Isolation and Characterization of Plant Growth-Promoting Compost Bacteria That Improved Physiological Characteristics in Tomato and Lettuce Seedlings
Round 1
Reviewer 1 Report
Dear authors, I thank you for your kind response and appreciate your tireless efforts in this manuscript
best wishes
Author Response
First, thank you for your comments that helped improve the writing of this article. On this occasion, a rigorous revision of the English language was carried out to correct grammatical errors, which was carried out by qualified personnel.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The author lacks the art of distinctively addressing the queries. However, the author has added a few data which has improved the quality of the manuscript. The author has enhanced the comprehensiveness of the title. Furthermore, it sounds better to replace enhanced with improved physiological characteristics.............in Title. Moreover, please rigorously check the article to correct grammatical errors.
Author Response
First, thank you for your comments that helped improve the writing of this article. On this occasion, a rigorous revision of the English language was carried out to correct grammatical errors, which was carried out by qualified personnel.
Also we replace enhanced with improved physiological characteristics... in Title.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The manuscript presents data from a strain bacteria of specie (Bacillus pumilus) widely exploited as a plant growth-promoting bacteria and antagonist of several pathogens. The manuscript only presents preliminary data on the initial development of the tested cultures, without presenting other complementary assessments, such as plant nutrition and in vivo biological control and evaluations of plants in the field (or even fruit development, in the case of tomatoes plants), which could present most promising data for the candidate inoculant. The manuscript presents only preliminary data of a possible inoculant.
Author Response
Point 1: The manuscript presents data from a strain bacteria of specie (Bacillus pumilus) widely exploited as a plant growth-promoting bacteria and antagonist of several pathogens. The manuscript only presents preliminary data on the initial development of the tested cultures, without presenting other complementary assessments, such as plant nutrition and in vivo biological control and evaluations of plants in the field (or even fruit development, in the case of tomatoes plants), which could present most promising data for the candidate inoculant. The manuscript presents only preliminary data of a possible inoculant.
Response 1: Indeed, the objective that was raised to this moment for this work was the isolation of a bacterial strain with plant growth-promoting properties from a mature compost. The use of compost extracts in agriculture has increased constantly and considerably since they offer an attractive way to improve plant growth and control some diseases that allow a replacement of agrochemicals. Therefore, this study sought to isolate and characterize a bacterium that exhibited mechanisms, mainly phosphate solubilization, from a compost produced from bovine manure. This strain was evaluated only for its use during the first stages of lettuce and tomato plant growth, germination and seedling, with promising results. To verify the effectiveness of the application of this bacterium in all the phenological stages of the field, we need to carry out a more extensive evaluation.
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript focuses on the aspect promoting of plant growth (tomato and lettuce seedlings) by Bacillus pumilus isolated from compost. The subject of this manuscript is consistent with the scope of the Journal. The issues concerning of promoting growth of plant are very interesting. However, I am concerned about the following remarks listed below.
- line 74 – 79 - Has pure culture been isolated? It does not follow from the description. I wonder about the consequences of the lack of pure culture in subsequent studies?
- line 85-87- NCBI is only a website, not an analysis tool. I am asking for a specific description and parameters of the analysis.
- line 124 – 125 - Please provide a specific temperature and write why it was chosen.
- line 136-142 - Why were such seedling growth conditions chosen?
- line 155-156 - Based on the Gram staining, taxonomic affiliation to the genus cannot be clearly established.
- line 158-160 - In my opinion, DNA analysis of the 16S rRNA gene is insufficient to determine the species Bacillus pumilus.
- There are many factual errors throughout the manuscript (e.g. Bacillus, in vitro italics) etc.
- Overall, I think the manuscript is short and would be better suited as shor communication rather than article.
Author Response
Point 1: - line 74 – 79 - Has pure culture been isolated? It does not follow from the description. I wonder about the consequences of the lack of pure culture in subsequent studies?
Response 1: Yes, there is a pure isolate that has been used during all the tests. The procedure followed to do so is added in detail to the text:
Line 94-96: Likewise, the selected colonies were streaked three times on fresh nutrient agar plates to obtain a pure isolate. The purified bacterial isolates were stored at 4 °C in 20% glycerol. Subsequently, some pure isolates were lyophilized for long-term storage.
Point 2: - line 85-87- NCBI is only a website, not an analysis tool. I am asking for a specific description and parameters of the analysis.
Response 2: Specifically, the NCBI BLAST nucleotide sequence program was used, in which the partial sequence of the 16S ribosomal gene obtained was compared with other similar sequences from the GenBank database. In the comparison, the isolate was identified as Bacillus pumilus according to DNA sequencing of the 16S ribosomal gene, with 99% identity with Bacillus pumilus strain MB411.
The wording in the methodology is modified to make it clearer what was done:
Line 102-105: The DNA sequencing of the 16S ribosomal gene obtained from the amplicon was compared with others in the GenBank database using the NCBI BLAST nucleotides program on the website https://blast.ncbi.nlm.nih.gov/Blast.cgi
Line 180: 99% similarity to Bacillus pumilus strain MB411 in BLASTn analysis.
Point 3: - line 124 – 125 - Please provide a specific temperature and write why it was chosen.
Response 3: The wording of the lines is corrected:
Line 143-144: At room temperature, approximately 20 °C ± 5 °C, to maintain the temperature conditions of the region where the experiments are performed.
Point 4: - line 136-142 - Why were such seedling growth conditions chosen?
Response 4: The conditions were established according to the temperature, humidity, and photoperiod of the greenhouses located in the community of Amazcala, El Marqués, Querétaro, Mexico. Those greenhouses are used for the germination and production of lettuce and tomato. The experimentation was carried out like this to evaluate the effect of bacterial inoculation in seedlings on production conditions in the region.
Point 5: - line 155-156 - Based on the Gram staining, taxonomic affiliation to the genus cannot be clearly established.
Response 5: The line is corrected:
Line 175-176: gram-positive bacilli.
Point 6: - line 158-160 - In my opinion, DNA analysis of the 16S rRNA gene is insufficient to determine the species Bacillus pumilus.
Response 6: Currently, 16S ribosomal RNA (rRNA) genes have been the most predominantly used molecular marker in bacterial classification, phylogeny, and taxonomy studies. Comparisons of the 16S rRNA sequences (or the genes that encode them) make it possible to establish the existing phylogenetic relationships between prokaryotic organisms. That has given rise to the current classification system and allows fast and accurate identification of bacteria.
Many investigations indicate that although there are alternative molecular makers to 16S rRNA, this gene has specific characteristics, which is why it is still widely used:
- It is an old molecule present in all current bacteria. Therefore, it constitutes a universal target for their identification.
- Changes occur slowly enough to provide information about all prokaryotes and, together with variations in 18S rRNAs, throughout the entire evolutionary scale. rRNAs do, however, contain enough variability to differentiate not only the most distant organisms but also the closest ones.
- The relatively long size of 16S rRNAs (1,500 nt) minimizes statistical fluctuations.
- Preservation in secondary structure can assist in comparisons, providing a basis for precise alignment.
- Since 16S rDNAs are relatively easy to sequence, there are extensive, ever-growing databases.
Point 7: - There are many factual errors throughout the manuscript (e.g. Bacillus, in vitro italics) etc.
Response 7: Errors in the manuscript are corrected, mainly italicized words that should not be italicized.
Point 8: - Overall, I think the manuscript is short and would be better suited as shor communication rather than article.
Author Response File: Author Response.pdf
Reviewer 3 Report
- The introduction did not include references to lettuce
- The isolation was done based on dissolving phosphate only, but there are many traits that promote plant growth and it was worth considering some of them, such as:
- Nitrogen fixation
- Indole-3-acetic acid production
- Ammonia production
- Siderophore production
- ACC-deaminase activity
- Cyanide production
- Extracellular enzyme activity
- Production of organic acids
- You run the antagonistic assay against Fusarium oxysporum and Sclerotium cepivorum , But there are a huge number of phyto pathogenic fungi as well as soilborne root pathogens such as:
Alternaria alternata, Aspergills niger, Botrytis cinerea, Pythium ultimum, Rhizoctonia solani.
It is better to conduct this test against more than two pathogens
- Regarding molecular characterization, you must add the phylogenetic tree
- Photos must be added to:
- Phosphate solubilization
- Antagonistic activity
- Germination assay
- Effect of Bacillus pumilus on Tomato and Lettuce Seedlings
- Is 75 days long enough to consider the compost mature and stable?
Line 56-58: you reported " the evaluation of a PGPB isolated from compost with phosphorus solubilizing activity, an antagonist of pathogens, and promotion of plant growth has not been reported"
, But many reports have already explored this point.
Line 67: Correct "75-day-old" to "75 day old"
Line 85: correct "16s" to "16S"
Line 107: correct the title "Effect Evaluation on Plant Growth of Bacillus pumilus" to be "evaluating the effect of B. Pumilus on plant growth"
Line 127: correct "with (4)" to be "with equation (4)"
Line 161: correct "complete" to "partial"
Line 177,178: you reported "the bacteria promoted the growth of Sclerotium cepivorum by 25% without finding a significant statistical difference with the control", You must add the results and photos to prove that
Line 189: You reported "the latency time varied by one day (Figure 1)", But the text does not include any information about the latency time, as well as the figure 1
Line 192: correct "devia-tion" to "deviation"
Line 234: "CSR", But the text does not contain any reference to that abbreviation
Line 277: "leaf area", the text does not contain any data related to the area of the leaf?
Author Response
Point 1: The introduction did not include references to lettuce
Response 1: A section about the effect of Bacillus pumilus inoculation on lettuce plants is added to the introduction. (Bibliographic references were corrected)
Line 63-65: And, the inoculation of B. pumilus in lettuce plants considerably increases vigor, average head weight, and height compared to other PGPB [20].
Point 2: The isolation was done based on dissolving phosphate only, but there are many traits that promote plant growth and it was worth considering some of them, such as:
- Nitrogen fixation
- Indole-3-acetic acid production
- Ammonia production
- Siderophore production
- ACC-deaminase activity
- Cyanide production
- Extracellular enzyme activity
- Production of organic acids
Response 2: What you mention is correct, other traits can be evaluated in bacterial strains to be considered PGPB; however, in this study, we are based on the importance of phosphate solubility due to the agronomic importance presented. Therefore, a paragraph is added to the introduction indicating the importance of the phosphate solubilizing activity in agriculture:
Line 38-45: In addition, some bacteria solubilize phosphate rock and other sources of inorganic phosphorus in the soil and are called phosphate-solubilizing bacteria. This activity is relevant because a large amount of the P contributed by phosphate fertilizers loses efficiency due to different biochemical processes such as sorption, fixation, and immobilization, which causes high rates of P application in crops. The inefficient use of phosphate fertilizers can modify the state of the nutrients in the soil, the availability for the absorption of the crops, and the microbial communities [4]. These bacteria can be an alternative to increase the phosphorus available to plants [5,6].
Point 3: You run the antagonistic assay against Fusarium oxysporum and Sclerotium cepivorum , But there are a huge number of phyto pathogenic fungi as well as soilborne root pathogens such as:
Alternaria alternata, Aspergills niger, Botrytis cinerea, Pythium ultimum, Rhizoctonia solani.
It is better to conduct this test against more than two pathogens
Response 3: This test was carried out using the important fungi of the region, however, the study can be expanded later by considering a greater number of fungi and evaluating the effect of the isolated bacteria.
Point 4: Regarding molecular characterization, you must add the phylogenetic tree
Photos must be added to:
Phosphate solubilization
Antagonistic activity
Germination assay
Effect of Bacillus pumilus on Tomato and Lettuce Seedlings
Response 4: The figure of the phylogenetic tree constructed from B. pumilus is added:
Line 187-190: Figure 1. Phylogenetic neighbor-binding tree reconstructed based on the 16S rRNA gene sequence showing the phylogenetic relationship between the isolated Bacillus pumilus strain (MN067217) and others like it.
Line 181-183: The phylogenetic position of B. pumilus was constructed by retrieving 16S rRNA gene sequences from closely related bacterial species using BLAST pairwise alignments at https://www.ncbi.nlm.nih.gov/blast/treeview/treeView.cgi (Figure 1).
Line 202-205: Figure 2. Antifungal in vitro activity of Bacillus pumilus. (a) Fusarium oxysporum control, (b) antifungal activity of B. pumilus against Fusarium oxysporum, (c) Sclerotium cepivorum control, (d) antifungal activity of B. pumilus against Sclerotium cepivorum.
Line 227-230: Figure 4. Effect of Bacillus pumilus on the germination of tomato and lettuce seeds. a) Tomato seeds germinated in the presence of B. pumilus and the uninoculated control. b) Lettuce seeds germinated in the presence of B. pumilus and the uninoculated control.
Point 5: Is 75 days long enough to consider the compost mature and stable?
Response 5: The maturity time of compost varies concerning its content: In the case of the compost used, different tests were used to evaluate its maturity:
* Sensory parameters of maturity: smell, color, and granulometry.
* Chemical indicators of maturity: carbon-nitrogen ratio (C/N)
Both indicators showed that the compost was in a mature state. With a C/N = 6.3, indicative of a stable compost.
Point 6: Line 56-58: you reported " the evaluation of a PGPB isolated from compost with phosphorus solubilizing activity, an antagonist of pathogens, and promotion of plant growth has not been reported", But many reports have already explored this point.
Response 6: The expression is corrected to indicate that the studies that have been done for the evaluation of PGPB, despite being many so far, few are based on obtaining these bacteria from a compost, taking into account that the composition, state of maturity, and conditions of compost preparation affect the bacterial composition of the same:
Line 69-72: Several studies have focused on evaluating the activities of PGPB with commercial interest; however, the evaluation of a PGPB isolated from compost with phosphorus solubilizing activity, an antagonist of pathogens, and promotion of plant growth has been little explored.
Point 7: Line 67: Correct "75-day-old" to "75 day old"
Response 7: The expression is corrected.
Point 8: Line 85: correct "16s" to "16S"
Response 8: The expression is corrected.
Point 9: Line 107: correct the title "Effect Evaluation on Plant Growth of Bacillus pumilus" to be "evaluating the effect of B. Pumilus on plant growth"
Response 9: The expression is corrected.
Point 10: Line 127: correct "with (4)" to be "with equation (4)"
Response 10: The expression is corrected.
Point 11: Line 161: correct "complete" to "partial"
Response 11: The expression is corrected.
Point 12: Line 177,178: you reported "the bacteria promoted the growth of Sclerotium cepivorum by 25% without finding a significant statistical difference with the control", You must add the results and photos to prove that
Response 12: The results of the comparison between B. pumilus and S. cepivorum are added in Table 1.
Point 13: Line 189: You reported "the latency time varied by one day (Figure 1)", But the text does not include any information about the latency time, as well as the figure 1
Response 13: According to you, latency time is not included, so this part is removed from the text.
Point 14: Line 192: correct "devia-tion" to "deviation"
Response 14: The expression is corrected.
Point 15: Line 234: "CSR", But the text does not contain any reference to that abbreviation
Response 15: Fix the CSR expression was a typing error. It is exchanged for RSE (Line: 271)
Point 16: Line 277: "leaf area", the text does not contain any data related to the area of the leaf?
Response 16: The expression is corrected:
Line 314: leaf number.
Author Response File: Author Response.pdf
Reviewer 4 Report
Overall, the research effort is fine. However, the author lacks a significant result to meet the impact and standard of the journal. More physiological and molecular data are required. I have a few other suggestions for the author which could enhance the quality and comprehensiveness of the manuscript.
The title is incomplete.
Bacillus pumilus isolated from compost with plant growth-promoting properties in tomato and lettuce seedlings
The author can consider the following terms for the broad title. Isolation/characterization/identification of plant growth-promoting rhizosheric/endophytic/compost fungus/bacteria that enhanced/modulated …physiological/molecular/proteomics…….in tomato/lettuce…
The structure of the abstract is organized well, however it could be improved further. The abstract structure contains Problems, necessities, scope, methodologies, results, conclusion, and recommendations.
Concise methodologies statement, efficient and impactful results, slight discussion, a brief conclusion, and pragmatic recommendation is required.
Line 23. The above suggests that……what to do you mean by above? Verb
is missing
Line 23-24: Re-check the conclusion and recommendation statement. Can be improved.
The Introduction section lacks the importance of compost and the limitation of PGPB.
The research design could be made broad including pathogenic tests and the beneficial role of selected isolates in plants.
The author has focused on P solubility as a major plant growth-promoting trait but did not show any mechanism and evidence. Here P quantification in either plant/soil/microbes inoculated pure culture is important for determining the microbe's potentiality. Moreover, the image of microbes in inhibiting fungal growth is required.
Line 221: The author directly jumped to a conclusion.
Avoid redundant statements.
Best Regards.
Author Response
Point 1: The title is incomplete.
Bacillus pumilus isolated from compost with plant growth-promoting properties in tomato and lettuce seedlings
The author can consider the following terms for the broad title. Isolation/characterization/identification of plant growth-promoting rhizosheric/endophytic/compost fungus/bacteria that enhanced/modulated …physiological/molecular/proteomics…….in tomato/lettuce…
Response 1: A modification of the title is made to expand and improve it concerning the content of the paper.
“Isolation and characterization of plant growth-promoting compost bacteria that enhanced physiological characteristics in tomato and lettuce seedlings”
Point 2: The structure of the abstract is organized well, however it could be improved further. The abstract structure contains Problems, necessities, scope, methodologies, results, conclusion, and recommendations.
Concise methodologies statement, efficient and impactful results, slight discussion, a brief conclusion, and pragmatic recommendation is required.
Response 2: According to the maximum length allowed by the journal, a description is made of the main steps of the methodology, the most relevant results obtained, and a general conclusion. The last sentence of the abstract is slightly modified as a pragmatic recommendation.
Line 24-26: The results suggest that Bacillus pumilus has properties as a plant growth promoter and can be used as a promising inoculant to enhance the growth of tomato and lettuce seedlings.
Point 3: Line 23. The above suggests that……what to do you mean by above? Verb is missing
Response 3: The expression is corrected:
Line 24-26: The results suggest that Bacillus pumilus has properties as a plant growth promoter and can be used as a promising inoculant to enhance the growth of tomato and lettuce seedlings.
Point 4: Line 23-24: Re-check the conclusion and recommendation statement. Can be improved.
Response 4: The expression is modified
Line 24-26: The results suggest that Bacillus pumilus has properties as a plant growth promoter and can be used as a promising inoculant to enhance the growth of tomato and lettuce seedlings.
Point 5: The Introduction section lacks the importance of compost and the limitation of PGPB.
Response 5: A paragraph is added indicating the importance of compost today:
Line 46-51: The use of compost is constantly increasing since it is considered a potential alternative to control soil pathogens and increase plant growth safer for health and the environment. Several studies have shown that the properties of compost are governed by mechanisms of microbial origin. That is, plant growth and phytopathogens control occur due to the presence of some PGPB in the bio-oxidative composting process of organic waste to produce a stable material rich in humic substances [7-9].
In addition, a paragraph is added indicating the limitations presented by PGPB (B. pumilus) to improve plant growth:
Line 65-68: However, the positive interaction of B. pumilus with crops depends on the plant genotype, soil nutrition, inoculant density, and environmental conditions, which significantly affects the capacity of PGPB to improve plant growth under field conditions [21].
Point 6: The research design could be made broad including pathogenic tests and the beneficial role of selected isolates in plants.
Response 6: The scope that we proposed for this research was mainly the isolation and characterization of a bacterial strain of compost with characteristics that promote plant growth, specifically phosphorus solubilization, due to the agronomic importance of phosphorus fertilization today. Finally, to test this strain in the first phenological stages of a crop, tomato, and lettuce, to evaluate the effect of the inoculation of the isolated bacteria.
Later, more tests will be carried out against other phytopathogens and throughout the cultivation of different vegetables.
Point 7: The author has focused on P solubility as a major plant growth-promoting trait but did not show any mechanism and evidence. Here P quantification in either plant/soil/microbes inoculated pure culture is important for determining the microbe's potentiality. Moreover, the image of microbes in inhibiting fungal growth is required.
Response 7: The property of the bacteria as a phosphate solubilizer was only evaluated in vitro.
Images of the pathogen inhibition, germination and seedling tests performed are added.
Line 202-205: Figure 2. Antifungal in vitro activity of Bacillus pumilus. (a) Fusarium oxysporum control, (b) antifungal activity of B. pumilus against Fusarium oxysporum, (c) Sclerotium cepivorum control, (d) antifungal activity of B. pumilus against Sclerotium cepivorum.
Point 8: Line 221: The author directly jumped to a conclusion.
Response 8: After line 259, a comparison is made with other studies concerning B. pumilus, and the discussion of the results found in this investigation continues. The results are developed by comparing and analyzing them with others already reported previously.
From line 317, a general conclusion of the work begins to be developed, as well as the studies that need to be carried out to complement this research.
Author Response File: Author Response.pdf
