Characterisation of Pectinolytic Bacillus pumilus and Paenibacillus amyloliticus Strains, New Pathogens of Potato in Tunisia
, Agata Motyka-Pomagruk 2,3, Natalia Kaczynska 2,3, Wojciech Sledz 2,3, Belhassen Tarhouni 4, Mohamed Rabeh Hajlaoui 5, Ewa Lojkowska 2,3,* and Najla Sadfi-Zouaoui 1,*Round 1
Reviewer 1 Report
The overall presentation is good, but some more information should be given in following ways
1. GPS location of the sites/farms from where the bacteria were isolated
2. Is it possible to grow plant pathogenic bacteria at 37 degree celsius?
3. Authors are requested to construct one phyllogenectic tree of the pectolytic isolates based on 16S rDNA sequences. Similarly a dendrogram can be drawn based on the biochemical assay results. And then advised to see some correlation if present.
4. Is there any symptomatological differences between potato soft rot caused by Pectobacterium sp and Bacillus sp.
Good luck
Good..but mistakes should be corrected.
Author Response
We are highly grateful to the Reviewers for evaluation of the manuscript and suggestions on improvement of our work.
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Reviewer 1
According to Reviewer 1’s suggestion minor English issues have been corrected in the resubmitted version of the manuscript.
The overall presentation is good, but some more information should be given in following ways
- GPS location of the sites/farms from where the bacteria were isolated
Ad 1. Thank you for this suggestion. The requested information has been added to the resubmitted version of the manuscript.
- Is it possible to grow plant pathogenic bacteria at 37 degree celsius?
Ad. 2. The plant pathogenic bacteria are able to grow at the temperature range between 14°C to 37°C, with an optimum at 28°C
- Authors are requested to construct one phyllogenectic tree of the pectolytic isolates based on 16S rDNA sequences. Similarly a dendrogram can be drawn based on the biochemical assay results. And then advised to see some correlation if present.
Ad 3. As we wrote in the manuscript all the examined B. pumilus strains had identical 16S rDNA sequences, therefore construction of such a phylogenetic tree would add nothing to the scientific contents of the manuscript. In our opinion the phenotypic data are easy to follow and interpret in the attached colored Table 3. As the 16 S rDNA sequences of B. pumilus are identical, there is also no point in looking for correlations between them and the phenotypic data.
- Is there any symptomatological differences between potato soft rot caused by Pectobacterium sp and Bacillus sp.
Ad. 4 See photo of Symptoms of brown soft rot of potato tubers caused by Bacillus sp. in attached file
Author Response File: 
Author Response.docx
Reviewer 2 Report
In the manuscript “Characterization of pectinolytic Bacillus pumilus and Paenibacillus amyloliticus strains, new pathogens of potato in Tunisia”, the authors proved that Bacillus pumilus and Paenibacillus amyloliticus are new pathogens of potato in Tunisian. 20 strains of Bacillus pumilus and Paenibacillus amyloliticus were isolated, cultured and identified. In addition, the authors analyzed the phenotypic characteristics of 20 strains and confirmed that they have the same or stronger pathogenicity than Dickeya and Pectobacterium. In my opinion, the experimental method adopted in this paper is scientific, and it is proved from many aspects that Bacillus pumilus and Paenibacillus amyloliticus are new pathogens of Tunisian potato. This manuscript provides an important basis for the prevention of potato soft rot in Tunisia. And it warns researchers to take maximum care to avoid using potentially pathogenic strains of bacteria to fight fungus-derived diseases or stimulate plant growth. In summary, the article can be accepted because its topic meets the requirements of agriculture.
Please see the detailed comments below.
Why is there no culture-medium photo for Strain A12? I think it's important to single out the only mycobacterium amyloid. Furthermore, Figures 2E and 2F don’t show that all 20 strains are pathogenic. The authors should inoculate each strain on potato tissue for observation and take photos of each strain infecting potato tissue. I think this will give readers a more intuitive idea of the pathogenicity of these 20 strains.
Other comments:
1, Please indicate the experimental group and the control group respectively in Figures 2E and 2F.
2, Please check the number of journals in the references. Some are not in italics.
Author Response
Reviewer 2
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In the manuscript “Characterization of pectinolytic Bacillus pumilus and Paenibacillus amyloliticus strains, new pathogens of potato in Tunisia”, the authors proved that Bacillus pumilus and Paenibacillus amyloliticus are new pathogens of potato in Tunisian. 20 strains of Bacillus pumilus and Paenibacillus amyloliticus were isolated, cultured and identified. In addition, the authors analyzed the phenotypic characteristics of 20 strains and confirmed that they have the same or stronger pathogenicity than Dickeya and Pectobacterium. In my opinion, the experimental method adopted in this paper is scientific, and it is proved from many aspects that Bacillus pumilus and Paenibacillus amyloliticus are new pathogens of Tunisian potato. This manuscript provides an important basis for the prevention of potato soft rot in Tunisia. And it warns researchers to take maximum care to avoid using potentially pathogenic strains of bacteria to fight fungus-derived diseases or stimulate plant growth. In summary, the article can be accepted because its topic meets the requirements of agriculture.
Thank you for appreciating our research work
Please see the detailed comments below.
Why is there no culture-medium photo for Strain A12? I think it's important to single out the only mycobacterium amyloid. Furthermore, Figures 2E and 2F don’t show that all 20 strains are pathogenic. The authors should inoculate each strain on potato tissue for observation and take photos of each strain infecting potato tissue. I think this will give readers a more intuitive idea of the pathogenicity of these 20 strains.
The photos depicted in Figures 2E and 2F are representative for the studied isolates. As listed in the manuscript the studied B. pumilus isolates exhibit similar morphology of the colonies in addition to have identical 16S rDNA sequences, therefore attaching a photo for each strain would not be beneficial for the manuscript. Though, to satisfy the Reviewer we included information which strains, as the representative ones, have been depicted in the figure panels.
Figure 2 (revised)
Phenotypic characterization of the selected pectinolytic bacterial isolates. A, B. Morphology of the colonies on TSA agar medium. C. Cavity formation on CVP agar medium inoculated with a pectinolytic isolate and incubated at 28°C for 48 h. D. Cell morphology post Gram staining. E. Bacterial soft rot on potato tuber slices of cv. Lilly after 24 and 48 h incubation at 28°C of the strain“A10”. F. Bacterial soft rot on potato tuber slices of cv. Spunta after 24 and 48 h incubation at 28°C of the strain “A9”.
See photo of the Colonies of the strain A12 on TSA medium in attached file
Other comments:
1, Please indicate the experimental group and the control group respectively in Figures 2E and 2F.
Ad 1. These panels represent the observed disease symptoms caused by the examined B. pumilus strains on potato. To satisfy the Reviewer the description of the applied control groups has been improved in the Materials and Methods section to look as follows:
“The holes designated as negative controls were filled with 50 µl of sterile 0.85% NaCl instead of the bacterial suspension. In terms of positive controls of efficient potato macerators, Dickeya solani IFB0099 and Pectobacterium parmentieri IFB5308 strains were applied.”
2, Please check the number of journals in the references. Some are not in italics.
Ad. 2. Thank you for this comment. The references have been revised according to the suggestion of the Reviewer.
Author Response File: Author Response.docx
