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Olea europaea L. Root Endophyte Bacillus velezensis OEE1 Counteracts Oomycete and Fungal Harmful Pathogens and Harbours a Large Repertoire of Secreted and Volatile Metabolites and Beneficial Functional Genes

Microorganisms 2019, 7(9), 314; https://doi.org/10.3390/microorganisms7090314

by Manel Cheffi¹

, Ali Chenari Bouket²

, Faizah N. Alenezi^3,†, Lenka Luptakova^3,4

, Marta Belka^5,6

, Armelle Vallat⁷, Mostafa E. Rateb⁸

, Slim Tounsi⁹, Mohamed Ali Triki¹

and Lassaad Belbahri^3,10,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Microorganisms 2019, 7(9), 314; https://doi.org/10.3390/microorganisms7090314

Submission received: 5 July 2019 / Revised: 30 August 2019 / Accepted: 1 September 2019 / Published: 3 September 2019

(This article belongs to the Special Issue Plant Control of Symbiotic Microbe Behavior and Reproduction)

Round 1

Reviewer 1 Report

Although the data reported in the article are potentially interesting and a great deal of work has been done, in my opinion the article cannot be published in the present form.

Several revisions are needed. The introduction is not clear in some points and throughout the article there are some formatting errors (for example the names of the strains in many cases are not in italic).

According to the introduction the “microbiome of olive trees under Phytophthora and fungal infection threat was use as a starting point for wide screening of root endophytes against large collection of Phytophthora and fungal pathogens”. But the procedure by which these endophytes were isolated and how and why the Bacillus velezensis strain was chosen among the others is not reported clearly.

The phenotypic data that should be reported in table 2 are missing (see my specific comments) and in general the order in which the results are reported is confusing. The quality of the figures is low, in many cases nothing can be read. A paragraph that should be in the materials and methods is found in the results.

My suggestion is to review the article, in particular the results, reviewing the order in which they are reported, summarizing them in a clearer way and defining better the purpose of each analysis.

Table 1 and many of the figures could be moved to additional materials, leaving only a few figures in the text that are fundamental to understand the results. Materials and methods can also be summarized. Many of the methods used has been already published and could be reported only as additional files to shorten the manuscript and facilitate the reading.

Specific comments

Introduction:

Line 65-68: this sentence is not very clear

Line 74-76: not clear

Line 84: strain among species… what it means?

Materials and methods

Line 100: why use 37°C for environmental strains?

Line 112: Check the names of microorganisms in many cases they are not in italic

Line 120: The number of cells used has been verified in some way?

Line 172: HCN define the abbreviation

Line 232: remove the point, remove through filter.

The materials and methods for the core genome analysis are missing

Results:

Table 1: move to supplementary materials

Line 371-375: these results are not reported in table 2 as indicate by the text and I could not find these results anywhere in the manuscript

Line 377-379: same thing the results are missing

Figure 3B: the figure is absolutely incomprehensible, improve also the quality of figure 3C

The meaning of the two heatmaps is not clear. The numbers are the copies of genes in the genome for each category? Add a legend

In general, the exposure of the results is confusing. First, the authors report the phenotypic characterization of the strain, then they move to the genomic analysis, then they return to the characterization of the molecules produced, then back to the genomes. The description of the pangenoma is also confusing ... it is better to first describe the pangenoma in general and then go into details (Paragraph 3.12 should be placed before paragraph 3.11)

Figure 6: move to supplementary files

Line 454-459 : materials and methods

Figure 7B: move to supplementary files

Author Response

Dear Editor in Chief of Microorganisms,

We are grateful to the anonymous reviewers; their comments were highly valuable for providing the best relevant data to the journal readers. We believe this highly improved the quality of the manuscript. Therefore, we followed the vast majority of their suggestions and made the suggested changes in the manuscript. Below we provide a list of reviewers’ recommendations and the changes operated in the revision.

Reviewers' comments:

Academic Editor Notes

If the reviewers have suggested that your manuscript should undergo extensive English editing, please address this during revision. We suggest that you have your manuscript checked by a native English-speaking colleague or use a professional English editing service. Alternatively, MDPI provides an English editing service checking grammar, spelling, punctuation and some improvement of style where necessary for an additional charge (extensive re-writing is not included), see details at https://www.mdpi.com/authors/english.

Reviewer #1:

Comments and Suggestions for Authors

In this article the authors reported an in-depth characterization of a Bacillus velezensis strain. A phenotypic and genomic characterization of this strain has been carried out, highlighting the ability to both inhibit several plant pathogens and promote plant growth. Although the data reported in the article are potentially interesting and a great deal of work has been done, in my opinion the article cannot be published in the present form. Several revisions are needed.

Corresponding Author: According to reviewer recommendation the paper have been improved.

The introduction is not clear in some points and throughout the article there are some formatting errors (for example the names of the strains in many cases are not in italic).

Corresponding Author: Scientific names of the strain are in italic now. The introduction has also been clarified according to reviewer recommendation.

Corresponding Author: The issue was resolved and the following text was inserted in the manuscript:

Isolation of endophytic bacteria and screening for antifungal activity

Isolation of bacterial endophytes was performed from healthy olive trees belong to the cultivar Chemlali located in Sfax (South Tunisia 34°43'50.7"N 10°44'08.6"E). The entire plant was washed under running water to remove soil and dust and endophytic bacteria were isolated from each plant part as described by [73]. To ensure the surface sterilization efficacy, 100 µL of rinsing water from the last washing was plated on TSA medium and incubated at 30 °C [74].

Antifungal activity of the isolated bacteria was evaluated using the dual culture test [17] against Rhizoctonia bataticola HQ392809.1, R. solani KU863546, Neofusicoccum australe EU375516.1, Nigrospora sp. JN 207298.1, Botryospheria sp., F. solani FJ874633.1, Pu: Pythium ultimum, F. oxysporum JN400698.1 and Cylindrocarpon sp. The isolate showing an ability to inhibit the most of pathogens and a strong activity against F. solani was selected for the rest of this study.

Results

A total of 42 bacterial strains were isolated from olive trees cv. Chemlali, a widely cultivated variety in this area [75] and highly sensitive to the majority of olive tree diseases [76]. All these strains were screened for their antagonistic activity using the dual culture method and 45 % of them showed more than 60% mycelial growth inhibition (Table S3). Interestingly, the root-derived strain OEE1 was able to reduce F. solani growth to 82.42%. Thus, this strain was selected to investigate its antifungal potential against F. solani and its plant growth promoting ability.

Table S3. Antifungal activity of endophytic bacteria against the olive trees pathogens using the dual culture method

	Rb	Pu	Rs	Na	B	N	Fs	Fo	C
Control	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
10R3I	86,14 ± 0.72^b	86,39 ± 0.26^b	16,22 ± 1.70ⁿ	21.36 ± 0.02^mn	63.32 ± 0.39^f	25.89 ± 0.88^m	60.24 ± 0.59^c	54.27 ± 0.58^g	70.35 ± 0.69^c
11R3I	13,30 ± 2.25^kl	20,98 ± 0.11^l	20,89 ± 1.58^m	33.83 ± 1.04^k	0.00	0.00	29.46 ± 0.36^h	0.00	0.00
12R3I	63,13 ± 2.24^d	32,22 ± 0.45^k	28,37 ± 1.73^j	36.21 ± 0.95^j	44.10 ± 2.54^h	0.00	54.92 ± 0.41^d	13.58 ± 0.65^o	64.91 ± 0.32^e
13R3I	26,50 ± 0.2^h	89,03 ± 0.07^a	50,44 ± 0.58^h	74.03 ± 0.32^bc	31.96 ± 1.37ⁱ	23.14 ± 0.31^m	0.00	8.29 ± 1.28^q	69.75 ± 1.20^c
14R3I	0.00	20,98 ± 1.01^l	35,92 ± 1.70ⁱ	28.23 ± 0.45^l	0.00	0.00	12.57 ± 0.85^m	0.00	4.32 ± 0.03^r
15R3I	30,42 ± 0.91^g	51,83 ± 0.21^h	19,20 ± 1.02^m	19.74 ± 0.09ⁿ	28.45 ± 0.21ⁱ	11.5 ± 0.67^o	28.62 ± 1.22^h	60.38 ± 0.31^ef	0.00
17B1I	51,89 ± 2.88e	73,17 ± 0.39^de	25,49 ± 0.91^kl	32.51 ± 1.23^k	82.37 ± 0.69^bc	72.15 ± 1.33^c	64.91 ± 0.09^b	57.36 ± 0.97^f	76.22 ± 0.52^b
OEE1	61,09 ± 0.65^d	56,47 ± 3.55^fg	84,30 ± 1.15^a	85.78 ± 0.42^a	86.38 ± 0.54^a	82.54 ± 0.25^a	82.42 ± 0.15^b	42.86 ± 0.64ⁱ	81.47 ± 0.47^a
1B2I	0.00	45,10 ± 1.81ⁱ	0,00	0.88 ± 0.63^s	0.00	0.00	0.00	13.82 ± 0.09^o	4.61 ± 0.61^r
1C3I	56,72 ± 1.01^d	7,49 ± 0.23^no	7,34 ± 1.30^o	16.06 ± 0.33^o	31.91 ± 2.14ⁱ	0.00	26.48 ± 0.37ⁱ	37.60 ± 0.22^j	44.35 ± 0.32^j
1F1I	0,00	9,00 ± 0.01ⁿ	0,00	0.00	0.00	0.00	4.36 ± 0.94^o	10.21 ± 0.03^p	0.00
1F2I	35,52 ± 0.44^fg	39,41 ± 3.01^j	45,17 ± 0.03^hi	58.57 ± 0.41^f	0.00	62.33 ± 0.89^fg	2.58 ± 0.60^p	46.24 ± 0.72^h	61.75 ± 0.08^fg
1R3I	79,78 ± 1^c	26,43 ± 0.11^k	68,93 ± 0.76^e	69.11 ± 1.12^c	83.20 ± 0.085^b	64.38 ± 0.37^e	46.21 ± 0.03^f	70.59 ± 0.83^c	47.21 ± 0.64^hi
1T1I	20,81 ± 0.04^j	61,78 ± 0.49^f	69,81 ± 0.24^d	72.65 ± 0.37^c	8.51 ± 0.61^m	35.26 ± 0.14^jk	28.24 ± 0.77^hi	66.35 ± 0.23^d	48.91 ± 0.01^h
20R3I	1,00 ± 1.73^m	11,15 ± 0.34ⁿ	0,00	0.00	43.71 ± 0.12^h	0.00	0.00	21.34 ± 0.94^m	0.00
20R3I'	0,00	19,49 ± 1.43^l	0,00	0.00	0.00	29.20 ± 2.97^l	0.00	0.00	5.36 ± 1.24^r
2B1F	23,10 ± 0.33ⁱ	15,55 ± 0.84^m	2,52 ± 1.03^p	6.46 ± 0.52^q	32.18 ± 0.66ⁱ	2.31 ± 2.32^q	73.26 ± 0.26^a	0.00	67.92 ± 0.68^d
2B1I	74,03 ± 0.57^cd	27,74 ± 0.64^k	66,78 ± 1.76^f	69.91 ± 0.69^c	68.92 ± 0.73^e	78.31 ± 0.90^ab	59.28 ± 0.42^c	66.84 ± 0.56^d	46.80 ± 0.07ⁱ
2B2I	0,00	3,00 ± 0.1^o	0,00	0.00	0.00	24.59 ± 0.38^m	0.00	0.00	0.00
2C3I	51,75 ± 2.31^e	7,52 ± 0.15^no	0,00	2.20 ± 0.35^rs	54.91 ± 2.39^g	46.2 ± 0.66ⁱ	27.13 ± 0.83ⁱ	0.00	33.56 ± 0.28^l
2F1I	0,00	8,15 ± 0.56^no	25,31 ± 1.03^kl	24.11 ± 1.08^m	31.96 ± 0.48ⁱ	19.67 ± 0.07ⁿ	20.61 ± 0.68^k	0.00	10.82 ± 0.33^p
2R3I	20,02 ± 2.31^j	76,02 ± 0.06^d	49,20 ± 1.45^h	53.98 ± 0.94^g	16.20 ± 0.90^k	37.29 ± 0.58^j	24.66 ± 0.49^j	41.09 ± 0.38ⁱ	7.34 ± 0.19^q
2T1I	0,00	87,81 ± 0.68^a	24,28 ± 1.21^l	36.90 ± 0.67^j	12.50 ± 1.50^l	0.00	39.88 ± 1.08^g	60.75 ± 2.06^ef	0.00
3B1I	58,45 ± 0.9^d	14,25 ± 0.15^m	69,54 ± 0.21^de	69.66 ± 0.28^c	81.60 ± 0.11^c	76.14 ± 0.21^b	38.09 ± 0.16^g	57.92 ± 0.33^f	69.83 ± 0.21^c
3B2I	0,00	68,78 ± 1.74^e	70,54 ± 1.82^d	67.82 ± 0.84^d	0.00	0.00	51.40 ± 0.73^e	74.36 ± 0.49^b	9.29 ± 0.43^p
3R3I	5,46 ± 0.46^lm	52,67 ± 3.04^g	53,86 ± 0.85^g	58.21 ± 0.12^f	0.00	46.97 ± 0.96ⁱ	23.50 ± 0.89^j	0.00	69.84 ± 0.97^c
3T1I	11,27 ± 0.04^l	20,16 ± 0.66^l	17,49 ± 1.39ⁿ	38.99 ± 1.61ⁱ	0.00	18.97 ± 0.72ⁿ	24.58 ± 0.44^j	3.21 ± 0.81^r	0.00
4B1I	0,00	0,00	53,02 ± 0.61^g	0.00	25.92 ± 0.82^ij	0.00	0.00	44.67 ± 0.66^h	0.00
4B2I	8,77 ± 0.03^l	6,97 ± 1.15^no	65,14 ± 1.33^f	71.17 ± 0.24^c	0.00	62.74 ± 0.02^fg	0.00	18.63 ± 0.32^mn	0.00
4C3I	14,13 ± 2.47^k	61,14 ± 0.8^f	0,97 ± 0.52^pq	11.94 ± 0.61^pq	19.82 ± 1.86^j	0.00	0.00	31.57 ± 0.91^k	16.80 ± 0.38ⁿ
4R3I	0,00	3,82 ± 0.05^o	69,93 ± 0.48^d	70.02 ± 1.33^c	0.00	49.65 ± 0.34^h	8.03 ± 0.04ⁿ	0.00	58.28 ± 0.45^g
5B2I	0,00	3,91 ± 0.6^o	0,00	57.44 ± 0.92	21.69 ± 0.49^j	37.19 ± 1.69^j	0.00	0.00	22.64 ± 0.97^m
5R3I	42,88 ± 0.94^f	7,27 ± 0.81^no	2,30 ± 1.00^p	12.6 ± 0.09^p	0.00	0.00	9.38 ± 0.13ⁿ	59.01 ± 0.64^f	0.00
6B1I	0,00	31,56 ± 4.1^k	0,86 ± 1.49^pq	8.14 ± 0.25^q	22.73 ± 0.72^j	6.01 ± 0.72^p	0.94 ± 0.81^q	0.00	0.00
6B2I	20,88 ± 0.16^j	82,78 ± 0.83^c	41,74 ± 0.85^hi	45.58 ± 0.86^h	0.00	0.00	46.85 ± 0.60^f	17.91 ± 0.82ⁿ	22.19 ± 0.83^m
6R3I	42,05 ± 0.89^f	75,60 ± 0.04^d	73,39 ± 0.42^c	75.89 ± 2.01^bc	0.00	51.48 ± 0.11^h	16.08 ± 0.72^l	20.09 ± 0.74^m	0.00
7B1I	0,00	19,43 ± 1.01^l	67,84 ± 0.58^ef	62.63 ± 0.43^e	21.65 ± 0.25^j	67.33 ± 0.91^d	0.00	0.00	40.97 ± 0.09^k
7R3I	90,93 ± 1.02^a	21,67 ± 0.41^l	26,92 ± 1.73^k	36.55 ± 0.66^j	62.82 ± 0.83^f	58.21 ± 0.83^g	0.00	24.94 ± 0.12^l	13.50 ± 0.50^o
8B1I	41,70 ± 1.65^f	68,96 ± 0.43^e	0,73 ± 0.15^pq	4.63 ± 0.28^r	0.00	63.48 ± 0.41^e	40.09 ± 0.36^g	61.83 ± 1.36^e	7.21 ± 0.26^q
8R3I	0,00	13,52 ± 0.22ⁿ	0,99 ± 0.2^pq	7.41 ± 0.94^q	0.00	0.00	12.30 ± 2.09^m	0.00	4.07 ± 0.05^r
9R3I	29,15 ± 0.33^g	44,41 ± 1.66ⁱ	77,24 ± 0.03^b	79.38 ± 1.20^b	0.00	34.81 ± 0.09^k	0.00	78.31 ± 0.64^a	0.00
B2Fr	47,69 ± 0.3^ef	29,07 ± 0.55^k	66,80 ± 1.85^f	54.01 ± 0.03^g	75.07 ± 1.22^d	74.95 ± 0.65^bc	26.04 ± 0.57ⁱ	61.99 ± 0.91^e	43.70 ± 0.93^j

Rb: Rhizoctonia bataticola HQ392809.1, Rs: Rhizoctonia solani KU863546, Na: Neofusicoccum australe EU375516.1, N: Nigrospora sp. JN 207298.1, B: Botryospheria sp., Fs: Fusarium solani FJ874633.1, Pu: Pythium ultimum, Fo: Fusarium oxysporum JN400698.1, C: Cylindrocarpon sp.

Corresponding author: We tried to follow reviewer recommendation as close as possible. Figures have been improved and some of them have been transferred to supplementary data. If some changes are still required, we can address them.

My suggestion is to review the article, in particular the results, reviewing the order in which they are reported, summarizing them in a clearer way and defining better the purpose of each analysis.

Corresponding author: Results part have been re-arranged. If specific reviewer changes are still required, we can revisit them.

Corresponding author: Table 1 is transferred to supplementary material. Figures 3 B and C, Figure 6, Figure 7B have also been transferred to supplementary material.

Specific comments

Introduction:

Line 65-68: this sentence is not very clear

Corresponding author: The sentence has been rewritten.

Line 74-76: not clear

Corresponding author: Reviewer recommendation have been addressed.

Line 84: strain among species… what it means?

Corresponding author: Reviewer recommendation have been addressed.

Materials and methods

Line 100: why use 37°C for environmental strains?

Corresponding author: The temperature used in the study is the prevalent temperature in the field. Therefore, it has been selected.

Line 112: Check the names of microorganisms in many cases they are not in italic

Corresponding author: All of them are italic now.

Line 120: The number of cells used has been verified in some way?

Corresponding author: Yes, different cell numbers have been tested and the reported cell number gave the best result.

Line 172: HCN define the abbreviation

Corresponding author: It is addressed.

Line 232: remove the point, remove through filter.

Corresponding author: They are addressed.

The materials and methods for the core genome analysis are missing

Corresponding author: It is addressed.

Results:

Table 1: move to supplementary materials

Corresponding author: It is addressed.

Line 371-375: these results are not reported in table 2 as indicate by the text and I could not find these results anywhere in the manuscript

Corresponding author: Reviewer is absolutely right and data have been added to supplementary material and reported in the text.

Line 377-379: same thing the results are missing

Corresponding author: Reviewer is absolutely right and data have been added to supplementary material and reported in the text.

Figure 3B: the figure is absolutely incomprehensible, improve also the quality of figure 3C

Corresponding author: They are transferred to supplementary material.

The meaning of the two heatmaps is not clear. The numbers are the copies of genes in the genome for each category? Add a legend

Corresponding author: Reviewer is absolutely right. Therefore, we addressed the issue and added a legend.

Corresponding author: While we understand reviewer point of view, we think that keeping the data as it is presented is better for the reader because it follows the way how the work has been conducted.

Figure 6: move to supplementary files

Corresponding author: It is addressed.

Line 454-459: materials and methods

Corresponding author: The suggested part has been removed to material and methods according to reviewer recommendation.

Figure 7B: move to supplementary files

Corresponding author: It is addressed.

Reviewer 2 Report

Cheffi et al presented an extensive manuscript about the beneficial properties of a Bacillus strain. The authors showed that this strain is able to protect olive plants from fungal and oomycetal pathogens as well as is also able to promote the olive growth and development. The authors performed the analyses both in vitro, in planta and in silico, which constitutes a proper approach and provide useful information if someone wants to use this strain in the future. Interestingly, the authors provide phylogenomics analyses in which they showed the taxonomic affiliation of this strain and defined its position among other isolates with available genomes.

However, I do have some concerns about the manuscript:

- The origin of the strain. Please, add information about the origin of the strain, place of isolation, identification, characterization….

- Figures are not where they should be within the text. You can get lost reading the manuscript. Figure and table legends should be improved as well as the quality of some of them. For example, it is impossible to read what is in figure 3 b and c. Moreover, some tables (table 1) and I think maybe some figures must be placed as supplementary data. Species column of Table 1 can be removed, as there is always the same species. Also, a column stating if the genome is complete or draft would improve the table.

-The production of CWDE is by itself considered as a PGP mechanism. There is no need to separate as endophytic traits. Moreover, the claim that this strain is an endophyte is very weak. Did the authors check if there are other strains present inside the roots (this is possible as the plants were not axenic), maybe there are strains with a natural resistance to the selected antibiotics?? Have you performed disinfection controls (i.e. surface-disinfected root passed through LB plates to see if the method was effective)? I would recommend microscopy analyses.

-Figure 1 showed a result, not a methodology.

-I would appreciate some more discussion about the taxonomic affiliation of the 4 different species that the authors suggest. It is quite interesting, and some criticism at least is expected.

- How do you extract VOCs? It is not clear, and I think not written properly in the text and them, there is a supplementary figure showing effcts of VOCs on several pathogens…

- Introduction should be improved (it is kind of short) and the methods employed should be resumed or rearranged.

Other comments:

Lines 112-120. Names must be italicized. Please, check this through the ms and also, tables and figures, as there are some errors on taxa names.

Figure 10a legends must be checked as it is difficult to follow. Panel c showed OEE1 mutant strain, only this panel? What is presented in 1, 2, 3 and 4 subpanels?

Line 537. …Mutant strain was designated with the same name as wild type?

Line 602. …rich weapons??

Please, check the species name very carefully through the ms as well as in tables and figures. Full name Bacillus velezensis should be written as B. velezensis after the first time it is named. Please, check italics as well.

Author Response

Dear Editor in Chief of Microorganisms,

Reviewers' comments:

Academic Editor Notes

Reviewer #2:

Comments and Suggestions for Authors

However, I do have some concerns about the manuscript:

- The origin of the strain. Please, add information about the origin of the strain, place of isolation, identification, characterization….

Corresponding author: It is addressed.

Corresponding author: The comments are addressed.

Corresponding author: Reviewer is absolutely right. Disinfection controls have been conducted and gave negative results. Strains with double resistance as the strain selected in the study are highly unlikely.

-Figure 1 showed a result, not a methodology.

Corresponding author: The position of Figure 1 is changed now.

-I would appreciate some more discussion about the taxonomic affiliation of the 4 different species that the authors suggest. It is quite interesting, and some criticism at least is expected.

Corresponding author: It is addressed.

- How do you extract VOCs? It is not clear, and I think not written properly in the text and them, there is a supplementary figure showing effects of VOCs on several pathogens…

Corresponding author: Corrected and explained more appropriately.

- Introduction should be improved (it is kind of short) and the methods employed should be resumed or rearranged.

Corresponding author: It is addressed.

Other comments:

Lines 112-120. Names must be italicized. Please, check this through the ms and also, tables and figures, as there are some errors on taxa names.

Corresponding author: It is addressed.

Figure 10a legends must be checked as it is difficult to follow. Panel c showed OEE1 mutant strain, only this panel? What is presented in 1, 2, 3 and 4 subpanels?

Corresponding author: Yes, reviewer is right for panel C. It is only the mutant strain.

Line 537. …Mutant strain was designated with the same name as wild type?

Corresponding author: No, it is designed by the naming OEE1 double mutant strain. In 1, 2, 3 and 4 subpanels re-isolation of the mutant strain OEE1’ from A: leaves and B: stems after root inoculation with mutant B. velezensis OEE1.

Line 602. …rich weapons??

Corresponding author: It is addressed.

We would like to thank the reviewers for their valuable comments, the manuscript has now been improved. All recommendations of the reviewers have been addressed.

We would like to thank the editor for his time handing our manuscript and looking for his positive response.

Best regards

Dr. Lassaad Belbahri

Round 2

Reviewer 1 Report

The manuscript have been improved and the authors responded to all comments.

The quality of the figure have been improved. The only figure which is not yet clear is figure 4. The font of the name of the strains can not be increased but that of functions can be increased to facilitate reading

Author Response

Reviewer 1

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )

Comments and Suggestions for Authors

The manuscript have been improved and the authors responded to all comments.

The quality of the figure have been improved. The only figure which is not yet clear is figure 4. The font of the name of the strains cannot be increased but that of functions can be increased to facilitate reading.

Corresponding Author: Thanks for the reviewer 1. We improved the quality of Figure 4.

Reviewer 2 Report

The manuscript is quite improved. However, there are some minor concerns that must be addressed, mostly regarding to the figures.

Figures 1, 2, 3 and 4 are out of place. Also, other figures such as figure 9 are out of place. Please, make sure that this is fixed in the last version. Figure 6 caption seems incomplete. Please, improve the caption of figure 9 a). Are there 6 strains of Fso? Or is it just one? I would recommend introducing some signal of which ones are belonging to preventive, curative and so on. There is no mention of Figure 1 in the text, just in the methods and discussion section. Figure 1 should be mentioned in section 3.1 or 3.2. Every figure must be placed after mentioned on text, in the results section, as all the figures described results and not methods. Please, fix it. Please, identify type strain(s) of B. velezensis in Figure 3, TableS2 and along the entire manuscript, when necessary. Please, clarify how the phylogenetic tree was obtained; did the authors use the core genome? The authors should indicate how many genes of the genome were considered for the tree. Why the authors did not consider all the genomes available for B. velezensis? What do the colours mean in figure 3? I suppose that every colour corresponds to a “sister clade” that represent a putative new species, but it is not written anywhere. Strain CBMB205 has two genome assemblies…is it necessary to include both in the analyses? In order to improve the interpretation and the discussion regarding to B. velezensis phylogenomic analyses, I strongly recommend the authors to check Dunlap et al 2016 (IJSEM doi: 10.1099/ijsem.0.000858).

Other comments:

There is no need of repeating the genus name “Bacillus” in the tree and in the rest of figures. Abbreviation as “B.” is best. Also, the word “strain” is not necessary to be repeated. In table 1, Toxipedia and Wikipedia references were given by the authors. I am sorry but I think this kind of references are not acceptable. Names on figure 4 are not easily readable. Could you increase letter size a bit? Line 64: “…for mean annual losses”. Do you mean “average annual losses”? Line 545: strain

Author Response

Reviewer 2

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
( ) English language and style are fine/minor spell check required
(x) I don't feel qualified to judge about the English language and style

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	( )	(x)	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )

Comments and Suggestions for Authors

The manuscript is quite improved. However, there are some minor concerns that must be addressed, mostly regarding to the figures.

Figures 1, 2, 3 and 4 are out of place. Also, other figures such as figure 9 are out of place. Please, make sure that this is fixed in the last version.

Corresponding Author: Yes Figures 1, 2, 3, 4 and 9 have been fixed according to reviewer recommendations.

Figure 6 caption seems incomplete.

Corresponding Author: Figure 6 caption is now complete following reviewer recommendation.

Please, improve the caption of figure 9 a).

Corresponding Author: Figure 9a caption is now complete following reviewer recommendation.

Are there 6 strains of Fso? Or is it just one?

Corresponding Author: There is just one (Fso1). It is addressed based on reviewer recommendation.

I would recommend introducing some signal of which ones are belonging to preventive, curative and so on.

Corresponding Author: It is improved in Figure 9a caption.

There is no mention of Figure 1 in the text, just in the methods and discussion section.

Corresponding Author: Figure 1 is now cited in the text (result section) following reviewer recommendation.

Figure 1 should be mentioned in section 3.1 or 3.2.

Corresponding Author: Figure 1 is now cited in section 3.1 and 3.2.

Every figure must be placed after mentioned on text, in the results section, as all the figures described results and not methods. Please, fix it.

Corresponding Author: Reviewer recommendation have been fulfilled. Each figure is now placed in the results section after mentioned.

Please, identify type strain(s) of B. velezensis in Figure 3, TableS2 and along the entire manuscript, when necessary.

Corresponding Author: Type strain have been identified now in Figure 3, Table S2 and along the entire manuscript following reviewer recommendation.

Please, clarify how the phylogenetic tree was obtained; did the authors use the core genome? The authors should indicate how many genes of the genome were considered for the tree.

Corresponding Author: The entire genomes parts that can align have been used for phylogenomic tree as described in the algorithm of realphy (Bertels, F.; Silander, O.K.; Pachkov, M.; Rainey, P.B.; van Nimwegen, E. Automated reconstruction of whole-genome phylogenies from short-sequence reads. Mol Biol Evol 2014, 31, 1077-1088).

Why the authors did not consider all the genomes available for B. velezensis?

Corresponding Author: At the time that the analysis has been conducted we considered all the available B. velezensis genomes. However, given the speed by which the genomes are released in the international databases, our analysis become partial soon after completion.

What do the colours mean in figure 3? I suppose that every colour corresponds to a “sister clade” that represent a putative new species, but it is not written anywhere.

Corresponding Author: Yes, reviewer is right. We indicated that in the text.

Strain CBMB205 has two genome assemblies…is it necessary to include both in the analyses?

Corresponding Author: Yes this was to check the veracity of the two assemblies and therefore validate the genome of the species.

In order to improve the interpretation and the discussion regarding to B. velezensis phylogenomic analyses, I strongly recommend the authors to check Dunlap et al 2016 (IJSEM doi: 10.1099/ijsem.0.000858).

Corresponding Author: We found this suggestion very constructive and cited this reference in the literature to meet reviewer recommendation.

Other comments:

There is no need of repeating the genus name “Bacillus” in the tree and in the rest of figures. Abbreviation as “B.” is best. Also, the word “strain” is not necessary to be repeated.

Corresponding Author: Reviewer recommendation have been fulfilled.

In table 1, Toxipedia and Wikipedia references were given by the authors. I am sorry but I think this kind of references are not acceptable.

Corresponding Author: Reviewer recommendation have been fulfilled.

Names on figure 4 are not easily readable. Could you increase letter size a bit?

Corresponding Author: Reviewer recommendation have been fulfilled.

Line 64: “…for mean annual losses”. Do you mean “average annual losses”?

Corresponding Author: Yes, reviewer is right. We replaced mean by average in the text to meet reviewer suggestion.

Line 545: strain

Corresponding Author: Reviewer recommendation have been fulfilled.

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Olea europaea L. Root Endophyte Bacillus velezensis OEE1 Counteracts Oomycete and Fungal Harmful Pathogens and Harbours a Large Repertoire of Secreted and Volatile Metabolites and Beneficial Functional Genes

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