Diversity and Benefits of Arbuscular Mycorrhizae in Restored Riparian Plantations
, Eduardo J. Azevedo Correa 2, Mónica A. Lugo 3,4 and Neimar F. Duarte 5
Round 1
Reviewer 1 Report
It is usual to use mycorrhizae as the plural for mycorrhiza, not mycorrhizas. Please change this throughout the manuscript.
L 34 - "Plantations affects the soil physical and chemical properties, modifying the number, diversity and activity of the soil microbiota, including both free and symbiotic fungal populations." - please add a reference.
L48-54 - Please add adequate references. Wherever you make a strong statement that should be supported by a reference.
Figure 1 - I can barely see the sampling sites in this figure. please consider enlarging the figure.
2.2. Root Staining - Does finding fungal mycelia in the root confirm its mycorrhizae? I don't think so. A plethora of fungi colonises the roots of a plant of which a part is symbiotic. Hence I believe this could have also impacted the calculation using the formula.
AMF spore isolation and identification - here you need to briefly describe the pipeline even if you give a reference.
Figure 3. Please use standard error instead of standard deviation.
The result section needs to have all the sub-headings listed in the M&M, in the same order. In M& M, you did staining, isolation, etc. Where is the data for them? All the details should be included. Plus, I was expecting to see some nice microscopic images, but there is just one very hazy picture of a root section in figure 4. Moreover, how do you know the species of these Glomeromycota? You have never used DNA-based identification. So how are you so sure of the IDs? I am expecting a lot more pictures and details in the section.
Figure 5. Ordination diagram - need to include a high-resolution image.
When reading your discussion I was lost because I kept wondering how to find all this information. After all, your result section was thin.
The manuscript is full of grammatical errors please correct them.
Author Response
REFEREE 1
We thank the Reviewer for all his/her contributions made to improve the manuscript, his/her detailed correction of the entire original version of this work, and his/her suggestions. Below, we respond to each of his/her queries, suggestions and complaints.
It is usual to use mycorrhizae as the plural for mycorrhiza, not mycorrhizas. Please change this
Answer: Mycorrhizas was replaced by mycorrhizae along the text.
Suggestions for Authors throughout the manuscript.
L 34 - "Plantations affects the soil physical and chemical properties, modifying the number, diversity and activity of the soil microbiota, including both free and symbiotic fungal populations." - please add a reference.
Answer: We have added the reference for this statement (now it is the reference 2: Wang, Y.; Jiao, P.; Guo, W.; Du, D.; Hu, Y.; Tan, X.; Liu, X. Changes in bulk and rhizosphere soil microbial diversity and composition along an age gradient of Chinese fir (Cunninghamia lanceolate) plantations in subtropical China. Front. Mi-crobiol. 2022, 12, 777862. doi: 10.3389/fmicb.2021.777862)
L48-54 - Please add adequate references. Wherever you make a strong statement that should be supported by a reference.
Answer: We have included three new references citations for support each statement included in the text. They are references 9 to 11:
- Solaiman, Z. M.; Mickan, B. Use of Mycorrhiza in Sustainable Agriculture and Land Restoration; Springer, 2014; pp. 1–15. doi:10.1007/978-3-662-45370-4_1
- Silva Matos, P.; Figueira da Silva, C.; Melo Damian, J.; Pellegrino Cerri, C.E.; Pereira, M.G.; Zonta, E. Beneficial services of Glomalin and Arbuscular Mycorrhizal fungi in degraded soils in Brazil. Sci. Agric. 2022, 79 (5), e20210064
- Wang, F. Arbuscular mycorrhizas and ecosystem restoration; Springer, 2017; pp. 245–292. doi:10.1007/978-981-10-4115-0_11
Figure 1 - I can barely see the sampling sites in this figure.
Answer: The Figure 1 was enlarged to better appreciate the location of the sampling sites.
Please consider enlarging the figure. 2.2.
Answer: It is done.
Root Staining - Does finding fungal mycelia in the root confirm its mycorrhizae? I don't think so. A plethora of fungi colonises the roots of a plant of which a part is symbiotic. Hence I believe this could have also impacted the calculation using the formula.
Answer: We agree with the referee that there is a great variety of fungi in addition to the AMF that inhabit the roots as a niche; these can also form very similar colonization such as AMF. However, the hyphal diameters, the size of the vesicles, etc. differ from those that characterize the AMF. In addition, we have taken into account to characterize the colonization not only the presence of hyphae generally without septation of the AMF and their vesicles, but also the presence of their typical arbuscules. We have also now added better illustrations of these structures in Figure 5.
AMF spore isolation and identification - here you need to briefly describe the pipeline even if you give a reference.
Answer: A brief description of the methodology to AMF spore isolation and identification was now included together with INVAM web link and the bibliographic reference (citation 28: Błaszkowski, J. Glomeromycota. W. Szafer Institute of Botany Polish Academy of Sciences, Kraków; 2012; pp. 304. ISBN: 978-83-89648-82-2) used for spore identification. Please see Lines 188 to 193.
Figure 3. Please use standard error instead of standard deviation.
Answer: In the Figure 3 standard deviation was replaced by standard error.
The result section needs to have all the sub-headings listed in the M&M, in the same order.
Answer: Now, the result section includes sub-headings like M&M, and that also follow the same order.
In M& M, you did staining, isolation, etc. Where is the data for them? All the details should be included.
Answer:
Rhizospheric soils were collected for analysis of AM spores. Spores were extracted from 100 g soil. AM spores were recovered from soil samples of each treatment in the field, separated by wet sieving, decanting and sucrose centrifugation (Pagano and Scotti 2009), and the analysed data were expressed as number of spores/1 g of dry soil. Only healthy spores were counted. Each spore type was mounted sequentially in PVLG (polyvinyl-lacto-glycerol) and Melzer’s reagent for identification. Identification was based on spore colour, size, surface ornamentation and wall structure, with reference to the descriptions provided by the International collection of vesicular and AMF (http://invam.caf.wvu.edu) and the original species descriptions. Numbers of species were counted and spore numbers were square rooted, transformed and statistically analyzed, and means were compared by the Tukey’s test (P < 0.05).
The roots of three or more plants randomly selected were excavated and only those attached to the plant were used; these were fixed in FAA solution (5 ml of formaldehyde, 5 ml of acetic acid, 90 ml of ethyl alcohol) until samples could be processed. Roots were stained and assessed for mycorrhizal infection as follows. Roots were taken from the FAA, washed several times in tap water and bleached in 10% (w/v) KOH overnight and then heated to approximately 90°C in a water bath for 1 h. The cooled root samples were washed and stained with 0.05% trypan blue (Pagano and Scotti 2009). Roots were cut into 1 cm segments
Plus, I was expecting to see some nice microscopic images, but there is just one very hazy picture of a root section in figure 4.
Answer: We agree with the Referee proposal and thus, we included more images of the isolated spores and improved also root colonization illustration. Furthermore, Figure 4 is now Figure 5, it is due to the Results item was reorganized according to the Referee suggestion.
Moreover, how do you know the species of these Glomeromycota? You have never used DNA-based identification. So how are you so sure of the IDs? I am expecting a lot more pictures and details in the section.
Answer: In previous researches in the studied sites our work group has identified the AMF by morphological approaches. These Glomeromycota taxa were also confirmed by means of DGGE method. Furthermore, we have included new pictures of the spores as it has been suggested by the Reviewer.
Figure 5. Ordination diagram - need to include a high-resolution image.
Answer: We implemented the resolution of this figure according to the following suggestions of the Reviewer.
When reading your discussion I was lost because I kept wondering how to find all this information. After all, your result section was thin. The manuscript is full of grammatical errors please correct them.
Answer: According to the Referee suggestions, we clarified the discussion redaction to a more fluid reading also following the same order than in Mat & Met and Results sections. Furthermore, grammatical errors were corrected.
Reviewer 2 Report
Dear Authors
i would like to take your attention toward removing the hogh lighted areas in the Ms and please in the section of materials and methods and reference add the identification keys used in identification of recorded taxa.
if you can add more photos related to identified taxa and wall layers it will be great.
Author Response
REFEREE 2
Suggestions for Authors
I would like to take your attention toward removing the highlighted areas in the Ms and please in the section of materials and methods and reference add the identification keys used in identification of recorded taxa. If you can add more photos related to identified taxa and wall layers it will be great.
Answer: We are grateful for the kind return of the Reviewer's criticisms, suggestions and corrections. In accordance with the correction of the Reviewer we have eliminated the highlights in yellow that by mistake were in the text.
Furthermore, the references of taxonomical keys to determine the species of Glomeromycota and web site (http://www.amf-phylogeny.com/, http://fungi.invam.wvu.edu/the-fungi/ spe-cies-descriptions.html) used were included in the text of Mat & Met also the Reference section (citation 28: Błaszkowski, J. Glomeromycota. W. Szafer Institute of Botany Polish Academy of Sciences, Kraków; 2012; pp. 304. ISBN: 978-83-89648-82-2). Please see also Lines 188 to 193.
Also more photos were added to illustrate the arbuscular mycorrhizal fungi found in this study, now in the new Figure 5.
Reviewer 3 Report
Overview and general recommendation
The manuscript authored by Pagano et al. evaluated the benefits of inoculation, the root colonization of inoculated seedlings, the soil aggregation, and arbuscular mycorrhizal fungi (AMF) diversity according to two field treatments (Fertilized with organic matter-OM/Fertilized with natural rock phosphate-P). Although the results showed that AMF are important components in riparian areas and bring information on inoculant production for restoration, the data is not enough to support the conclusion, and the experimental design lacks rigor (e.g., climate change and soil conditions may affect the results). Therefore, I suggest this manuscript can not be published as it does not meet the high standard required.
Comments:
L202-203: “In general, OM and P addition did not affect root colonisation by AMF.” Could you provide some discussion on why the results were different from previous studies? A lot of researchers confirm that P addition or high P concentration can decrease AMF colonization in the roots of plants.
Fig. 4 (a) is not clear, could you please put a clearer figure with AMF structure included?
Comments for author File: 
Comments.pdf
Author Response
REFEREE 2
Suggestions for Authors
I would like to take your attention toward removing the highlighted areas in the Ms and please in the section of materials and methods and reference add the identification keys used in identification of recorded taxa. If you can add more photos related to identified taxa and wall layers it will be great.
Answer: We are grateful for the kind return of the Reviewer's criticisms, suggestions and corrections. In accordance with the correction of the Reviewer we have eliminated the highlights in yellow that by mistake were in the text.
Furthermore, the references of taxonomical keys to determine the species of Glomeromycota and web site (http://www.amf-phylogeny.com/, http://fungi.invam.wvu.edu/the-fungi/ spe-cies-descriptions.html) used were included in the text of Mat & Met also the Reference section (citation 28: Błaszkowski, J. Glomeromycota. W. Szafer Institute of Botany Polish Academy of Sciences, Kraków; 2012; pp. 304. ISBN: 978-83-89648-82-2). Please see also Lines 188 to 193.
Also more photos were added to illustrate the arbuscular mycorrhizal fungi found in this study, now in the new Figure 5.
Round 2
Reviewer 1 Report
Dear Authors,
Thank you for considering my suggestions for the previous version of the manuscript. I'm satisfied with the improvements. I have no additional suggestions.
Best wishes
Reviewer 3 Report
L153: "coole0d" should be "cooled".
