreeding of Vitis vinifera and Vitis labrusca. This variety is characterized by its dark purple to black, seedless berries, which are primarily utilized for fresh consumption and wine production. While ‘Summer Black’ grapes exhibit strong stress resistance and high yield, while their root systems tend to be shallower compared to those of other varieties. Hence, we hypothesized that AMF inoculation would significantly enhance the rooting success rate and early growth performance of ‘Summer Black’ grape cuttings. Therefore, the present study was aimed to evaluate the mycorrhizal colonization capabilities of each AMF strain in grape roots. Simultaneously quantify their impact on overall growth performance and detailed root system architecture of grape cuttings, as well as elucidate their impact on the physiological activities regulation of nutrient absorption and accumulation in grape tissues. Thus, highly efficient AMF strains with great practical application potential in sustainable viticulture could be identified. The findings will provide a crucial theoretical and practical foundation for optimizing AMF technology in grape production, ultimately contributing to establish more resilient and environmentally friendly cultivation systems.

 

Material and Methods

Generally clear, but several methodological details are missing or need clarification for reproducibility:
Comment 5: L124–131: Indicate the exact propagation date of the cuttings and clarify how uniformity was ensured.

Author Response: Thank you. We collected the mother branches in December of the previous year and stored them in sand for 8 weeks at 4-7 ° C, with stable humidity and relative humidity maintained at around 80%. Cutting will be carried out in February of the following year. Before cutting, we checked all the cuttings and found no signs of aging. We have provided a detailed description of this information in the revised manuscript.

 

Comment 6: L133–134: Provide a more precise description of substrate sterilization protocol, e.g., confirm whether a single or repeated autoclaving cycle was used.

Author Response: Thank you, we have made the corresponding changes in the revised manuscript. The content is as follows: To eliminate indigenous arbuscular mycorrhizal fungi (AMF) spores and other soil-borne pathogens, the substrate was sterilized by autoclaved at 121°C and 0.1 MPa for 2 hours with each cycle lasting 1 hour and performed twice.

 

Comment 7: L142–147: It is unclear if the inoculation layer was directly in contact with cuttings or if any separation barrier was present. Also, were non-AMF pots inoculated with a sterilized inoculum of the same matrix?
Author Response: Thank you. In this study, we made detailed modifications as follows: For AMF treatments, 80 g of the respective AMF inoculum was applied using a “layered inoculation method” during planting [‎21]. This method involved placing approximately half of the sterilized substrate into each pot, then evenly spreading the 80 g of AMF inoculum, placing the grape cutting on top, and finally covering it with the remaining sterilized substrate. This ensures optimal contact between the inoculum and the developing root system, facilitating early colonization. For the non-AMF control, 80 g of sterilized inoculum (prepared by autoclaving the mixed inoculum at 120°C, 0.1 MPa for 2 hours to inactivate viable spores) was added to each pot to standardize the organic matter input and exclude viable AMF spores while minimizing microbial community differences.

 

Comment 8: L165–170: Please clarify whether the LI-6400 chamber settings (e.g., CO₂ concentration, PAR) were standardized across measurements.

Author Response: Thanks, we standardized all LI-6400 chamber settings prior to measurements and maintained these parameters consistently throughout the experiment. Prior to harvest, on a sunny day between 9:00 and 11:00 AM, photosynthetic parameters were measured. The fifth fully expanded functional leaf below the apex of each cutting was selected for measurement. A portable photosynthesis system (LI-6400, LI-COR, USA) was used to determine net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci) and stomatal conductance (Gs) under the condition of 400 µmol/mol [CO₂] on a sunny morning [23].

 

Comment 9: L188–198: Confirm how root sampling locations were randomized or standardized, as root colonization may vary spatially.

Author Response: Thank you for your suggestion—we confirm that root sampling locations were determined through random sampling. We have already noted this in the revised draft.

 

Results

Comment 10: The main weakness of the results section is excessive detail and statistical granularity, which makes it hard to extract key findings.

Author Response: We have rewritten the results section to simplify the description as much as possible, in order to highlight the key points and enable readers to quickly access the core content. Due to too many modifications, it is not convenient to display them here. Please review the revised draft. thank you.

 

Comment 11:

3.1 Mycorrhizal Colonization
L225–247: The description of colonization success is clear. However, linking this immediately to downstream performance (e.g., colonization vs. root growth) would add value.
Author Response: Thank you for your valuable suggestion. We have added a correlation analysis between infection rate and root growth in the revised manuscript. The specific content is as follows:

3.8 Pearson correlations

The Pearson correlation analysis conducted on the dataset, aimed to identifying cor-relations unique to each treatment group concerning mycorrhizal development and root growth parameters, variables Physiological index (Figure 5). On plant growth and root morphology, mycorrhizal colonization was significantly and positively correlated with total root length and average diameter, soil hyphae density was positively correlated with 2^nd-order lateral root. Meanwhile, root morphology parameters, especially the number of adventitious roots, are closely related to most growth indicators (p ≤ 0.05 or p ≤ 0.01).

In terms of plant physiology, mycorrhizal colonization was significantly positively correlated with Gs, but negatively correlated with leaf SOD. Meanwhile, there were a positive relationship exist between soil hyphae density and Ci and leaf SOD, similarly, a negatively relationship was also appeared between soil hyphae density and leaf soluble sugar content, Pn, and CAT (p ≤ 0.05 or p ≤ 0.01).

For nutrient absorption, mycorrhizal development may have a better effect on the ab-sorption of large amounts of nutrients than on the promotion of trace elements, although there is a negative correlation between soil hyphae density and the content of some ele-ments, mainly because mycorrhizal infection has maintained the absorption of host plant nutrients at a stable level.

Figure 5. Pearson correlations between mycorrhizal development and root morphology traits (a), Physio-logical index (b), and mineral element contents (c) of Vitis vinifera L. ‘Summer Black’  cuttings inoculated with different AMF species. Red and blue colors indicate positive and negative relationships, respectively. Circle sizes indicate the correlation strength. *, p < 0.05, means significant; **, p < 0.01, means extremely significant.

 

Comment 12:

3.2–3.3 Growth and Root Traits
Tables 1–2 & Figures 3–5: Consider summarizing the most impactful traits in bar graphs for visual clarity (e.g., shoot length, root volume, adventitious root number).
Suggest consolidating data presentation:
Present only significantly different traits.
Avoid reporting decimal-level precision when biologically irrelevant.
Consider using principal component analysis (PCA) or heatmaps to show the differentiation among AMF treatments.
Author Response: Thank you very much for your valuable suggestion. We attempted to create a bar chart for the data of new shoot length, root volume, and adventitious root quantity, but the presentation was not as intuitive as a table. Therefore, we continued to choose to present it in the form of a table. We have also further integrated and optimized Figure 3-5. In addition, based on your suggestion, we have removed some redundant indicators without biological significance such as root mean diameter and projected area, and further optimized the numerical accuracy. In the results section, we added a correlation analysis heatmap (Figure 5). Due to the extensive modifications, it is not convenient to present them here. Please refer to the revised draft for details.

 

Comment 13:

3.4 Mineral Uptake
While comprehensive, the presentation is too complex for a general reader. Group nutrients into categories (e.g., macro vs. micro) and summarize trends.
It’s unclear whether the authors assessed the nutrient content per gram DW or per organ — this should be clarified.

Author Response: Thank you for your valuable suggestions. We have implemented these changes in the revised manuscript. In this study, all nutritional composition were calculated based on unit dry weight, as detailed in the revised manuscript..

 

Comment 14:3.5–3.7 Physiological Data
Figures 6–7 and Table 4: Improve clarity by presenting treatment comparisons for each physiological trait side-by-side.

Author Response: Thank you, we have made the necessary changes based on your suggestions. Please review the revised draft.

 

Comment 15: L387–406: Leaf temperature (Tl) changes are minor and their interpretation is weak. Either remove or explain the relevance more clearly.

Author Response: OK, done.

 

Discussion

The discussion is long and repeats results extensively. It needs to be refocused to:
Comment 16: Synthesize patterns rather than restate numbers.

Author Response: Thank you for your suggestion. We have organized and simplified the entire discussion section, deleting repetitive sentences and unnecessary parts. Due to the significant changes made, it is not convenient to present them here. Please refer to the revised draft for details.

 

Comment 17: Clearly identify which AMF strains are agronomically promising.

Author Response: Thanks. In the revised manuscript, we made a detailed comparison of the physiological effects of different bacterial strains, especially the F. mosseae and P. occultum treatments. In the conclusion, we also made a summary comparison between these two strains and clearly pointed out that F. mosseae has the potential for agricultural promotion.

 

Comment 18: Explicitly contrast positive vs. inhibitory effects (e.g., F. mosseae vs. P. occultum).

Author Response: In the revised manuscript, we made a detailed comparison of the physiological effects of different bacterial strains, especially the F. mosseae and P. occultum treatments. In the conclusion, we also made a summary comparison between these two strains and clearly pointed out that F. mosseae has the potential for agricultural promotion. Specifically, as follows: This comprehensive study confirms the successful symbiotic establishment of all four inoculated AMF strains with ‘Summer Black’ grapevine roots, F. mosseae consistently demonstrated superior efficacy, leading to the highest root colonization, significant improvements in overall root system development (total root length, surface area, volume), and the most pronounced increases in P absorption by both leaves and roots. This strain, also significantly enhanced above-ground growth parameters, photosynthetic rate (Pn, Gs, and Tr). Furthermore, all tested AMF species to varying degrees increased antioxidant enzyme activities (SOD, POD, CAT) in both roots and leaves, as well as soluble protein and soluble sugar contents. This multifaceted physiological enhancement collectively im-proved grapevine resilience and promoted robust growth and development. Conversely, P. occultum, despite forming symbiosis, exhibited inhibitory effects on several growth metrics and specific root architectural traits, underscoring the potential for less compatible or even detrimental interactions depending on the specific AMF-host combination. Therefore, F. mosseae is recommended candidates for AMF-based inoculation in grapevine propagation.

 

Comment 19: L449–466: Discussion on nutrient uptake should connect more with field relevance (e.g., do these effects persist in soil vs. sterile media?).

Author Response: Thank you for your valuable feedback. We have made detailed revisions based on your feedback. Specific content is as follows: In addition, in this study, we observed different responses of AMF inoculation to various mineral elements, which may be related to the nutritional level of the cultivation substrate itself, as additional NPK fertilization may inhibit AMF infection and thus affect nutrient absorption [43-44].

we observed differential responses of AMF inoculation to various mineral elements, potentially due to the interplay between N, P, K fertilization and AMF colonization, especially the content of N, P, K. Previous field research has demonstrated that some bio stimulants such as Ausuma, Bioilsa, and BF-Ecomix can enhance the abundance and species richness of mycorrhizal fungi in the grapevine rhizosphere [43-44].

43. Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.; Frąc, M.; Sitarek, M.; Przybył, M. Occurrence of Arbuscular Mycorrhizal Fungi in the Roots of Two Grapevine Cultivars in Response to Bioproducts. Afr. J. Enol. Vitic. 2019, 40(1), 1-4, doi: 10.21548/40-1-3115.
44. Sas-Paszt, L.; Głuszek, S.; Derkowska, E.; Sumorok, B.; Lisek, J.; Trzciński, P.; Lisek, A.; Frąc, M.; Sitarek, M.; Przybył, M.; Górnik, K. Diversity of Arbuscular Mycorrhizal Fungi in the Rhizosphere of Solaris and Regent Grapevine Plants Treated with Bioproducts. Afr. J. Enol. Vitic 2020, 41(1), 83-89, doi: 10.21548/41-1-3725.

 

Comment 20: L467–499: Antioxidant and sugar/protein results are well contextualized but would benefit from reference to field stress mitigation, particularly under water or salt stress.

Author Response: Thank you for your valuable feedback. We have made detailed revisions based on your feedback. Specific study as follows:

AMF inoculation significantly elevated soluble sugars and soluble protein levels, thereby enhancing grapevine resilience to drought stress and bolstering defense against biotic stressors [43].

Wen, Y.; Zhou, L.J.; Xu, Y.J.; Hashem, A. Abd_Allah, E.F.; Wu, Q.S. Growth Performance and Osmolyte Regulation of Drought-stressed Walnut Plants are Improved by Mycorrhiza. Agriculture 2024, 14:367. doi: 10.3390/agriculture14030367.

 

Conclusion
Comment 21: A strong summary of findings, but could be condensed.
Suggest concluding with a clearer recommendation: e.g., “F. mosseae and D. versiformis are recommended candidates for AMF-based inoculation in grapevine propagation.”

Author Response: Thanks. Based on your suggestion, we will end the results section with F. mosseae and D. versiformes are recommended candidates for AMF inoculation in grape propagation.

 

Comment 22:

Minor Issues and Language
Scientific names (e.g., Vitis vinifera, Funneliformis mosseae) must be italicized consistently throughout.
Acronyms (e.g., AMF) should be defined only once.
Tables should use superscript letters for significance for readability.
There are minor English issues that require copyediting, including overly long sentences and passive constructions.

Author Response: Thank you. Based on your suggestion, we have maintained italicization for Vitis vinifera and Funneliformes mosseae in the article, and defined the acronym (AMF) only once. The table has undergone detailed changes using superscripts, etc. Please refer to the revised draft for details.

 

In addition, we also do the needful editing according to the editor’s comments and author guidelines. All changes are marked in highlighted. Please see the revised manuscript for details.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I have thoroughly evaluated the authors' responses to my previous rejection and the revised manuscript. While the authors have made considerable efforts to address my concerns, I must acknowledge that they have successfully resolved the majority of critical issues that led to my initial rejection.
The improvements are notable: the title and abstract have been appropriately simplified, the introduction has been tightened to reduce redundancy, and several methodological details have been clarified including cutting propagation procedures and sterilization protocols. The addition of correlation analysis between AMF colonization and root growth parameters is a valuable enhancement that strengthens the manuscript's analytical depth. The discussion has been condensed and better focused on synthesizing patterns rather than restating numerical results.
However, several issues still require attention before the manuscript can be considered for publication. The root sampling protocol remains insufficiently detailed - "random sampling" needs specification regarding sampling locations, depths, and number of sampling points to ensure reproducibility. While the authors claim extensive revisions to the Results section, the actual changes are not demonstrated in their response, leaving uncertainty about whether the statistical presentation has been adequately simplified as requested. The methodology section still lacks clarity on standardized measurement conditions for photosynthetic parameters. Additionally, the discussion of nutrient uptake results must benefit from explicit consideration of how findings from sterile substrate conditions translate to field applications.
The scientific merit of the work is evident, and the strain-specific effects of AMF on grapevine cuttings represent a valuable contribution to sustainable viticulture practices. However, the remaining methodological ambiguities and presentation issues prevent immediate acceptance.
My Decision: Minor Revision Required
The manuscript shows significant improvement and addresses most fundamental concerns. With the specific clarifications requested above, this work will provide meaningful insights for AMF applications in grape propagation.

Author Response

Response to Reviewer

 

Comment 1: I have thoroughly evaluated the authors' responses to my previous rejection and the revised manuscript. While the authors have made considerable efforts to address my concerns, I must acknowledge that they have successfully resolved the majority of critical issues that led to my initial rejection.
The improvements are notable: the title and abstract have been appropriately simplified, the introduction has been tightened to reduce redundancy, and several methodological details have been clarified including cutting propagation procedures and sterilization protocols. The addition of correlation analysis between AMF colonization and root growth parameters is a valuable enhancement that strengthens the manuscript's analytical depth. The discussion has been condensed and better focused on synthesizing patterns rather than restating numerical results.

Author Response: Thank you very much for your recognition. Your valuable suggestions have provided us with great help.

However, several issues still require attention before the manuscript can be considered for publication. Comment 2: The root sampling protocol remains insufficiently detailed - "random sampling" needs specification regarding sampling locations, depths, and number of sampling points to ensure reproducibility.

Author Response: Thank you very much for your suggestion. Regarding the selection of root systems, we have provided detailed explanations in the revised manuscript, as follows: Remove the root tip (approximately 0-0.5cm), from the root hair zone, specifically 2–6 cm from the root tip, sixty 1-cm-long fresh root segments from each whole cutting root system, categorized by class (ten primary adventitious root, twenty 1^st-order lateral adventitious roots, twenty 2^nd-order lateral adventitious roots and ten 3^rd-order lateral adventitious roots) per treatment, were random selected for mycorrhizal colonization determination.

 

Comment 3: While the authors claim extensive revisions to the Results section, the actual changes are not demonstrated in their response, leaving uncertainty about whether the statistical presentation has been adequately simplified as requested.

Author Response: We are very sorry that we did not provide a detailed display of the revised "discussion" in our response earlier, which caused inconvenience to your review work. The following is our revised detailed discussion content:

This study comprehensively investigated the diverse impacts of four distinct AMF specie, D. versiformis, D. spurca, F. mosseae, and P. occultum, on the growth, root architecture, mineral nutrition, and physiological functions of ‘Summer Black’ grapevine cuttings, the result robustly demonstrate that inoculation with different AMF strains elicits distinct and measurable responses in the host plant.

The foundation of AMF-induced benefits lies in the successful establishment of the symbiotic relationship. Our results confirmed that all four inoculated AMF species successfully colonized the roots of ‘Summer Black’ grapevine cuttings (Figure 1), this initial compatibility is a prerequisite for any subsequent beneficial effects. Among the tested strains, F. mosseae exhibited the highest mycorrhizal colonization, which was obviously surpassed than other stains, suggesting a high degree of compatibility with ‘Summer Black’ grapevine under the experimental conditions. In contrast, D. versiformis, despite its comparatively lower intraradical colonization, developed the most extensive soil extra-radical mycelial density 24.87cm g⁻¹. This divergence highlights that the functional effectiveness of an AMF strain is not solely dictated by its internal root colonization percentage but also by its capacity for prolific external hyphal growth, which is critical for soil exploration and nutrient uptake beyond the root depletion zone [‎35]. This differential emphasis on internal vs. external fungal biomass development likely contributes to the varied plant responses observed across the strains.

The most conspicuous outcome of successful AMF symbiosis is often enhanced plant growth [‎36]. Our results demonstrated that F. mosseae and D. versiformis consistently pro-moted the above-ground growth of ‘Summer Black’ grape cuttings, significantly increasing shoot length and biomass (Table 1). This generalized growth enhancement is a direct consequence of improved resource acquisition and optimized plant physiological processes by AMF [‎37]. Plant root system is the main organ for obtaining nutrient resources from soil. In this study, the superior total root length, root surface area, root volume and primary and 1st-order adventitious root numbers were observed in F. mosseae-inoculated cuttings, which was very beneficial for promoting robust above-ground growth as it pro-vided a greater absorptive surface. In addition, D. versiformis and D. spurca showed inconsistent effects on root system architecture of grape cuttings, indicating that different AMF strains can “engineer” distinct root architectures tailored to different soil resource foraging strategies. Conversely, P. occultum intensely exhibited the inhibitory effects on several growth parameters and adventitious root development, suggesting that while symbiosis was established, it may impose a metabolic cost or be less functionally compatible with ‘Summer Black’ grapevine under these specific conditions, highlighting the inherent variability in AMF-host interactions [‎38].

Enhanced nutrient uptake, particularly P, is a cornerstone of AMF benefits. Our study consistently revealed a significant increase in P content in both grape leaves and roots across all AMF treatments (Table 3), with F. mosseae and D. spurca exhibiting the most pronounced effects in leaves and F. mosseae in roots. This confirms the well-established role of AMF in facilitating P acquisition by extending the effective absorption zone beyond the root depletion zone and potentially accessing less available P forms [‎30]. The impact on other mineral elements, however, was highly variable and strain-dependent, reflecting the complex interplay between host demand, fungal transport capabilities, and soil nutrient dynamics. For instance, while D. spurca, F. mosseae, and P. occultum generally in-creased leaf Zn and Cu, D. versiformis led to their reduction. Similarly, root Fe content generally increased, but P. occultum treatment resulted in a decrease. This nuanced selectivity in nutrient absorption and translocation among different AMF species underscores that AMF do not simply enhance general nutrient uptake but actively modulate the plant’s mineral profile in a highly specific manner [‎39]. Such differential nutrient foraging strategies could be linked to the varied requirements of the fungal strains or their specific transporter systems, providing a competitive advantage for certain elements [40-41]. In addition, in this study, we observed different responses of AMF inoculation to various mineral elements, which may be related to the nutritional level of the cultivation substrate itself, as additional N, P, K fertilization may inhibit AMF infection and thus affect nutrient absorption under field conditions [43-44]. Furthermore, previous study also showed that field application of AMF (Acauloapora scrobiculata, Diversispora spurca, D. versiformis) was able to improve the fruit external quality of Citrus sinensis via increasing soil ammonium N, nitrate N, Olsen P and available K content [45]. Subsequently, similar results were obtained on Camellia oleifera after application of the endophytic fungus Serendipita indica in field conditions, as well as the content of P, K, and Ca significantly increasing in Camellia oleifera leaves and roots [46]. These results indicated that microorganisms in the soil, especially endophytic fungi, can interact with mineral elements in the soil, thereby promoting the absorption and utilization of nutrients by host plants.

Previous studies have demonstrated that AMF inoculation significantly elevated soluble sugars and soluble protein levels, thereby enhancing host resilience to drought stress and bolstering defense against biotic stressors, including grape [‎47]. In this study, the four different AMF strains tested in the present study was all dramatically altered the content of soluble protein in varying degrees, but we all know that this is mainly attributed to the improvement of nutrient conditions, which enhances the resistance of grape cut-tings. we observed differential responses of AMF inoculation to various mineral elements, potentially due to the interplay between N, P, K fertilization and AMF colonization, especially the content of N, P, K. Previous field experiment has demonstrated that some bio stimulants such as Ausuma, Bioilsa, and BF-Ecomix can enhance the abundance and species richness of mycorrhizal fungi in the grapevine rhizosphere [43-44].

Beyond macronutrients, AMF symbiosis also significantly modulated plant physio-logical resilience. Our study revealed notable increases in the activities of key antioxidant enzymes, SOD, POD, and CAT, in both grape roots and leaves following AMF inoculation (Figure 4). These enzymes are vital components of the plant’s defense system against reactive oxygen species (ROS), which are naturally produced during metabolism and amplified under stress conditions [47]. The elevation in antioxidant enzyme activities across multiple AMF treatments suggests an enhanced capacity for oxidative stress mitigation, even under the relatively optimal conditions of this experiment. This pre-priming or strengthened basal defense mechanism could confer greater intrinsic resilience to future environmental challenges such as drought, salinity, or pathogen attack encountered in field settings [‎34,‎48].

The benefits of AMF symbiosis also extended to the photosynthetic machinery of ‘Summer Black’ grape cuttings (Figure 5). All four AMF species significantly enhanced Pn, Gs, and Tr, indicating improved carbon assimilation efficiency and water flux through the plant. F. mosseae demonstrated the most pronounced positive effects on Pn, Gs, and Tr, consistent with its superior root colonization and overall growth promotion. The in-creased Gs facilitates greater CO₂ influx into the leaves, supporting higher photosynthetic rates. Changes in Ci were more variable: D. versiformis and D. spurca increased Ci, suggesting that photosynthetic carboxylation capacity might have become more limiting than stomatal opening. Conversely, P. occultum reduced Ci, indicating potential stomatal limitations. The overall enhancement of photosynthetic efficiency, combined with improved nutrient availability, directly contributes to increased biomass production by providing more photosynthates for plant growth and for supporting the fungal symbiont. This is further corroborated by the elevated soluble protein and soluble sugar contents in both leaves and roots of AMF-inoculated plants (Table 4), signifying enhanced metabolic activ-ity and efficient carbon partitioning. These soluble compounds also serve as crucial osmolytes, energy reserves, and building blocks for various physiological processes and stress responses [34].

In conclusion, our study provides robust evidence for the significant and highly species-specific benefits of AMF symbiosis for ‘Summer Black’ grapevine cuttings. We con-firm that different AMF strains vary markedly in their colonization patterns (intraradical vs. extraradical development), their capacity to promote specific aspects of plant growth and root architecture, their selective influence on mineral nutrient profiles, and their differential enhancement of antioxidant defenses and photosynthetic efficiency. F. mosseae and D. versiformis emerged as particularly effective strains for promoting general growth and physiological vigor in grapevines, largely attributed to their strong capacities for nutrient uptake and enhanced photosynthetic performance. The complex and nuanced responses observed across different AMF strains underscore that selecting the appropriate AMF strain is crucial for maximizing the benefits in viticulture and achieving desired outcomes under specific cultivation conditions.

 

Comment 4: The methodology section still lacks clarity on standardized measurement conditions for photosynthetic parameters.

Author Response: Thank you for your suggestion. We have revised in Materials and Methods as follows: A portable CO₂/H₂O analysis system LI-6400 (Li-COR, USA) was used to determine net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO₂ concentration (Ci) and stomatal conductance (Gs). The effective radiation of the light source was PAR 1000 m² mol/ (m². s), the leaf chamber used was 2 × 3 cm, and the gas flow rate was 500 mmol. s^-1. Three groups of leaves were determined randomly at each layer of each plant [26].

 

Comment 5: Additionally, the discussion of nutrient uptake results must benefit from explicit consideration of how findings from sterile substrate conditions translate to field applications.

Author Response: Thank you. In the experimental process, we created a sterile environment to eliminate the influence of other microorganisms in the soil. Evaluating the role of the fungi we applied in plant growth alone does not change the nutrient content and level in the cultivation substrate. In fact, under field cultivation conditions, the content of mineral elements in the soil can interact with soil microorganisms, especially endophytic fungi, to improve nutrient availability. We have supplemented these contents in our discussion, as follows: Enhanced nutrient uptake, particularly P, is a cornerstone of AMF benefits. Our study consistently revealed a significant increase in P content in both grape leaves and roots across all AMF treatments (Table 3), with F. mosseae and D. spurca exhibiting the most pronounced effects in leaves and F. mosseae in roots. This confirms the well-established role of AMF in facilitating P acquisition by extending the effective absorption zone beyond the root depletion zone and potentially accessing less available P forms [‎30]. The impact on other mineral elements, however, was highly variable and strain-dependent, reflecting the complex interplay between host demand, fungal transport capabilities, and soil nu-trient dynamics. For instance, while D. spurca, F. mosseae, and P. occultum generally in-creased leaf Zn and Cu, D. versiformis led to their reduction. Similarly, root Fe content generally increased, but P. occultum treatment resulted in a decrease. This nuanced selectivity in nutrient absorption and translocation among different AMF species underscores that AMF do not simply enhance general nutrient uptake but actively modulate the plant’s mineral profile in a highly specific manner [‎39]. Such differential nutrient foraging strategies could be linked to the varied requirements of the fungal strains or their specific transporter systems, providing a competitive advantage for certain elements [40-41]. In addition, in this study, we observed different responses of AMF inoculation to various mineral elements, which may be related to the nutritional level of the cultivation substrate itself, as additional N, P, K fertilization may inhibit AMF infection and thus affect nutrient absorption under field conditions [43-44]. Furthermore, previous study also showed that field application of AMF (Acauloapora scrobiculata, Diversispora spurca, D. versiformis) was able to improve the fruit external quality of Citrus sinensis via increasing soil ammonium N, nitrate N, Olsen P and available K content [45]. Subsequently, similar results were obtained on Camellia oleifera after application of the endophytic fungus Serendipita indica in field conditions, as well as the content of P, K, and Ca significantly increasing in Camellia oleifera leaves and roots [46]. These results indicated that microorganisms in the soil, especially endophytic fungi, can interact with mineral elements in the soil, thereby promoting the absorption and utilization of nutrients by host plants.

 

Comment 6: The scientific merit of the work is evident, and the strain-specific effects of AMF on grapevine cuttings represent a valuable contribution to sustainable viticulture practices. However, the remaining methodological ambiguities and presentation issues prevent immediate acceptance.
My Decision: Minor Revision Required
The manuscript shows significant improvement and addresses most fundamental concerns. With the specific clarifications requested above, this work will provide meaningful insights for AMF applications in grape propagation.

Author Response: Thank you very much for your recognition of our work and for your valuable suggestions. We will further improve our paper.

 

In addition, we also do the needful editing according to the editor’s comments and author guidelines. All changes are marked in highlighted. Please see the revised manuscript for details.

 

Author Response File: Author Response.pdf