Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1

Round 1

Reviewer 1 Report

The manuscript "Identification, biocontrol activity, and field application effect of Bacillus strain Yb-1" is devoted to the important problem of the scientific researches: selection of microorganisms as the biocontrol  agents and using them for plant-growth promoting effects. The authors performed a complex study including laboratory and field experiments.  They obtained interesting and informative results although some minor corrections can improve their understanding and significance.

Section 2.4.

-       It is better to define the primer concentrations used in PCR then just the volume.

-       How PCR products were purified, define the kit.

-       Please also provide the information about sequencing technology (method).

-       Define the version of MEGA software.

-       Why the authors used the maximum likelihood method instead of Neighbor Joining that is more often used for such type of data. 

-       What the program or internet resource was used for sequences concatenation?

The germination of seeds can depend on the pepper variety. If the pepper variety in the lab experiment was the same as in the field one (section 2.8.1.) then it should be mention: add the information in section 2.6.

Section 3.2.

-       Was it only one colony chosen for the following research? Is there a possibility that not only Bacillus velezensis but also other species or strains could be isolated from cucumber leaves? If yes, why authors analyzed only one?

-       Provide the information about the size of sequenced genes fragments (16S rRNA, gyrA, gyrB).

-       What was the highest percent of similarity and to what species according to blastn analysis for 16S rRNA, gyrA, gyrB genes?

-       Provide the accession numbers (in GenBank) of bacterial sequences used for dendrogram construction. The type strains of Bacillus species should be added to the phylogenetic tree and the number of strains should be increased (4-6 of each species).

Figure 3 should be slightly edited: if it is possibly to present the better photo of seedlings, they are too curvy and it is difficult to compare them.

The results of the lab experiments showed strong antagonistic activity of isolate Yb-1 against Colletotrichum capsici and Botrytis cinerea at 10⁸ CFU but at the same time, this concentration inhibited seed germination and growth. Could the authors comment it (probably in Discussion section)?

 In section 3.5: explanation of what are treatment A, B, C and D should be repeated for better understanding (might be in short form without concentration).

According to the Table 2 both the bacterial suspension and difenoconazole had almost the effect and only treatment with their mix demonstrated the beneficial effect. It would be interesting to know the authors’ opinion about it (probably in Discussion section).

In section 4 (same Discussion) the author’ suggestion about the reason why the bacterial concentration of 10⁸ CFU inhibited the seed germination and growth should be extended. The explanation of “the seeds are treated differently” seems unclear and hard to understand.

The manuscript "Identification, biocontrol activity, and field application effect of Bacillus strain Yb-1" is devoted to the important problem of the scientific researches: selection of microorganisms as the biocontrol  agents and using them for plant-growth promoting effects. The authors performed a complex study including laboratory and field experiments.  They obtained interesting and informative results although some minor corrections can improve their understanding and significance.

Section 2.4.

-       It is better to define the primer concentrations used in PCR then just the volume.

-       How PCR products were purified, define the kit.

-       Please also provide the information about sequencing technology (method).

-       Define the version of MEGA software.

-       Why the authors used the maximum likelihood method instead of Neighbor Joining that is more often used for such type of data. 

-       What the program or internet resource was used for sequences concatenation?

The germination of seeds can depend on the pepper variety. If the pepper variety in the lab experiment was the same as in the field one (section 2.8.1.) then it should be mention: add the information in section 2.6.

Section 3.2.

-       Was it only one colony chosen for the following research? Is there a possibility that not only Bacillus velezensis but also other species or strains could be isolated from cucumber leaves? If yes, why authors analyzed only one?

-       Provide the information about the size of sequenced genes fragments (16S rRNA, gyrA, gyrB).

-       What was the highest percent of similarity and to what species according to blastn analysis for 16S rRNA, gyrA, gyrB genes?

-       Provide the accession numbers (in GenBank) of bacterial sequences used for dendrogram construction. The type strains of Bacillus species should be added to the phylogenetic tree and the number of strains should be increased (4-6 of each species).

Figure 3 should be slightly edited: if it is possibly to present the better photo of seedlings, they are too curvy and it is difficult to compare them.

The results of the lab experiments showed strong antagonistic activity of isolate Yb-1 against Colletotrichum capsici and Botrytis cinerea at 10⁸ CFU but at the same time, this concentration inhibited seed germination and growth. Could the authors comment it (probably in Discussion section)?

 In section 3.5: explanation of what are treatment A, B, C and D should be repeated for better understanding (might be in short form without concentration).

According to the Table 2 both the bacterial suspension and difenoconazole had almost the effect and only treatment with their mix demonstrated the beneficial effect. It would be interesting to know the authors’ opinion about it (probably in Discussion section).

In section 4 (same Discussion) the author’ suggestion about the reason why the bacterial concentration of 10⁸ CFU inhibited the seed germination and growth should be extended. The explanation of “the seeds are treated differently” seems unclear and hard to understand.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript titled "Identification, biocontrol activity, and field application effect of Bacillus strain Yb-1 in Horticulture" investigates the potential use of a Bacillus strain Yb-1 as a biocontrol agent in horticulture. The study employs a combination of morphological, biochemical, and molecular techniques to identify the strain, evaluates its antifungal activity against different plant pathogens in vitro, and tests its efficacy in vivo in controlling fungal diseases in tomato plants. Overall, the study is well-conducted and provides valuable insights into the potential use of Bacillus strain Yb-1 as a biocontrol agent in horticulture. 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

In general, the work is good and interesting. In the present paper, the data presented were solely dedicated to the bacteria isolate Yb-1.  There are two minor points related this issue as follows:

1. There was no need then to have Section 2.1. Besides, the isolating method was for the endophyte; was this correct? If so, the endophytic feature of this bacterial strain should be mentioned. The brief summary of this bacterial isolate can also provided in the initial part of the Results.

2. In the Results, most of the data given were related to the Yb-1 and no other bacterial isolates were mentioned. Therefore, revise some contents especially those in Section 3.1. Otherwise, comparative activity of the bacterial isolates investigated should be discussed.

Author Response

Plesase see the attchment.

Author Response File: Author Response.pdf