Boron and Zinc Diminish Grey Necrosis Incidence by the Promotion of Desirable Microorganisms on Hazelnut Orchards
, Patricio Javier Barra 1,2, MarÃa de la Luz Mora 1, Adriano Nunes-Nesi 3 and Cristian Merino-Gergichevich 1,4,*Round 1
Reviewer 1 Report
The manuscript titled "Boron and Zinc diminish grey necrosis incidence by the promotion of desirable microorganisms on hazelnut orchards." is focused on the relationships between grey necrosis, pathogens, bacteria, and nutrients that can diminish the symptoms. This study showed that B and Zn can affect the bacteria and improve microbial environments surrounding the hazelnut clearly. However I found 3 serious weaknesses that authors should address.
- DGGE is one of the method which can show the structure of the microbial communities. Authors also used this method, but the detail of the DGGE condition was not provided on the method section. Authors should show the equipment used in this study, the condition of electrophoresis, and its citations.
- In figure 3, OTUs identified as the same genus was not placed in the same clades. Did authors truly realize the phylogenetic analysis? What did the figure mean?
- In figure 4A, authors noted "Dendrogram of Diaporthe and Neofusicoccum( not Neofusiccoum) associated to similar strains of pure fungal in vitro." But it was not the dendrogram of Diaporthe and Neofusicoccum. Authors should explain the detail of the fig 4A.
I am looking forward to seeing the improved manuscript.
Author Response
We thanks to Revisor 1 for the time involving in the improvement of the present manuscript (please found in red the author responses).
- DGGE is one of the method which can show the structure of the microbial communities. Authors also used this method, but the detail of the DGGE condition was not provided on the method section. Authors should show the equipment used in this study, the condition of electrophoresis, and its citations
Author response: The information was added in lines 180-184: DGGE analysis was performed using a DCode system (Bio-Rad Laboratories, Inc.). Twenty-five microliters of PCR product was loaded onto a 6% (w/v) polyacrylamide gel with a 40–70% gradient (urea and formamide). The electrophoresis was run for 16 h at 75 V. The gel was then stained with SYBR Gold (Molecular Probes, Invitrogen Co.) for 30 min and photographed on a UV transilluminator
2. In figure 3, OTUs identified as the same genus was not placed in the same clades. Did authors truly realize the phylogenetic analysis? What did the figure mean?
Author response: We decide remove figure 3 in order to avoid confusion to readers. This is because the figure showed the same information of table 5, where we highlighted that all strains isolated from BCNf have been linked with biocontrol activity.
3. In figure 4A, authors noted "Dendrogram of Diaporthe and Neofusicoccum( not Neofusiccoum) associated to similar strains of pure fungal in vitro." But it was not the dendrogram of Diaporthe and Neofusicoccum. Authors should explain the detail of the fig 4A.
Author response: Figure Legend was amended, lines 623-627 (Hazelnut plants cv Barcelona analyzed by MALDI-TOF/MS. (A) Dendrogram of microbial involve on grey necro-sis associated to similar strains of pure fungal in vitro. (B) Protein profiles of leaves protein spectra associated to pathogens, plant, plant + pathogens, plant+pathogens and plant+pathogens+bacterial consortium, n=3. (C) Symp-toms of dieback in hazelnut plants (plant+fungal) and effect of inoculated bacteria (plant+fungi+bacteria consortia) and control healthy plant (without inoculation) n=3)
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper evaluates the effect of boron and zinc fertilization on beneficial microorganisms to diminish the incidence of grey necrosis on hazelnut cultivar Barcelona in Chile. It is needed contribution to the field because of the high losses in some of the major hazelnut growing areas in the world and complex and uncertain controlling options available.
The same team reported earlier the first report of grey necrosis, so this research is logical extension of their work - here they evaluate the effect of B and Zn fertilization on beneficial microorganisms to diminish the incidence of the disease. The study reveals influence and possible applicability of microbial diversity changes on treated hazelnut plants in combination with micronutrients addition.
I found no important weaknesses, nor methodological inaccuracies. Major controls were included, and experimental design was appropriate. Details on procedures are given in the methods section (as well as statistical methods and databases used) and data presentation is appropriate and consistent through the results and discussion sections. The manuscript is clear and well-structured.
Specific comments
- Line 81: the space between words B- and tolerant should be omitted.
Line 23: consider to use method name MALDI-TOF/TOF instead of MALDITOF-TOF; the same is applicable throughout the manuscript and figure's descriptions
Line 121: "different cardinal points of the plants" should be described in more details
Line 147: more data on the manufacturer of the thermal cycler should be added
Line 150: symbol ° for Celsius degree should be used (not e.g. 93â—¦C); moreover, one space after the number of Celsius degrees should be input; the same should be checked in all parts of the manuscript (e. g. correct will be 93 °C)
Lines 196-209: the font in this lines is not uniform
Line 207: greenhouse conditions should be specified
Lines 212-213: margins should be adjusted
Line 250: the abbreviation "Al sat" was not introduced (the same appears in Table 2.)
Line 267: it is not clear "50% of thirty nuts (51 nuts)"; please reconsider
Line 442: please, reconsider if it is Neofussicoccum sp. or spp.
Line 484: . after sp (twice); to be checked in whole MS
Line 508-509: to reconsider linguistically
Line 538: try to format species names (in Latin) in literature list and elsewhere in text into italic (e.g. Corylus avellana - Corylus avellana)
Line 544: page numbers are missing - try to check all references in literature list
Line 560: the name of the last author in your publication is missing
Author Response
We thanks to revisor 2 for the commentaries and suggestion in order to improve the present manuscript. All suggestions were amended and clarified in red
Line 81: the space between words B- and tolerant should be omitted.
Author response: change was made (line 92)
Line 23: consider to use method name MALDI-TOF/TOF instead of MALDITOF-TOF; the same is applicable throughout the manuscript and figure's descriptions
Author response: changes were made in all document
Line 121: "different cardinal points of the plants" should be described in more details
Author response: information and reference were added (line 131)
Line 147: more data on the manufacturer of the thermal cycler should be added
Author response: information was added (line 162-163), using Swift Maxi PCR thermal cycler (ESCO Technologies Inc., USA)
Line 150: symbol ° for Celsius degree should be used (not e.g. 93â—¦C); moreover, one space after the number of Celsius degrees should be input; the same should be checked in all parts of the manuscript (e. g. correct will be 93 °C)
Author response: changes were made in all document
Lines 196-209: the font in this lines is not uniform
Author response: done, lines 237-254
Line 207: greenhouse conditions should be specified
Author response: information was added (line 249-250), under greenhouse conditions (16/8 h photoperiod at 25/16 °C (day/night) and relative humidity range of 60-70% )
Lines 212-213: margins should be adjusted
Author response: done lines 256-276
Line 250: the abbreviation "Al sat" was not introduced (the same appears in Table 2.)
Author response: information was added, line 297
Line 267: it is not clear "50% of thirty nuts (51 nuts)"; please reconsider
Author response: 51 nut was removed to avoid confusion to readers
Line 442: please, reconsider if it is Neofussicoccum sp. or spp.
Author response: we changed to spp in order to continue with the same nomenclature of strains
Line 484: . after sp (twice); to be checked in whole MS
Author response: change was done in all document
Line 508-509: to reconsider linguistically
Author response: Paragraph was amended (lines 704-706)
Line 538: try to format species names (in Latin) in literature list and elsewhere in text into italic (e.g. Corylus avellana - Corylus avellana)
Author response: changes were done in all document
Line 544: page numbers are missing - try to check all references in literature list
Author response: the template not include page numbers, all references were checked
Line 560: the name of the last author in your publication is missing
Author response: the name was added, line 762
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The manuscript still need some improvement. In DEEG method, authors should add the process of analysis on the result of DEEG. How authors obtain the data from the result of DEEG? In addition, authors should improve the explanation of Fig. 4 (A). This is not dendrogram of microbial involve on grey necrosis. I think it was produced on the basis of the result of Protein profile. Does authors truly realize the each analysis?
Author Response
We thanks to Revisor for the time involving in the improvement of the present manuscript (please found in red the author responses).
In DEEG method, authors should add the process of analysis on the result of DEEG. How authors obtain the data from the result of DEEG?
Author response
Please see lines 175-179 and 261-269:
DGGE analysis was performed using a DCode system (Bio-Rad Laboratories, Inc.). Twenty-five microliters of PCR product was loaded onto a 6% (w/v) polyacrylamide gel with a 40–70% gradient (urea and formamide). The electrophoresis was run for 16 h at 75 V. The gel was then stained with SYBR Gold (Molecular Probes, Invitrogen Co.) for 30 min and photographed on a UV transilluminator [33,34]
The similarities between fungal and bacterial communities between BCNf and BCNc obtained from DGGE analyses were visualized by non-metric multidimensional scaling analysis (MDS), using Primer 7 software (Primer-E Ltd., Ivybridge, UK), which showed a Bray-Curtis similarity index greater than 80% and 0.14 stress values (Clarke 1993). Values were given as mean ± standard error. We considered differences significant when the P value was lower than or equal to 0.01. The in silico analysis was also used to estimate the microbial diversity by richness (S) and the Shannon-Wiener index and dominance by the Simpson Index (D) represented by 1- D or 1- λ [40]. The neighbour-joining trees (Phylogenetic trees) were constructed with Molecular Evolutionary Genetic Analysis (Mega 7).
In addition, authors should improve the explanation of Fig. 4 (A). This is not dendrogram of microbial involve on grey necrosis. I think it was produced on the basis of the result of Protein profile. Does authors truly realize the each analysis?
Author response please see lines 484-487
Hazelnut plants cv Barcelona analyzed by MALDI-TOF/MS. (A) Dendogram and (B) Spectra of protein profiles associated to fungal, plant, plant + fungal, and plant+fungal+bacterial consortium, n=3. (C) Symptoms of dieback in hazelnut plants (plant+fungal) and effect of inoculated bacteria (plant+fungi+bacteria consortia) and control healthy plant (without inoculation) n=3
Author Response File: Author Response.pdf
