Multiple Potential Plant Growth Promotion Activities of Endemic Streptomyces spp. from Moroccan Sugar Beet Fields with Their Inhibitory Activities against Fusarium spp.

Round 1

Reviewer 1 Report

The manuscript is dedicated to the investigation of actinomycetes of genus Streptomyces as promising Plant Growth-Promoting Rhizobacteria (PGPR) for agricultural applications. The article presents the results of a comprehensive study of the activity of Streptomyces strains according to various parameters: solubilization of potassium for better uptake by the plant; secretion of indoleacetic acid (IAA), the most important phytohormone for plant growth and development; and antimicrobial activity of actinomycetes – for suppression of development of pathogenic microogranisms that reduce the quality of agricultural products. The manuscript contains interesting data, however, the level of scientific presentation is insufficient. It requires substantial revision to be accepted for publication.

The remarks are as follows:

1. The combined presentation of both biomass accumulation and pH changes on Fig. 2 is irrelevant. Either the discussion related to pH needs to be moved to section 3.1, where this diagram is located, or the biomass accumulation and pH change data need to be split into separate figures. In addition, it may be worth developing a less cumbersome way to illustrate the data obtained. Biomass accumulation plots for all strains should be combined into a single plot, and so should pH change plots, with the plots moved to the appropriate sections of Discussion.
2. In caption to Fig. 3, there is no description of symbols A-E. Accumulation of biomass would be more clearly shown using a second column on the diagram.
3. 4. Indole Acetic Acid (IAA) production. – remove the dot after the title
4. Captions with names of the test strains should be added to Fig. 6, and it should be moved to Supporting Information, because it duplicates the data in Table 1.
5. Table 2. The scoring for the studied strains are very arbitrary. Every strain is rated on a scale of 1 to 10 on completely different parameters, calculation of the “final score” does not apply any weights to various parameters. For example, assigning equal importance to antimicrobial activity and the ability to produce siderophores is very questionable in the context of biocontrol applications, as is equal importance of other mentioned parameters. A serious justification of the scoring approach with references to the methodology being used needs to be added, or this section needs to be reworked.
6. The list of references is sloppy and needs to be thoroughly rechecked.

Author Response

Point 1 : The combined presentation of both biomass accumulation and pH changes on Fig. 2 is irrelevant. Either the discussion related to pH needs to be moved to section 3.1, where this diagram is located, or the biomass accumulation and pH change data need to be split into separate figures. In addition, it may be worth developing a less cumbersome way to illustrate the data obtained. Biomass accumulation plots for all strains should be combined into a single plot, and so should pH change plots, with the plots moved to the appropriate sections of Discussion.

 

Response 1 : according to the reviewer’s suggestion the biomass accumulation and pH change data were separated into two figures (Fig. 2 for Biomass and Fig. 4 for pH evolution).

 

Point 2 : In caption to Fig. 3, there is no description of symbols A-E. Accumulation of biomass would be more clearly shown using a second column on the diagram.

 

Response 2 : Corrected

 

Point 3 : 4. Indole Acetic Acid (IAA) production. – remove the dot after the title

Response 3 : Done

 

Point 4 : Captions with names of the test strains should be added to Fig. 6, and it should be moved to Supporting Information, because it duplicates the data in Table 1.

Response 4 : Corrected

 

Point 5 : Table 2. The scoring for the studied strains are very arbitrary. Every strain is rated on a scale of 1 to 10 on completely different parameters, calculation of the “final score” does not apply any weights to various parameters. For example, assigning equal importance to antimicrobial activity and the ability to produce siderophores is very questionable in the context of biocontrol applications, as is equal importance of other mentioned parameters. A serious justification of the scoring approach with references to the methodology being used needs to be added, or this section needs to be reworked.

 

Response 5 :

We agree that the non-weighted scoring approach was poorly justified in light of the central aim of the project: to identify the most promising strains for combined biofertilizer and biocontrol applications. To approach this aim, the rating of phosphate solubilisation, potassium solubilisation, indole acetic acid production and suppression of indigenous Fusarium strains were doubled (highest activity strain that was rated by 10 => value 20). Table 2 was revised and the weighted scoring was also described in the Materials and methods section.

 

Materials and methods, line 231

 

To select the most promising streptomycete isolates for combined biofertilizer and biocontrol applications, their relative activities in rock phosphate, tricalcium phosphate and potassium solubilization, antifungal activity against Fusarium, antibacterial activity, production of IAA and siderophores were cross-compared between the strains and used to grade them from 1 (lowest) to 10 (highest activity). In order to follow the aim of selecting for combined biofertilizer and biocontrol agents, the rating values for rock phosphate and potassium solubilisation, IAA production and Fusarium inhibition, were weighted by 2 (doubled).

 

Point 6 : The list of references is sloppy and needs to be thoroughly rechecked.

 

Response 6 : We regret the multiple mistakes that occurred in the References section. The section has been carefully edited.

 

Author Response File: Author Response.docx

Reviewer 2 Report

The work is devoted to the development of methods for sustainable and environmentally friendly agriculture. The objects of the study were 10 strains of actinomycetes isolated from the rhizosphere of sugar beet. The specific task of the authors was to analyze the ability of these actinomycetes to dissolve the potassium-containing mineral orthoclase, which was the only source of potassium in the nutrient medium. The ability of actinomycetes to synthesize Indole Acetic Acid and antimicrobial substances was also analyzed. As a result, the most promising strains for field trials were selected.

The disadvantages of this work are shown in the table below. They can be overcome by the authors as a result of careful reading and editing of the text.

I would like to note that when re-mentioning species names (binary nomenclature), the genus designation should be abbreviated. This rule is violated throughout the text.

Special attention is drawn to the discrepancy between the data of figure 3 and the text. This needs to be corrected, after which the work can be published.

 

Line	Original text	Change please on
2	PGP	Plant growth-promotion? Give the full name, there is no decryption anywhere in the text.
22	PGPR	Plant growth-promoting rhizobacteria? Give the full title once in the text.
37-38	Fusarium equiseti and two Fusarium fujikuroi	Fusarium equiseti and two F. fujikuroi
54-55	nitrogen (N), phosphorus and potassium (K).	nitrogen (N), phosphorus (Р) and potassium (K).
90-91	From the 10,000 antimicrobial molecules produced by microorganisms, over than 50 % were isolated from Actinomycetes [40].	Work [40] is devoted to streptomycetes. In actinomycetes, about 10,000 antimicrobial compounds was known. See for example Berdy, 2005.  doi: 10.1038/ja.2005.1.
133	L tryptophan	L-tryptophan
159-160	on potato dextrose agar medium (PDA).	Provide the composition or reference.
185	and 50ng of DNA	and 50 ng of DNA
204	of the 10 selected strains [54] against the Gram negative bacteria: Escherichia coli	of the 10 selected strains [54] against the Gram negative bacteria: Escherichia coli
224	culture and S is radial growth of the fungal in the presence of Actinomycete	culture and S is radial growth of the fungus (?) in the presence of Actinomycete
248	grown in AMM+ orthoclase	grown in AMM + orthoclase
256	greatly varied from strain to strain and ranged from 3.8 mg/L to 216.6 mg/L.	Figure 3 shows other values.
253-280		Please check the text and its compliance with figure 3.
291-293	AZ, AYD, DE2 and BYC released a highest amount of K from insoluble orthoclase (216.6, 155.6 and 125.4 mg/L, respectively).	Please clarify.
315	CHAMP1, CHAMP2 and CHAMP3) belongs to the genus Fusarium. Macroscopically,	CHAMP1, CHAMP2 and CHAMP3) belongs to the genus Fusarium. Macroscopically,
332	Fusarium fujikuroi and the isolate CHAMP2 was related to Fusarium equiseti.	F. fujikuroi and the isolate CHAMP2 was related to F.equiseti.
350-353		Mark clearly on the figure a-e and in the caption A, B.
354-355	None of the 10 tested Streptomyces sp. showed an activity against the Gram positive bacteria Bacillus subtilis, Streptococcus sp. or Pseudomonas aeruginosa.	Change the phrase, for example, add "or Gram-negative bacteria Pseudomonas aeruginosa”.
356	against Staphylococus aureus.	against Staphylococcus aureus.
392-393	source. Interestingly, in the broth AMM, the amount of soluble K ranged from 3.8 mg/L to 216.6 mg/L after 5 days of cultivation.	Does not match Figure 3.

 

Author Response

Point 1 : The disadvantages of this work are shown in the table below. They can be overcome by the authors as a result of careful reading and editing of the text.

Response 1 : Correction done in the text

Point 2 : I would like to note that when re-mentioning species names (binary nomenclature), the genus designation should be abbreviated. This rule is violated throughout the text.

Response 2 : Correction done in the text

Point 3 : Special attention is drawn to the discrepancy between the data of figure 3 and the text. This needs to be corrected, after which the work can be published.

Response 3 : Correction done in the Figure 3.

Line	Original text	Change please on
2	PGP	Plant growth-promotion? Give the full name, there is no decryption anywhere in the text. Correction Done in the text: line 2
22	PGPR	Plant growth-promoting rhizobacteria? Give the full title once in the text. Correction done in the text: line 22
37-38	Fusarium equiseti and two Fusarium fujikuroi	Fusarium equiseti and two F. fujikuroi Done
54-55	nitrogen (N), phosphorus and potassium (K).	nitrogen (N), phosphorus (Р) and potassium (K). Done
90-91	From the 10,000 antimicrobial molecules produced by microorganisms, over than 50 % were isolated from Actinomycetes [40].	Work [40] is devoted to streptomycetes. In actinomycetes, about 10,000 antimicrobial compounds was known. See for example Berdy, 2005.  doi: 10.1038/ja.2005.1. Correction done in the text: line 90-91 : From the 10,000 antimicrobial molecules produced by Actinomycetes, over than 50 % were isolated from Streptomycetes [40].
133	L tryptophan	L-tryptophan Done line 133
159-160	on potato dextrose agar medium (PDA).	Provide the composition or reference. Composition provided, line 159
185	and 50ng of DNA	and 50 ng of DNA Done, line 186
204	of the 10 selected strains [54] against the Gram negative bacteria: Escherichia coli	of the 10 selected strains [54] against the Gram negative bacteria: Escherichia coli Correction done in the text: line 204
224	culture and S is radial growth of the fungal in the presence of Actinomycete	culture and S is radial growth of the fungus (?) in the presence of Actinomycete Correction done in the text: line 222-226
248	grown in AMM+ orthoclase	grown in AMM + orthoclase Done
256	greatly varied from strain to strain and ranged from 3.8 mg/L to 216.6 mg/L.	Figure 3 shows other values. Sorry, the Figure 3 had a too low value for the strain AZ. It has been changed.
253-280		Please check the text and its compliance with figure 3.
291-293	AZ, AYD, DE2 and BYC released a highest amount of K from insoluble orthoclase (216.6, 155.6 and 125.4 mg/L, respectively).	Please clarify. Correction done in the text: line 280
315	CHAMP1, CHAMP2 and CHAMP3) belongs to the genus Fusarium. Macroscopically,	CHAMP1, CHAMP2 and CHAMP3) belongs to the genus Fusarium. Macroscopically, Correction done in the text: line 327
332	Fusarium fujikuroi and the isolate CHAMP2 was related to Fusarium equiseti.	F. fujikuroi and the isolate CHAMP2 was related to F.equiseti. Correction done in the text: line 343
350-353		Mark clearly on the figure a-e and in the caption A, B. Correction done in the figure 7
354-355	None of the 10 tested Streptomyces sp. showed an activity against the Gram positive bacteria Bacillus subtilis, Streptococcus sp. or Pseudomonas aeruginosa.	Change the phrase, for example, add "or Gram-negative bacteria Pseudomonas aeruginosa”. Correction done in the text: line 377
356	against Staphylococus aureus.	against Staphylococcus aureus. Done
392-393	source. Interestingly, in the broth AMM, the amount of soluble K ranged from 3.8 mg/L to 216.6 mg/L after 5 days of cultivation.	Does not match Figure 3. Sorry, Figure 3 value of strain AZ was wrong. It has been changed.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors addressed all the suggested issues. In present form the manuscript is suitable for publishing in Microorganisms.