Bacillus spp.-Mediated Growth Promotion of Rice Seedlings and Suppression of Bacterial Blight Disease under Greenhouse Conditions

Round 1

Reviewer 1 Report

In this article, Rajer and colleagues report the isolation and characterization as potential biocontrol agents for phytopathogenic bacteria, of two rhizospheric isolates from rice belonging to the Bacillus genus. The strains were selected on the basis of a screen for in vitro antagonistic activity against a panel of phytopathogenic fungi and bacteria. FA12 and FA26 showed a broad range of antagonistic activity and were further tested in their ability to promote seedling growth and to protect rice plants from Xanthomonas oryzae. Finally, authors identified the lipopeptides produced by these two isolates. Overall, the study was been well designed and conducted. There are however a number of issues that must be addressed to improve the robustness of the work. These are listed below:

1. Taxonomic assignment of isolate FA26: the species assignment is incorrect. Based on the 16S and gyrB sequences available for this strain in Genbank, BlastN analysis against type species results in 100% similarity of strain FA26 with the type strain TE3 of B. cabrialesi. Same for gyrB, although with a slightly lower % of similarity (98.5%). This means that the assignment made to the B. subtilis species as shown in Figure 3 is incorrect. This has to be corrected.

2. Figure 2 must shown the statistical significance of the differences mentioned in the text.

3. Figure 1 and Table 1: lenght of shoots and roots are not faithful indicators of plant growth promotion. Actually, fresh or/and dry weigth are more robust indicators,a nd must be shown.

4. Lines 349-352. I don't think is correct to say that the strains "increased the colonization up to...", because the comparison is made against the control plants whose seeds had not been inoculated, so there should not be any CFU count there. Additionally, how do the colonization level detected (about 10e5 CFU/g) compares to other studies on rice with other strains? Such CFU counts do not appear to be that high, considering that the substrate did not contain natural microflora (autoclaved peat soil). Also, it is not clear how long were plants incubated in the pots before harvesting.

5. Authors must be more cautious in the discussion/conclusion about two topics: 1) the strains may have the potential to produce other lipopeptides, in different growth conditions. 2) the strains must be challenged under real agronomical conditions; otherwise the results presented here only provide a potential biocontrol feature.

6. Methods:

Lines 110-112: how did authors preserve the isolates to avoid genetic changes? (long-term storage)

Lines 129: which % of agar did authors use in the bacterial antagonism assay?

Lines 230-231: does this mean that the two isolates are naturally resistant to both antibiotics? If so, this has to be clearly stated.

Lines 234-235: have authors checked that the heat-shock treatment is able to recover 100% of the viable cells present in the samples before the heat-shock? Induction of sporulation may not be 100% efficient and if so, CFU counts may be understimating the abundance of cells.

Lines 234-235: authors must clearly define how did thel obtain the "rhizospheric sample solution". The rhizosphere is defined technically.

7. The text requires improvement of grammar and punctuation.

Minor isuues:

Line 389: Pseudomonas carotovorum is not a valid genus/species name. I think authors refer to Pectobacterium carotovorum.

References 14 and 20 are incomplete. Authors must check all citations.

Author Response

Dear Editor,

Thank you very much for your attention and reviewers’ appreciations on our manuscript (Manuscript ID: pathogens-1921367). We highly acknowledge reviewers’ comments and suggestions, which were valuable in improving the quality of our manuscript. The detailed point-by-point responses to the referees’ comments are provided herewith. The manuscript has been revised accordingly using the “Track Changes” function in MS Word and a marked-up version of the manuscript is uploaded.

We sincerely hope that our manuscript will be finally acceptable for publication. Thank you very much for all your help and looking forward to your response.

Yours sincerely,

Prof. Xuewen Gao

Nanjing Agricultural University, Nanjing 210095, China.

E-mail: [email protected]

Reviewers’ comments:

Reviewer 1

In this article, Rajer and colleagues report the isolation and characterization as potential biocontrol agents for phytopathogenic bacteria, of two rhizospheric isolates from rice belonging to the Bacillus genus. The strains were selected on the basis of a screen for in vitro antagonistic activity against a panel of phytopathogenic fungi and bacteria. FA12 and FA26 showed a broad range of antagonistic activity and were further tested in their ability to promote seedling growth and to protect rice plants from Xanthomonas oryzae. Finally, authors identified the lipopeptides produced by these two isolates. Overall, the study was been well designed and conducted. There are however a number of issues that must be addressed to improve the robustness of the work. These are listed below:

1. Taxonomic assignment of isolate FA26: the species assignment is incorrect. Based on the 16S and gyrB sequences available for this strain in Genbank, BlastN analysis against type species results in 100% similarity of strain FA26 with the type strain TE3 of B. cabrialesi. Same for gyrB, although with a slightly lower % of similarity (98.5%). This means that the assignment made to the B. subtilis species as shown in Figure 3 is incorrect. This has to be corrected.

Response: Thank you very much for your suggestion. We have again searched it for 16S rRNA and gyrB gene sequences analyses. You are right that for 16S rRNA, strain FA26 (accession no. KJ646017.1) shows 100% similarity to Bacillus cabrialesii strain TE3 (accession no. MK462260.1). In addition, strain FA26 also shows 100% similarity to Bacillus subtilis strain G16 (accession no. MK049903.1) coming ahead of strain TE3.

However, for gyrB gene sequence analysis, we reconfirmed that strain FA26 (accession no. MF116377.1) belongs more closely to Bacillus subtilis strain STC51 (accession no. HM585073.1) with 98.81% (we considered 99%) similarity than that of Bacillus cabrialesi strain TE3, which shows 97.05% (97%) similarity.

Hence, based on the results of 16S rRNA and gyrB gene sequences, strain FA26 was again assigned as Bacillus subtilis. Bacillus cabrialesi strain TE3 has also been added into the phylogenetic tree in Figure 3.

2. Figure 2 must shown the statistical significance of the differences mentioned in the text.

Response: Thank you for your suggestion. Significant differences have been added in the figure (This is actually Figure 1).

3. Figure 1 and Table 1: lenght of shoots and roots are not faithful indicators of plant growth promotion. Actually, fresh or/and dry weigth are more robust indicators, and must be shown.

Response: Thank you for your suggestion. You are right. We noted these parameters too during the experiment. We have added wet and dry weights of plants in Table 1 and their description in the result of the revised manuscript.

4. Lines 349-352. I don't think is correct to say that the strains "increased the colonization up to...", because the comparison is made against the control plants whose seeds had not been inoculated, so there should not be any CFU count there. Additionally, how do the colonization level detected (about 10e5 CFU/g) compares to other studies on rice with other strains? Such CFU counts do not appear to be that high, considering that the substrate did not contain natural microflora (autoclaved peat soil). Also, it is not clear how long were plants incubated in the pots before harvesting.

Response: Thank you for your suggestion. We used already reported method for determining the colonization capacity of isolated bacterial strains (Xue et al., 2009) where they checked colonization in tomato seedlings. We did some modifications by applying our antagonists as seed inoculants. Our results are in line with their study. Our results showed that after seed treatment, isolated bacterial strains successfully colonized the rice roots indicating their excellent colonization ability. The colony counts might be high because we used autoclaved peat soil which does not have other microbes as competitors for niche so inoculated Bacillus strains had higher growth rate in that free environment. We have changed the expression of results in the revised manuscript according to your suggestions. In addition, we have added more information to clearly describe the methods as you suggested. And plants were harvested after 14 days of seed inoculations, which has been described too.

Reference:

Xue, Q.-Y.; Chen, Y.; Li, S.-M.; Chen, L.-F.; Ding, G.-C.; Guo, D.-W.; Guo, J.-H. Evaluation of the Strains of Acinetobacter and Enterobacter as Potential Biocontrol Agents against Ralstonia Wilt of Tomato. Biol. Control 2009, 48, 252–258, doi:10.1016/j.biocontrol.2008.11.004.

5. Authors must be more cautious in the discussion/conclusion about two topics: 1) the strains may have the potential to produce other lipopeptides, in different growth conditions. 2) the strains must be challenged under real agronomical conditions; otherwise, the results presented here only provide a potential biocontrol feature.

Response: Thank you for your suggestion. It is true that the production of metabolites by microbial strains can change with the change in growth conditions and newly isolated strains must be evaluated under real agronomical conditions to reveal their true potential. This study describes the initial results of newly isolated Bacillus strains, which produced Iturin and fengycin antimicrobials in Landy medium at 28°C and later, we performed greenhouse experiment using peat soil. Our strains showed excellent potential of inhibiting several pathogens in vitro and rice bacterial blight disease suppression in planta. Further experiments are in progress to evaluate newly isolated strains under real conditions.  

6. Methods:

Lines 110-112: how did authors preserve the isolates to avoid genetic changes? (long-term storage)

Response: For long-term storage, they were preserved in nutrient broth (NB) supplemented with 30% glycerol at -80 °C. We have added this to the text.

Lines 129: which % of agar did authors use in the bacterial antagonism assay?

Response: Antibacterial activity was determined in a NA medium containing 15g of agar (1.5% as the final concentration in the medium).

Lines 230-231: does this mean that the two isolates are naturally resistant to both antibiotics? If so, this has to be clearly stated.

Response: Yes, in preliminary experiments, strains were found tolerant to test antibiotics so those were used to select for strains during CFU calculation. We have now stated this in the text.

Lines 234-235: have authors checked that the heat-shock treatment is able to recover 100% of the viable cells present in the samples before the heat-shock? Induction of sporulation may not be 100% efficient and if so, CFU counts may be underestimating the abundance of cells.

Response: The experiment was done in green house so to avoid contamination during CFU calculation, samples were heat shocked so that only Bacillus could be counted. Samples were heat shocked at 65 °C not at 80 °C to save viable Bacillus cells other than spores. Because at 65 °C, almost all E. coli cells are killed.

Lines 234-235: authors must clearly define how did thel obtain the "rhizospheric sample solution". The rhizosphere is defined technically.

Response: We have now described the method in more detail in the text.

7. The text requires improvement of grammar and punctuation.

Response: Thank you for your valuable suggestion. We have got a more thorough proofreading of the text by a native speaker with Plant Pathology background.

Minor issues:

Line 389: Pseudomonas carotovorum is not a valid genus/species name. I think authors refer to Pectobacterium carotovorum.

Response: Thank you for your attention. We have corrected the name as Pectobacterium carotovorum ssp. carotovorum.

References 14 and 20 are incomplete. Authors must check all citations.

Response: Thank you for your attention. All references have been rechecked and corrected wherever necessary.

Reviewer 2 Report

I have evaluated the manuscript (pathogens-1921367) titled “Bacillus spp. mediated growth promotion of rice seedlings and suppression of bacterial blight disease under greenhouse conditions”. This manuscript focused on the isolation and identification of Bacillus strains from the rice rhizosphere and evaluation of their potential growth promotion for rice seedlings and suppression of bacterial blight disease under greenhouse conditions as well as determination of their antifungal and antibacterial activity against various phytopathogens.

The manuscript shows new results, and the objectives are very clear. However, I have some points that need to be addressed in the whole paper.

My revision suggestion includes:

1. Line 92: It is better to add some information about isolating Bacillus strains from rice rhizospheres.

2. Line 98: Please give detailed information about the isolation, identification, and pathogenicity of the fungal and bacterial phytopathogens.

3. Line 218: More details are required about the leaf-clipping method.

4. The obtained results need more discussion in depth (e.g., growth promotion of rice seedlings and suppression of bacterial blight disease by Bacillus strains FZB42, and FA12).

Author Response

Dear Editor,

Thank you very much for your attention and reviewers’ appreciations on our manuscript (Manuscript ID: pathogens-1921367). We highly acknowledge reviewers’ comments and suggestions, which were valuable in improving the quality of our manuscript. The detailed and point-by-point responses to the referees’ comments are provided herewith. The manuscript has been revised accordingly using the “Track Changes” function in MS Word and a marked-up version of the manuscript is uploaded.

We sincerely hope that our manuscript will be finally acceptable for publication. Thank you very much for all your help and looking forward to your response.

Yours sincerely,

Prof. Xuewen Gao

Nanjing Agricultural University, Nanjing 210095, China.

E-mail: [email protected]

Reviewers’ comments:

Reviewer 2

I have evaluated the manuscript (pathogens-1921367) titled “Bacillus spp. mediated growth promotion of rice seedlings and suppression of bacterial blight disease under greenhouse conditions”. This manuscript focused on the isolation and identification of Bacillus strains from the rice rhizosphere and evaluation of their potential growth promotion for rice seedlings and suppression of bacterial blight disease under greenhouse conditions as well as determination of their antifungal and antibacterial activity against various phytopathogens.

The manuscript shows new results, and the objectives are very clear. However, I have some points that need to be addressed in the whole paper.

My revision suggestion includes:

1. Line 92: It is better to add some information about isolating Bacillus strains from rice rhizospheres.

Response: Thanks, we have now added more detail on isolating Bacillus strains from the rice rhizospheres accordingly and the reference has also been added.

2. Line 98: Please give detailed information about the isolation, identification, and pathogenicity of the fungal and bacterial phytopathogens.

Response: Thanks, All the phytopathogens were actually obtained from their respective laboratory culture stocks, hence, were not required to be reconfirmed for their identification and pathogenicity. However, the text was not previously addressed about their sources. We have now modified the text and provided the sources of phytopathogens accordingly in the manuscript.

3. Line 218: More details are required about the leaf-clipping method.

Response: Thanks, the leaf-clipping method has now been described briefly according to the reported protocol.

4. The obtained results need more discussion in depth (e.g., growth promotion of rice seedlings and suppression of bacterial blight disease by Bacillus strains FZB42, and FA12).

Response: Thanks, the results particularly about growth promotion of rice seedlings and suppression of BB disease have been compared and discussed in detail.

Round 2

Reviewer 1 Report

The new version of the manuscript has addressed several suggestions made by both reviewers.

There are three points, already detailed in my previous revision, that they still need to be clarified/solved by authors.

1) Taxonomy of strain FA26: the highest similarity of the gyrB sequence of strain FA26 to that of B. subtilis STC51, DOES NOT mean that strain FA26 is indeed a representative of the B. subtilis species, because strain STC51b is NOT the B. subtilis type species strain. Instead, TE3 is the type strain of the B. cabrialesii species. So, authors have to build phylogenetic trees using the 16S and gyrB sequences of the closest type strains in order to assign the species to isolate FA26. Additionally, the type strains need to be clearly distinguished in the phylogenetic trees, by using bold type names or adding the superscript "T" (for Type strain) to each strain name in the trees.

2) With regards to the colonization ability of both strains (lines 449-453), again, I would not use the term "excellent" to describe the colonization, and just attach to the CFU numbers. The substrate was sterilized, so there was no competing microbes for the rhizosphere niche. So, from this point of view, 10e5 CFU/g sounds a rather weak colonization performance.

3) Conclusions: again, in line with my previous comment on the necessity to tone-down the potential of these microorganims until positive results are demonstrated under real agronomical conditions, I suggest modifying the last sentence of the conclusions as follows: "Overall results of this study suggested that FA12 and FA26 are effective BCAs against BB under greenhouse conditions, therefore, they could be tested in formulated inoculants under natural agronomical conditions".

Author Response

Dear Editor,

Thank you very much for your attention and reviewers’ appreciations on our manuscript (Manuscript ID: pathogens-1921367). We highly acknowledge reviewer’s comments and suggestions, which were valuable in improving the quality of our manuscript. The detailed point-by-point responses to the referees’ comments are provided herewith. The manuscript has been revised accordingly using the “Track Changes” function in MS Word and a marked-up version of the manuscript is uploaded.

We sincerely hope that our manuscript will be finally acceptable for publication. Thank you very much for all your help and looking forward to your response.

Yours sincerely,

Prof. Xuewen Gao

Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095, China.

Phone/Fax: 86-25-84395268

E-mail: [email protected]

Reviewer’s comments:

The new version of the manuscript has addressed several suggestions made by both reviewers.

There are three points, already detailed in my previous revision, that they still need to be clarified/solved by authors.

1) Taxonomy of strain FA26: the highest similarity of the gyrB sequence of strain FA26 to that of B. subtilis STC51, DOES NOT mean that strain FA26 is indeed a representative of the B. subtilis species, because strain STC51b is NOT the B. subtilis type species strain. Instead, TE3 is the type strain of the B. cabrialesii species. So, authors have to build phylogenetic trees using the 16S and gyrB sequences of the closest type strains in order to assign the species to isolate FA26. Additionally, the type strains need to be clearly distinguished in the phylogenetic trees, by using bold type names or adding the superscript "T" (for Type strain) to each strain name in the trees.

Response: Thank you very much for your valuable suggestion. Phylogenetic trees and the manuscript have been modified according to your suggestions (see line 394-413). Please also check figure 3.

2) With regards to the colonization ability of both strains (lines 449-453), again, I would not use the term "excellent" to describe the colonization, and just attach to the CFU numbers. The substrate was sterilized, so there was no competing microbes for the rhizosphere niche. So, from this point of view, 10e5 CFU/g sounds a rather weak colonization performance.

Response: Thanks. We have now revised it and removed the term, “excellent” considering your suggestion (see line 496-498).

3) Conclusions: again, in line with my previous comment on the necessity to tone-down the potential of these microorganims until positive results are demonstrated under real agronomical conditions, I suggest modifying the last sentence of the conclusions as follows: "Overall results of this study suggested that FA12 and FA26 are effective BCAs against BB under greenhouse conditions, therefore, they could be tested in formulated inoculants under natural agronomical conditions".

Response: Thank you. We have now modified the last sentence of the conclusions according to your suggestion (see line 569-572).