Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and Metabolic Profile of Saffron in Soilless Cultivation
, Sonia Demasi 1,†, Íris Marisa Maxaieie Victorino 2,3,4, Dario Donno 1, Antonella Faccio 2, Erica Lumini 2, Valeria Bianciotto 2 and Valentina Scariot 1,2
Round 1
Reviewer 1 Report
L2-L4 The general topic seems interesting to me. However, the current version contains important drawback that prevent me to understand fully the core of the study. I provided some comments to improve these aspects.
L20 I hardly believe that a mixture of Rhizofagus intraradices and Funneliformis mosseae is a “multispecies mixture”. Ideally, I would recommend inoculating at least five different AM fungal families. However, I understand the technical limitations underneath. Please, consider soften a bit the adjective “multispecies”.
L25-L27 are surprising. What is their connection with the introduction (L16-L20)? Please, consider adding a sentence in the abstract explaining why you chose these specific explanatory variables.
L28-L30 I cannot see a direct connexion between the results and the conclusion.
L38 I would add Spain, and probably other Mediterranean countries.
L35-L70 Information about the plant species is too wordy. Please, try to synthesize and put the emphasis on the relationship AM fungi-plant.
L73 Please, explain what “soilless cultivation” means exactly.
L76 Could you please clarify the temperature interval? I do not understand why you consider a range (23-27 ºC) as a starting point.
L84 Please, define biostimulant.
Please, delete “symbiotic”.
Please, add more citations after many crops. For instance, García de León et al 2018 Applied Soil Ecology 124, 289-297 studied the effect of AM fungi on soybean.
L96-101 I cannot understand the aim of the study. Please, clearly formulate the hypotheses and expectations. Plus, this paragraph does not seem well supported in the current introduction. Therefore, I do not dare to comment on methods, results or discussion at this moment. If the editor allows resubmitting this manuscript, and invite me to review it again, I will focus on these sections –if and only if the abstract and introduction are clearly written and attractive to many readers.
Author Response
April, 19th 2019To: Editorial Office Agronomy
we hereby submit a revised version of the article “Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and Metabolic Profile of Saffron in Soilless Cultivation.” by Matteo Caser, Sonia Demasi, Íris Marisa Maxaieie Victorino, Dario Donno, Antonella Faccio, Erica Lumini, Valeria Bianciotto and Valentina Scariot for publication in Agronomy, Section “Horticultural and Floricultural Crops”, Special Issue “Toward a Sustainable Agriculture Through Plant Biostimulants: From Experimental Data to Practical Applications”.
We thank for the comments and suggestions that were very helpful to further improve clarity of the manuscript.
For the preparation of the revised manuscript, we followed all the comments and suggestions of the reviewers as stated below. To highlight the corrections made to the text we have maintained the track changes.
Reviewer 1
We thank reviewer for useful comments and suggestions.
Rev.
“L2-L4 The general topic seems interesting to me. However, the current version contains important drawback that prevent me to understand fully the core of the study. I provided some comments to improve these aspects”.
Authors
We thank you for useful comments and suggestions.
Rev.
“L20 I hardly believe that a mixture of Rhizofagus intraradices and Funneliformis mosseae is a “multispecies mixture”. Ideally, I would recommend inoculating at least five different AM fungal families. However, I understand the technical limitations underneath. Please, consider soften a bit the adjective “multispecies”. L25-L27 are surprising. What is their connection with the introduction (L16-L20)? Please, consider adding a sentence in the abstract explaining why you chose these specific explanatory variables. L28-L30 I cannot see a direct connexion between the results and the conclusion”.
Authors
The Abstract was revised as suggested. We totally agree with you and we have changed the term “multispecies”. For clarity matter we have chosen to use the term “multispecies” because of the MycoAgro company inocula name itself which is denominated “mix inoculum”.
Rev.
“L38 I would add Spain, and probably other Mediterranean countries. L35-L70 Information about the plant species is too wordy. Please, try to synthesize and put the emphasis on the relationship AM fungi-plant. L73 Please, explain what “soilless cultivation” means exactly. L76 Could you please clarify the temperature interval? I do not understand why you consider a range (23-27 ºC) as a starting point. L84 Please, define biostimulant. Please, delete “symbiotic”. Please, add more citations after many crops. For instance, García de León et al 2018 Applied Soil Ecology 124, 289-297 studied the effect of AM fungi on soybean. L96-101 I cannot understand the aim of the study. Please, clearly formulate the hypotheses and expectations. Plus, this paragraph does not seem well supported in the current introduction. Therefore, I do not dare to comment on methods, results or discussion at this moment. If the editor allows resubmitting this manuscript, and invite me to review it again, I will focus on these sections –if and only if the abstract and introduction are clearly written and attractive to many readers”.
Authors
The Introduction section was rephrased and modified according to Reviewer suggestion. We have cut part of the text to make it more even and brief. We prefer not to cite articles referring to specific crop plants but instead insert two reviews were AMF influence on different crop plants are fully described. The objective of this research was clarified.
We remain available to clarify any issue or answer that Reviewers or Editors may raise.
Best regards
Sincerely Yours
Matteo Caser,
Sonia Demasi,
Íris Marisa Maxaieie Victorino,
Dario Donno,
Antonella Faccio,
Erica Lumini,
Valeria Bianciotto
and Valentina Scariot
Department of Agricultural, Forest and Food Sciences
University of Turin
Largo Paolo Braccini, 2
10095, Grugliasco (TO)
Italy
Phone number: +039-011/6708935
Fax number: +039-011/6708798
e-mail: [email protected]
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript “Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and
Metabolic Profile of Saffron in Soilless Cultivation” submitted for publication in Agronomy aim to investigate the crop performance, and spice yield, and quality, of saffron in a soilless system, using AMF as biostimulants.
The paper is a follow-up of an early work, already published in Agronomy (Caser et al. 2019), which missed data for spice yield, and quality, of the soilless experiment, allegedly due to lack of flowering, because of the small size of the corms used.
Therefore, one of the main objective of the manuscript, to compare soilless cultivation with open field crop (sees Fig. 4 and 5), is flawed by the fact that it discusses results of production obtained in two different environmental conditions, with two different plant density, in different years of cultivation (if data from Caser et al. are those of the first year of cultivation, this is not clearly stated).
The second main objective, investigating AMF role on saffron cultivation, is impaired by the lack of data on colonization rate, the paragraph on AMF evaluation being dedicated only on qualitative analysis of the occurrence of fungal structures in roots. However, one can wonder how relevant this information is to an agronomy journal.
Another questionable aspect of this paper is the evaluation of the data of bioactive compounds in the context of a dietary intervention (lines 255-356), considering the amount normally consumed of thos spice. Maybe, the authors should stress the possible interest for an industrial application (lines 94-95).
Some more specific comments.
Bibliography is not always appropriate.
some examples:
line 37 “This species is widely cultivated in Iran, Italy, Morocco, France, Greece, China, India and Mexico [3].”
Line 60 “It is traditionally cultivated in small and flat plots in which mechanisation is not economically sustainable due to the harvest type and short flowering period [8].”
Lines 61-62 “Five hundred hand labour hours are needed to obtain 1 kg of dried saffron [4].”
Lines 79-80 “Sourell and Weathers [27] and Mollafilabi et al. [25] concluded that open field cultivation more strongly affected the saffron yield…”: the first citation is not correct (in addition, edit the author name as “Souret”)
The experimental design should be made clear.
The authors say that “two inocula were used, and only one control” (lines 111-113), and this is in contrast with line 119 (what is the fourth treatment?)
It follows that is not clear how many pots were used.
Again, how many pots per treatments? Explain the sentence “(8 pots per treatment), for a total of 16 pots per treatment”); in fact, what are the two experimental plots units, and how they are integrated in the complete randomised block design?
Please, add the number of statistical units in the table and figure legends.
It would be strongly encouraged that fungal inocula be better described (adding the name of fungal strain/isolate, and possibly its origin and type (spores, root fragments, crude inoculum), especially coping with agronomic journals.
Data are not clearly reported and some important information are missing.
As an example, description of data reported in Table 3 is misleading and inconsistent:
“AMF inoculum composed of Ri+Fm significantly increased the contents of isoquercitrin …, and the total phenolic content (TPC)” it is not true when compared with treatment AMF-.
Actually, it is not clear if the authors compare the mycorrhizal treatments with control (then their description of result is misleading also for isoquercitrin and AOA) or between them (so that control is of little avail).
Another point which should be made clear has to do with the anthocyanin results which are amazing. Are there two orders of magnitude difference between the lower and the higher value ?
It is surprising that authors did not measured mycorrhizal colonization, since they did in the previous paper (Caser et al. 2019).
Instead of corm size and weight percent variation between the end and beginning of the trial, the actual data should be presented, as in Caser et al. 2019.
In order to let reading more comfortable, and realistic, I suggest that most production data should be compared per area and not per corm.
Other minor comments
Lines 439-440 “such as polyphenols, anthocyanins, vitamin C and elevated antioxidant activity”. It seems not to be true for vitamin C and possibly for anthocyanins.
Lines 314-315“Very few reports about the effective role of AMF in saffron flowering and yield are available in the literature, and only under open field conditions”, pay attention that this statement
is contradictory with
lines 223-224 “ flowering could be modulated, extended and considerably increased, compared with open field cultivation” credited to Maggio et al. and Gresta et al.
Line 210 emend “uranyl”
Author Response
April, 19th 2019To:Editorial Office Agronomy
Dear Editorial Office,
we hereby submit a revised version of the article “Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and Metabolic Profile of Saffron in Soilless Cultivation.” by Matteo Caser, Sonia Demasi, Íris Marisa Maxaieie Victorino, Dario Donno, Antonella Faccio, Erica Lumini, Valeria Bianciotto and Valentina Scariot for publication in Agronomy, Section “Horticultural and Floricultural Crops”, Special Issue “Toward a Sustainable Agriculture Through Plant Biostimulants: From Experimental Data to Practical Applications”.
We thank for the comments and suggestions that were very helpful to further improve clarity of the manuscript.
For the preparation of the revised manuscript, we followed all the comments and suggestions of the reviewers as stated below. To highlight the corrections made to the text we have maintained the track changes.
Reviewer 2
We thank reviewer for useful comments and suggestions.
Rev.
“The manuscript “Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and Metabolic Profile of Saffron in Soilless Cultivation” submitted for publication in Agronomy aim to investigate the crop performance, and spice yield, and quality, of saffron in a soilless system, using AMF as biostimulants.
The paper is a follow-up of an early work, already published in Agronomy (Caser et al. 2019), which missed data for spice yield, and quality, of the soilless experiment, allegedly due to lack of flowering, because of the small size of the corms used.
Therefore, one of the main objective of the manuscript, to compare soilless cultivation with open field crop (sees Fig. 4 and 5), is flawed by the fact that it discusses results of production obtained in two different environmental conditions, with two different plant density, in different years of cultivation (if data from Caser et al. are those of the first year of cultivation, this is not clearly stated)”.
Authors
The Introduction and Results and Discussion Sections were rephrased and modified according to Reviewer suggestion. Therefore, the aim of this research and the comparison with open-field trial was clarified in the text.
Rev.
“The second main objective, investigating AMF role on saffron cultivation, is impaired by the lack of data on colonization rate, the paragraph on AMF evaluation being dedicated only on qualitative analysis of the occurrence of fungal structures in roots. However, one can wonder how relevant this information is to an agronomy journal”.
Authors
We have included a brief description and a reference to our previous work (Caser et al 2019) in which are summarized the mycorrhizal frequencies obtained by using Trouvelot method on saffron roots in soilless conditions.
Rev.
“Another questionable aspect of this paper is the evaluation of the data of bioactive compounds in the context of a dietary intervention (lines 255-356), considering the amount normally consumed of thos spice. Maybe, the authors should stress the possible interest for an industrial application (lines 94-95)”.
Authors
We rephrased these sentences according to Reviewer suggestions.
Rev.
“Some more specific comments.
Bibliography is not always appropriate.
some examples:
line 37 “This species is widely cultivated in Iran, Italy, Morocco, France, Greece, China, India and Mexico [3].”
Line 60 “It is traditionally cultivated in small and flat plots in which mechanisation is not economically sustainable due to the harvest type and short flowering period [8].”
Lines 61-62 “Five hundred hand labour hours are needed to obtain 1 kg of dried saffron [4].”
Lines 79-80 “Sourell and Weathers [27] and Mollafilabi et al. [25] concluded that open field cultivation more strongly affected the saffron yield…”: the first citation is not correct (in addition, edit the author name as “Souret”)”.
Authors
We checked references and we modified the bibliography according to the Reviewer suggestions.
Rev.
“The experimental design should be made clear.
The authors say that “two inocula were used, and only one control” (lines 111-113), and this is in contrast with line 119 (what is the fourth treatment?)
It follows that is not clear how many pots were used.
Again, how many pots per treatments? Explain the sentence “(8 pots per treatment), for a total of 16 pots per treatment”); in fact, what are the two experimental plots units, and how they are integrated in the complete randomised block design?
Please, add the number of statistical units in the table and figure legends.
It would be strongly encouraged that fungal inocula be better described (adding the name of fungal strain/isolate, and possibly its origin and type (spores, root fragments, crude inoculum), especially coping with agronomic journals”.
Authors
We modified the Material and Method section according to the Reviewer suggestions. We have inserted some information about AMF but unfortunately the company selling the commercial inoculum does not provide a fully detailed information on spore density.
Rev.
“Data are not clearly reported and some important information are missing. As an example, description of data reported in Table 3 is misleading and inconsistent: “AMF inoculum composed of Ri+Fm significantly increased the contents of isoquercitrin …, and the total phenolic content (TPC)” it is not true when compared with treatment AMF-. Actually, it is not clear if the authors compare the mycorrhizal treatments with control (then their description of result is misleading also for isoquercitrin and AOA) or between them (so that control is of little avail). Another point which should be made clear has to do with the anthocyanin results which are amazing. Are there two orders of magnitude difference between the lower and the higher value ? It is surprising that authors did not measured mycorrhizal colonization, since they did in the previous paper (Caser et al. 2019). Instead of corm size and weight percent variation between the end and beginning of the trial, the actual data should be presented, as in Caser et al. 2019. In order to let reading more comfortable, and realistic, I suggest that most production data should be compared per area and not per corm”.
Authors
We modified Results and Discussion section according to the Reviewer suggestions, where it is possible. The data on anthocyanins were changed.
Rev.
“Other minor comments
Lines 439-440 “such as polyphenols, anthocyanins, vitamin C and elevated antioxidant activity”. It seems not to be true for vitamin C and possibly for anthocyanins.
Lines 314-315“Very few reports about the effective role of AMF in saffron flowering and yield are available in the literature, and only under open field conditions”, pay attention that this statement is contradictory with lines 223-224 “ flowering could be modulated, extended and considerably increased, compared with open field cultivation” credited to Maggio et al. and Gresta et al.
Line 210 emend “uranyl”
Authors
We modified the text according to the Reviewer suggestions. The data reported by Maggio et al. and Gresta et al. are not referred to AMF application in saffron cultivation in soilless systems. But only on saffron cultivation in soilless systems.
We remain available to clarify any issue or answer that Reviewers or Editors may raise.
Best regards
Sincerely Yours
Matteo Caser,
Sonia Demasi,
Íris Marisa Maxaieie Victorino,
Dario Donno,
Antonella Faccio,
Erica Lumini,
Valeria Bianciotto
and Valentina Scariot
Department of Agricultural, Forest and Food Sciences
University of Turin
Largo Paolo Braccini, 2
10095, Grugliasco (TO)
Italy
Phone number: +039-011/6708935
Fax number: +039-011/6708798
e-mail: [email protected]
Author Response File: Author Response.pdf
Reviewer 3 Report
LANGUAGE:
Quite good
TITLE
My suggestion is that you provide a striking result on the title, but it is also ok as it is
ABSTRACT
Please note that I revised the abstract before reading the whole manuscript. Additional comments on the abstract after reading the manuscript, if any, can be found at the end of this revision (Additional suggestions)
L16-17: Just a simple suggestion, I’d rather say that C. sativus can overflower in soilless compared to soil-bearing systems. I leave up to you the choice. I do not personally strongly like the line 17-18. This is because a reader is expected to know the reasons for exploring an interaction (i.e. soil presence vs AMF presence) so that a suitable beginning could take pave the way to the research by saying that absence of an actual soil (such as in soilless system) can provide the plants with a wealth of nutrients without competition with pathogens or problems related to nutrient-soil interaction, but can also consist in stresses for the plants for the establishment of important soil microbial flora that can improve the saffron quality, as you also explored in previous experiment. This may be a way of justifying the interaction you studied. Else, if this interaction was not studied and you simply studied the AMF effect (compared to a not inoculated control) under a soilless system, please strike to the aim of the experiment. Indeed, simply saying that “Further improvements may be obtained by…” can be applied to lot of treatments and is not a reason to make an experiment
L22: specify the high quality compared to what? A standard?
L24-25: plase avoid italianicised sentences. Just say “Rhizophagus intraradices performed better than the mix in term of….”
L25-26: it is ok to cite papers in the abstract if these are strongly related, but these should be cited in brackets (journal, year, issue, pages). My personal point of view is that the previous paper you are referring to is not so strongly related (but is indeed related!) so that there is no need of making such a comparison in the abstract since open field cultivation trials can have dramatic differences compared to the greenhouse ones, especially those in soilless conditions
L28: unclear if the AMF had less influence here or in the field experiment.
General comment: that abstract is puzzling. I suggest to strike more on the results and its implication with few or no hints to the previous in field trial, that can be fully debated in the discussion. In my humble opinion, here you should emphasize on inocula differences, not on growing conditions since it seem you only had one condition, i.e. the soilless cropping system.
INTRODUCTION
L42-43: I suggest not to use “not only…, but also…”. Instead directly say “These compounds confer the saffron’s unique colour, taste, and aroma, and can also have positive biological effects”
L47: this sentence is quite useless
L53-54: please contextualize the sentence “Commercially, a 2.5–3.5 cm diameter corm appears to be the most common size used for planting [20].” What is that for? Is that enough to have full flowering per unit area in field conditions? Does it compensate for environmental effects on the the flowing time and flushes?
L56-58: this is ok, but should be placed soon after the ref [5, 15] in line 49
L62-63: the sentence “Saffron cultivation can be carried out on an annual or multi-year cycle [22,23].” Should be placed earlier, when describing the crop in field conditions
L72: delete “recently”
L76: you may find suitable to say that corms has a verbalization requirement of XXX °Chhing hours at threshould temperature of XXXXX °C (if this information exists, but I don’t think so) or from 23–27 °C to 17 °C for XXX days.
L80-82: good, but clarify. Do you mean that field systems are more variable or more yielding? In the first instance, it is ok. In the second instance, it may be for unsuitable glasshouse conditions as you point in line 83
L90: please specify what you mean with “without the negative effects on biomass production [33,34].”
General comment: the introduction is quite good, but still lacking of a connection between the need of exploring the AMF effect in the greenhouse conditions. My suggestion is to add a sentence by specifying that the lack of information on the glasshouse conditions for saffron, as pointed in lines 80-83, may depend on a range of factors among of which the regulation of the plant nutrient uptake and nutrient ration in the soil or soil solution or nutritive solution and presence in the soilless conditions of oxidative stresses for the roots. Then specify that AMF fungi can regulate the nutrient concentration and availability for the plant, by temporarily subtracting some nutrient (e.g. reduced N) and preferentially delivering other (e.g. P, Zn, and B) and this can stimulate flowering.
MATERIALS AND METHODS
L109: when using various substrate and describing the volume, please also give the bulk density. Sterile quarts is usually around 1.6 kg/L, expanded clay has variable bulk density. Please also give pot size (diameter, height). Else give the amount in weight.
L112-113: please briefly describe the inocula (organic matter content, spore density, other inoculation means such as colonized root fragments). Just few words are enought
L118-119: it is unclear. You have 3 treatments (Ri, Ri+Fm and not inoculated) replicated 8 times, that is 24 replicate. If you made a whole experiment replication twice, you should have had 48 pots. Which is the fourth treatment you are referring to?
L121: please give the concentration of elements in the fertigation solution
L137-138: in any case, the spectrophotometer does not measure bitterness, flavouring strength, and colouring strength. Else say that you measured the concentration of picrocrocin, safranal, and crocin to have information on the bitterness, flavouring strength, and colouring strength, respectively, of the product. So, I’m just asking you to reverse the construct of the sentence, with regards to the sentence in lines 139-141
L185-192: it is still unclear to me the methodology of measuring AMF in roots. You detailed the methodology of root treatment, but which fraction of the AM colonization were counted and why? Please add details.
RESULTS AND DISCUSSION
L216-218: please highlight that data in 12 were coming from the experiment in ref 12 and not from that described here. Also, detail the aim of this analysis in the aim section, by the end of the introduction.
L226: if the Caser et al. experiment used the same plant and fungal inocula, please state it since it strengthen the results. With regards to the forwarding of the crop cycle, you should briefly discuss the differences in term of growing degrees day accumulation in the 2 experiments and any conditions or stress that may have anticipated flowering in the field (drought? Light? Vernalization requirements? Diurnal temperature excursions? Other?). In line 228 you spell that “in a glasshouse, the lowering of seasonal temperatures takes place more slowly than in an open field” and this is indeed correct, but point to the physiological aspect entailed and debate on any other potential factor. Also, compute your GDD from establishment to flowering here and in ref. 12 and add reference, if any, on the relationship between the factors inducing the flowering and your conditions
L235-236: it is unclear to me how you compute the yield per hectare in a pot experiment. It is possible but should be described in M&M. Also, please refer to the yield per unit area of the greenhouse so to provide the reader a practical implication in these conditions, which are indeed very interesting
L238: correct the brackets after Gresta et al. :
L236-241: I agree with these comments, but the discussion needs of a contextualization. Did Gresta et al (ref. 6) used the same genotypes? Please state it. Also, which are the potential contributors to these differences between field and greenhouse? Did any of the refs. 16, 24 and 51 used different genotypes? I agree that saffron flowering may depend on “a not yet fully understood combination of temperature and soil water content”, but you should at least describe the similarities and differences between your conditions and those of the others with regards to these potential main contributor and other potential strong contributors (e.g. the initial bulb size, the conditions inducing the ethylene release or any ethylene treatment?).
L242-250: please make the same inferences as those suggested for lines 236-241
L265-2268: move this part to the conclusion of the work
L266: delete “however”, since the sentence is not adversative to the previous one.
L268-270: this is methodology and is not needed here. Please delete. I agree that you discussed the mean yield and now you are going to discuss the AMF effect, but it is not needed to bring back the aim in the very middle of the R&D section unless you are treating this section in term of sub-aims (that you don’t have).
L272-275: this is a very general aspect not interesting for the discussion. I suggest to delete it.
L278-279: what is a well colonization? In wheat 30% of the root length colonized can be high, wereas in many legumes or in Tomato, 30% is low. Do you have a measure or a reference or simply a picture (that is indeed interesting)? Please state here en in the relevant section of the M&M.
Can you provide a bridge of debate between your measurements and the most used ones such as the the GIM of Giovannetti and Mosse, the microscope A-F-m of Trouvelot, the genetic expression measurements and even that used by Asuncion Morte in palms?). The method you used is very interesting and such a discussion should be brought out.
L292-295: too general, please delete. This has nothing to do with the discussion of your data despite you point something out in lines 303-305
L318-322: this is a R&D, not 2 separate sections. So please describe the results and then discuss it. This means that this section should be moved back soon after line 313 and the section from line 314 to 318 moved to lines 332 or 336.
L320: what do you mean with “a minimum”?
L313-351: this whole section is important but scarcely handled. You have differences (in the secondary metabolites and corm size) that are scarcely discussed. Also, lack of differences in the other traits (e.g. flower number, saffron yield, corm number and weight) can also be discussed and an hypothesis for the lack of effect put on the table. I suggest you to better deal with this section
L356: well, I’d not say dietary intervention with a product eaten at those scarce amount. Maybe better pointing to any industrial/pharmaceutical use.
L359: avoid the “not only…, but also…”. Just say “both in roots, shoots and flowers”. You may say that such alteration is not consistent among plant organs
L365: just say “under field conditions in a temperate mountain area (north-west Italy)”. Avoid “Italian open field conditions”
L366: the reader may not be familiar with “superior quality” definition. Please add a very brief discussion in brackets, e.g. an “(i.e. quality compared to the ISO standards)”.
L368: what is that however for? Also, which is this increase compared to? Is that compared to not inoculated or to Ri alone?
L373-375: I suggest to delete “in other plant species,” and also describe a difference between your experiment and those cited in refs. 69-70-71 such as, apart of the plant species, the P availability, the AMF genotypes, some other stress, etc.
L386-387: higher compared to what? Please don’t leave the reader disoriented and puzzled. Also, which are the percentage in lines 388-390 referred to? More 1000% compared to what?). Lastly, my suggestion is not use wording such as “superfoods”. Superfoods do not exist, we only have equilibrated diets and life-styles. You may suitably highlight these traits for their potential transformation in industrial uses, since the secondary metabolite (low) concentration poses a wealth of problems in the industrial processes
L391-392: delete it. Really, it is not needed!
L352: I guess this whole section could be named “3.3.2. Saffron metabolic profiling comparing to other foods” instead of simply “3.3.2. Saffron metabolic profiling”. I just leave up to you the choice
L408: were they made with the same method? If not, please debate on methodology differences
L411: change “this cultivation type” to “this latter cultivation system”
General comment: some parts of the R&D should be better described and the discussion sentences better contextualized to the results. However, in general, information provided is good
CONCLUSIONS
L436: say “appeared as an effective” instead of “is an effective”
L437-440: I’d not stress on the nutritional benefit of a product that is eaten in few mg/week or month on average. Instead stress on its suitability for industriall transformation and implication of th system difference (field vs greenhouse) for the profitability (total per per unit product depending on its quality) and ability to expand the cultivation period out of the growing cycle in the agricultural condition. Also, you can refear to any difference in the workload, that may have implication for the workers’ health.
L441: just use the past tense (colonized). Also, I can’t easily score the successfulness of this trait from your R&D
L446-447: this can be deleted without losing the interest in the (or of the) work
NOVELTY, LIMITATION AND ADDITIONAL SUGGESTION
Please better describe the implication of the results. In the R&D, make inferences and debates after having presented a result.
Regards.
Sergio Saia
Author Response
April, 19th 2019To:Editorial Office Agronomy
Dear Editorial Office,
we hereby submit a revised version of the article “Arbuscular Mycorrhizal Fungi Modulate the Crop Performance and Metabolic Profile of Saffron in Soilless Cultivation.” by Matteo Caser, Sonia Demasi, Íris Marisa Maxaieie Victorino, Dario Donno, Antonella Faccio, Erica Lumini, Valeria Bianciotto and Valentina Scariot for publication in Agronomy, Section “Horticultural and Floricultural Crops”, Special Issue “Toward a Sustainable Agriculture Through Plant Biostimulants: From Experimental Data to Practical Applications”.
We thank for the comments and suggestions that were very helpful to further improve clarity of the manuscript.
For the preparation of the revised manuscript, we followed all the comments and suggestions of the reviewers as stated below. To highlight the corrections made to the text we have maintained the track changes.
Reviewer 3
We thank reviewer for useful comments and suggestions.
Rev.
“TITLE
My suggestion is that you provide a striking result on the title, but it is also ok as it is”
Authors
We prefer to maintain this title because AMF are indeed effective in modulation of some morphological and chemical aspects.
Rev.
“ABSTRACT
Please note that I revised the abstract before reading the whole manuscript. Additional comments on the abstract after reading the manuscript, if any, can be found at the end of this revision (Additional suggestions)
L16-17: Just a simple suggestion, I’d rather say that C. sativus can overflower in soilless compared to soil-bearing systems. I leave up to you the choice. I do not personally strongly like the line 17-18. This is because a reader is expected to know the reasons for exploring an interaction (i.e. soil presence vs AMF presence) so that a suitable beginning could take pave the way to the research by saying that absence of an actual soil (such as in soilless system) can provide the plants with a wealth of nutrients without competition with pathogens or problems related to nutrient-soil interaction, but can also consist in stresses for the plants for the establishment of important soil microbial flora that can improve the saffron quality, as you also explored in previous experiment. This may be a way of justifying the interaction you studied. Else, if this interaction was not studied and you simply studied the AMF effect (compared to a not inoculated control) under a soilless system, please strike to the aim of the experiment. Indeed, simply saying that “Further improvements may be obtained by…” can be applied to lot of treatments and is not a reason to make an experiment
L22: specify the high quality compared to what? A standard?
L24-25: plase avoid italianicised sentences. Just say “Rhizophagus intraradices performed better than the mix in term of….”
L25-26: it is ok to cite papers in the abstract if these are strongly related, but these should be cited in brackets (journal, year, issue, pages). My personal point of view is that the previous paper you are referring to is not so strongly related (but is indeed related!) so that there is no need of making such a comparison in the abstract since open field cultivation trials can have dramatic differences compared to the greenhouse ones, especially those in soilless conditions
L28: unclear if the AMF had less influence here or in the field experiment.
General comment: that abstract is puzzling. I suggest to strike more on the results and its implication with few or no hints to the previous in field trial, that can be fully debated in the discussion. In my humble opinion, here you should emphasize on inocula differences, not on growing conditions since it seem you only had one condition, i.e. the soilless cropping system”.
Authors
We entirely rephrased and modified the Abstract according to the Reviewer suggestions.
Rev.
“INTRODUCTION
L42-43: I suggest not to use “not only…, but also…”. Instead directly say “These compounds confer the saffron’s unique colour, taste, and aroma, and can also have positive biological effects”
L47: this sentence is quite useless
L53-54: please contextualize the sentence “Commercially, a 2.5–3.5 cm diameter corm appears to be the most common size used for planting [20].” What is that for? Is that enough to have full flowering per unit area in field conditions? Does it compensate for environmental effects on the the flowing time and flushes?
L56-58: this is ok, but should be placed soon after the ref [5, 15] in line 49
L62-63: the sentence “Saffron cultivation can be carried out on an annual or multi-year cycle [22,23].” Should be placed earlier, when describing the crop in field conditions
L72: delete “recently”
L76: you may find suitable to say that corms has a verbalization requirement of XXX °Chhing hours at threshould temperature of XXXXX °C (if this information exists, but I don’t think so) or from 23–27 °C to 17 °C for XXX days.
L80-82: good, but clarify. Do you mean that field systems are more variable or more yielding? In the first instance, it is ok. In the second instance, it may be for unsuitable glasshouse conditions as you point in line 83
L90: please specify what you mean with “without the negative effects on biomass production [33,34].”
General comment: the introduction is quite good, but still lacking of a connection between the need of exploring the AMF effect in the greenhouse conditions. My suggestion is to add a sentence by specifying that the lack of information on the glasshouse conditions for saffron, as pointed in lines 80-83, may depend on a range of factors among of which the regulation of the plant nutrient uptake and nutrient ration in the soil or soil solution or nutritive solution and presence in the soilless conditions of oxidative stresses for the roots. Then specify that AMF fungi can regulate the nutrient concentration and availability for the plant, by temporarily subtracting some nutrient (e.g. reduced N) and preferentially delivering other (e.g. P, Zn, and B) and this can stimulate flowering”.
Authors
We modified the Introduction section according to the Reviewer suggestions.
Rev.
“MATERIALS AND METHODS
L109: when using various substrate and describing the volume, please also give the bulk density. Sterile quarts is usually around 1.6 kg/L, expanded clay has variable bulk density. Please also give pot size (diameter, height). Else give the amount in weight.
L112-113: please briefly describe the inocula (organic matter content, spore density, other inoculation means such as colonized root fragments). Just few words are enought
L118-119: it is unclear. You have 3 treatments (Ri, Ri+Fm and not inoculated) replicated 8 times, that is 24 replicate. If you made a whole experiment replication twice, you should have had 48 pots. Which is the fourth treatment you are referring to?
L121: please give the concentration of elements in the fertigation solution
L137-138: in any case, the spectrophotometer does not measure bitterness, flavouring strength, and colouring strength. Else say that you measured the concentration of picrocrocin, safranal, and crocin to have information on the bitterness, flavouring strength, and colouring strength, respectively, of the product. So, I’m just asking you to reverse the construct of the sentence, with regards to the sentence in lines 139-141
L185-192: it is still unclear to me the methodology of measuring AMF in roots. You detailed the methodology of root treatment, but which fraction of the AM colonization were counted and why? Please add details”.
Authors
We modified the Introduction section according to the Reviewer suggestions. We have inserted some information in AMF but unfortunately the company selling the commercial inoculum does not provide a fully detailed information on spore density. Moreover, we have included a brief description and a reference to our previous work (Caser et al 2019) in which are summarized the mycorrhizal frequencies obtained by using Trouvelot method on saffron roots in soilless conditions.
Rev.
“RESULTS AND DISCUSSION
L216-218: please highlight that data in 12 were coming from the experiment in ref 12 and not from that described here. Also, detail the aim of this analysis in the aim section, by the end of the introduction.
L226: if the Caser et al. experiment used the same plant and fungal inocula, please state it since it strengthen the results. With regards to the forwarding of the crop cycle, you should briefly discuss the differences in term of growing degrees day accumulation in the 2 experiments and any conditions or stress that may have anticipated flowering in the field (drought? Light? Vernalization requirements? Diurnal temperature excursions? Other?). In line 228 you spell that “in a glasshouse, the lowering of seasonal temperatures takes place more slowly than in an open field” and this is indeed correct, but point to the physiological aspect entailed and debate on any other potential factor. Also, compute your GDD from establishment to flowering here and in ref. 12 and add reference, if any, on the relationship between the factors inducing the flowering and your conditions
L235-236: it is unclear to me how you compute the yield per hectare in a pot experiment. It is possible but should be described in M&M. Also, please refer to the yield per unit area of the greenhouse so to provide the reader a practical implication in these conditions, which are indeed very interesting
L238: correct the brackets after Gresta et al. :
L236-241: I agree with these comments, but the discussion needs of a contextualization. Did Gresta et al (ref. 6) used the same genotypes? Please state it. Also, which are the potential contributors to these differences between field and greenhouse? Did any of the refs. 16, 24 and 51 used different genotypes? I agree that saffron flowering may depend on “a not yet fully understood combination of temperature and soil water content”, but you should at least describe the similarities and differences between your conditions and those of the others with regards to these potential main contributor and other potential strong contributors (e.g. the initial bulb size, the conditions inducing the ethylene release or any ethylene treatment?).
L242-250: please make the same inferences as those suggested for lines 236-241
L265-2268: move this part to the conclusion of the work
L266: delete “however”, since the sentence is not adversative to the previous one.
L268-270: this is methodology and is not needed here. Please delete. I agree that you discussed the mean yield and now you are going to discuss the AMF effect, but it is not needed to bring back the aim in the very middle of the R&D section unless you are treating this section in term of sub-aims (that you don’t have).
L272-275: this is a very general aspect not interesting for the discussion. I suggest to delete it.
L278-279: what is a well colonization? In wheat 30% of the root length colonized can be high, wereas in many legumes or in Tomato, 30% is low. Do you have a measure or a reference or simply a picture (that is indeed interesting)? Please state here en in the relevant section of the M&M.
Can you provide a bridge of debate between your measurements and the most used ones such as the the GIM of Giovannetti and Mosse, the microscope A-F-m of Trouvelot, the genetic expression measurements and even that used by Asuncion Morte in palms?). The method you used is very interesting and such a discussion should be brought out.
L292-295: too general, please delete. This has nothing to do with the discussion of your data despite you point something out in lines 303-305
L318-322: this is a R&D, not 2 separate sections. So please describe the results and then discuss it. This means that this section should be moved back soon after line 313 and the section from line 314 to 318 moved to lines 332 or 336.
L320: what do you mean with “a minimum”?
L313-351: this whole section is important but scarcely handled. You have differences (in the secondary metabolites and corm size) that are scarcely discussed. Also, lack of differences in the other traits (e.g. flower number, saffron yield, corm number and weight) can also be discussed and an hypothesis for the lack of effect put on the table. I suggest you to better deal with this section
L356: well, I’d not say dietary intervention with a product eaten at those scarce amount. Maybe better pointing to any industrial/pharmaceutical use.
L359: avoid the “not only…, but also…”. Just say “both in roots, shoots and flowers”. You may say that such alteration is not consistent among plant organs
L365: just say “under field conditions in a temperate mountain area (north-west Italy)”. Avoid “Italian open field conditions”
L366: the reader may not be familiar with “superior quality” definition. Please add a very brief discussion in brackets, e.g. an “(i.e. quality compared to the ISO standards)”.
L368: what is that however for? Also, which is this increase compared to? Is that compared to not inoculated or to Ri alone?
L373-375: I suggest to delete “in other plant species,” and also describe a difference between your experiment and those cited in refs. 69-70-71 such as, apart of the plant species, the P availability, the AMF genotypes, some other stress, etc.
L386-387: higher compared to what? Please don’t leave the reader disoriented and puzzled. Also, which are the percentage in lines 388-390 referred to? More 1000% compared to what?). Lastly, my suggestion is not use wording such as “superfoods”. Superfoods do not exist, we only have equilibrated diets and life-styles. You may suitably highlight these traits for their potential transformation in industrial uses, since the secondary metabolite (low) concentration poses a wealth of problems in the industrial processes
L391-392: delete it. Really, it is not needed!
L352: I guess this whole section could be named “3.3.2. Saffron metabolic profiling comparing to other foods” instead of simply “3.3.2. Saffron metabolic profiling”. I just leave up to you the choice
L408: were they made with the same method? If not, please debate on methodology differences
L411: change “this cultivation type” to “this latter cultivation system”
General comment: some parts of the R&D should be better described and the discussion sentences better contextualized to the results. However, in general, information provided is good
Authors
We deeply modified and contextualized the Results and Discussion section according to Reviewer suggestion. We have reorganized the text and included on M&M section a reference regarding mycorrhizal frequencies obtained by using Trouvelot method on saffron roots in soilless conditions (Caser et al 2019). Regarding the suggestion made by Rev. 3 “Can you provide a bridge of debate between your measurements and the most used ones such as the the GIM of Giovannetti and Mosse, the microscope A-F-m of Trouvelot, the genetic expression measurements and even that used by Asuncion Morte in palms?). The method you used is very interesting and such a discussion should be brought out”. It is indeed a very interesting methodology however it allows us to see only if the AMF is active inside the cortical cell. No measurement can be obtained with this method. For this reason we cannot provide a bridge of debate between our work and the most used ones that you refer.
Rev.
“CONCLUSIONS
L436: say “appeared as an effective” instead of “is an effective”
L437-440: I’d not stress on the nutritional benefit of a product that is eaten in few mg/week or month on average. Instead stress on its suitability for industriall transformation and implication of th system difference (field vs greenhouse) for the profitability (total per per unit product depending on its quality) and ability to expand the cultivation period out of the growing cycle in the agricultural condition. Also, you can refear to any difference in the workload, that may have implication for the workers’ health.
L441: just use the past tense (colonized). Also, I can’t easily score the successfulness of this trait from your R&D
L446-447: this can be deleted without losing the interest in the (or of the) work”.
Authors
We modified the work conclusions to better describe the implication of the results as suggested by the Reviewer.
We remain available to clarify any issue or answer that Reviewers or Editors may raise.
Best regards
Sincerely Yours
Matteo Caser,
Sonia Demasi,
Íris Marisa Maxaieie Victorino,
Dario Donno,
Antonella Faccio,
Erica Lumini,
Valeria Bianciotto
and Valentina Scariot
Department of Agricultural, Forest and Food Sciences
University of Turin
Largo Paolo Braccini, 2
10095, Grugliasco (TO)
Italy
Phone number: +039-011/6708935
Fax number: +039-011/6708798
e-mail: [email protected]
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Authors have addressed all the comments of previous review round. I do not have further comments.
Author Response
Dear Reviewer 1,
thank you for your comments.
