Ecofriendly Bioagents, Parthenocissus quinquefolia, and Plectranthus neochilus Extracts to Control the Early Blight Pathogen (Alternaria solani) in Tomato
, Hayssam M. Ali 4,*, Dunia A. Al Farraj 4, Ashraf A. Hatamleh 4, Mohamed Z. M. Salem 5 and Nader A. Ashmawy 6Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The authors investigated the isolation, identification and characterization of Alternaria solani isolates, through traditional and molecular PCR-based ITS region sequence analysis methods. The aim of the study was to evaluate and control of early blight disease using some fungal and bacterial biocontrol agents, as well as Plectranthus neochilus and Parthenocissus quinquefolia methanol extracts, were performed in vitro and in vivo, and to compare the obtained results to those for chemical fungicides. The experimental methodology seems to be sufficient, however, the interpretation of the results and discussions should be more elaborated.
General comments:
The manuscript presents a multi-interdisciplinary approach to an important issue the early blight disease caused by Alternaria solani in tomatoes. The obtained results obtained are due to fundamental research but have a strong applicative character. However, there some identified problems, particularly in the discussion part, by linking the results with complementary studies presented in the main flow of information available worldwide. In the introductory part of the manuscript, the hypothesis, the general objective and/or the specific objectives must be explicitly presented.
The authors conducted all the research correctly, recorded the results, but the statistical processing and analysis could have been performed much more professionally, which would have highlighted the results much better. The conclusions are very succinctly presented, require better structuring and detailing.
English language used could be improved, I suggest the proof reading of a native English speaker.
Overall, the manuscript is valuable, but necessary for careful review and completion, especially in the points outlined above.
Detailed comments:
lines 1-3: Title: if you use the common name of the disease the scientific name is placed in parentheses, or you can only use the scientific name, without the popular one,
lines 20-38: there is no need to specify; background, methods, results or conclusions, but is very important to explain the hypothesis and objective(s),
lines 39- 40: keywords- it is not recommended to use the same terms in the title,
lines 103-104: those photos (fig1) are not necessary... symptoms of the disease in tomatoes being well known, otherwise are not clear either,
line 212: in table 3 it is not necessary to present the chemical structure of flavonoids compounds, it is already known,
line 213: figure 7 is not clear, needs further processing,
lines 216-217: it is not necessary to specify the location of the equipment,
lines 221-222: this sentence is not necessary,
lines 222-225: this phrase is complicated, it needs to be reformulated, please reword,
line 246: in this study... it's obvious, please reword,
line 252: in the present study... please reword,
lines 463-468: Conclusions are very general, please reword.
Author Response
Rev. 1#
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
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Yes |
Can be improved |
Must be improved |
Not applicable |
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Does the introduction provide sufficient background and include all relevant references? |
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Is the research design appropriate? |
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Are the methods adequately described? |
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Are the results clearly presented? |
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Are the conclusions supported by the results? |
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Comments and Suggestions for Authors
Comments and Suggestions for Authors
The authors investigated the isolation, identification and characterization of Alternaria solani isolates, through traditional and molecular PCR-based ITS region sequence analysis methods. The aim of the study was to evaluate and control of early blight disease using some fungal and bacterial biocontrol agents, as well as Plectranthus neochilus and Parthenocissus quinquefolia methanol extracts, were performed in vitro and in vivo, and to compare the obtained results to those for chemical fungicides. The experimental methodology seems to be sufficient, however, the interpretation of the results and discussions should be more elaborated.
Answer: All the parts of the manuscript were amended and several corrections have been done.
General comments:
The manuscript presents a multi-interdisciplinary approach to an important issue the early blight disease caused by Alternaria solani in tomatoes. The obtained results obtained are due to fundamental research but have a strong applicative character. However, there some identified problems, particularly in the discussion part, by linking the results with complementary studies presented in the main flow of information available worldwide. In the introductory part of the manuscript, the hypothesis, the general objective and/or the specific objectives must be explicitly presented.
Answer:
- The discussion section was amended with more recent citations.
- In the introduction section we showed the cleared the research hypothesis as “This study is designed and carried out for the documentation and evaluation the activity of two biogents Trichoderma viride and Pseudomonas fluorescens as well as natural extracts from P. neochilus and P. quinquefolia against the growth of molecularly identified Alternaria solani isolates, the causal pathogen of tomato early blight in vitro and in vivo. The obtained resulted were compared to those for chemical fungicide (chlorothalonil). Furthermore, and for the characterization of two main flavonoid compounds, rutin and myricetin, were identified by chromatographic analysis, HPLC.
The authors conducted all the research correctly, recorded the results, but the statistical processing and analysis could have been performed much more professionally, which would have highlighted the results much better. The conclusions are very succinctly presented, require better structuring and detailing.
Answer:
- Statistical analysis was rerun for more accurate and the means were compared using compared using minimum significant different measured by Tukey's Studentized Range (HSD) Test at Alpha 0.05. Also, letters were inserted to show differences among the means.
- we rewrote the conclusion section as “In agriculture, there is an important need for alternate ecofriendly materials to control plant diseases. This study provides insights for the development of new phytosanitary products based on plant extracts of Plectranthus neochilus and Parthenocissus quinquefolia and on the bioagents Trichoderma viride and Pseudomonas fluorescens for the control of A. solani in tomato plants. The tested bioagents, plant extracts and the fungicide chlorothalonil were significantly reduced disease index of A. solani. T. viride and chlorothalonil were suggested to be superior in the reduction of disease severity of A. solani follwied by P. fluorescens extract. In vivo, and with spray application of the tested agents, T. viride, chlorothalonil followed by P. fluorescens extract were observed the most reduction in the disease index of early blight. This study suggested and recommended alternatives to chemical pesticides to achieve organic production.”
English language used could be improved, I suggest the proof reading of a native English speaker.
Answer; We appreciate you suggesting, but please we already sent it for English-editing before submission
Overall, the manuscript is valuable, but necessary for careful review and completion, especially in the points outlined above.
Detailed comments:
lines 1-3: Title: if you use the common name of the disease the scientific name is placed in parentheses, or you can only use the scientific name, without the popular one,
Answer: We placed the scientific name in parentheses
lines 20-38: there is no need to specify; background, methods, results or conclusions, but is very important to explain the hypothesis and objective(s),
Answer: this structure is from the format style of structured abstract of the Journal, however we improved the abstracted as suggested.
lines 39- 40: keywords- it is not recommended to use the same terms in the title,
Answer: Some of them are deleted.
lines 103-104: those photos (fig1) are not necessary... symptoms of the disease in tomatoes being well known, otherwise are not clear either,
Answer: We deleted Figure 1.
line 212: in table 3 it is not necessary to present the chemical structure of flavonoids compounds, it is already known,
Answer: Thank you for the comment, we deleted them
line 213: figure 7 is not clear, needs further processing,
Answer; Thanks for the comment, we amended this Figure.
lines 216-217: it is not necessary to specify the location of the equipment,
Answer: we deleted these information
lines 221-222: this sentence is not necessary,
Answer: we deleted this sentence
lines 222-225: this phrase is complicated, it needs to be reformulated, please reword,
Answer: we corrected and rewrote this sentence “Table 4 shows the efficacy of the bioagents T. viride and P. fluorescens, as well as the P. neochilus and P. quinquefolia plant extracts and the fungicide chlorothalonil, in reducing the severity of early blight disease in vivo and in vitro.”
line 246: in this study... it's obvious, please reword,
Answer: Corrected.
line 252: in the present study... please reword,
Answer; Corrected.
lines 463-468: Conclusions are very general, please reword.
Answer: we rewrote the conclusion section as “In agriculture, there is an important need for alternate ecofriendly materials to control plant diseases. This study provides insights for the development of new phytosanitary products based on plant extracts of Plectranthus neochilus and Parthenocissus quinquefolia and on the bioagents Trichoderma viride and Pseudomonas fluorescens for the control of A. solani in tomato plants. The tested bioagents, plant extracts and the fungicide chlorothalonil were significantly reduced disease index of A. solani. T. viride and chlorothalonil were suggested to be superior in the reduction of disease severity of A. solani follwied by P. fluorescens extract. In vivo, and with spray application of the tested agents, T. viride, chlorothalonil followed by P. fluorescens extract were observed the most reduction in the disease index of early blight. This study suggested and recommended alternatives to chemical pesticides to achieve organic production.”
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Dear authors,
My comments are as follows.
Abstract: No problem.
Introduction:
- Line 44: add a citation.
- Line 54: besides reference 8 and 9, suggest to find more update reference for support.
- Line 68: Suggest to find a proper citation to replace this No. 22 citation.
- Line 80: need one citation to support this sentence.
- Suggest exchange the two paragraph order (Line 67 and Line 80).
Results:
First, 2.3.2: You need to do major revised for this ITS phylogeny. NJ method without BP is not accepted here. You should use parsimony, ML or Bayesian to establish this phylogenetic tree, including bootstrap.
Second: Table 3 format has to be edited.
Discussion
Line 245: Need citations.
Line 263-267: Need citations.
Line 268-273: Could you find more recent citation for those?
Line 284: Need citation.
Line 286-289: Suggest to add some more recent reference.
Line 290-Line 296: Suggest to add some more recent reference.
Reference
No 43: format error.
Please check other than the format error.
Methods
Please Edit phylogenetics section. 4.4.
Author Response
Rev. 2#
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
( ) |
(x) |
( ) |
( ) |
|
Is the research design appropriate? |
( ) |
(x) |
( ) |
( ) |
|
Are the methods adequately described? |
( ) |
(x) |
( ) |
( ) |
|
Are the results clearly presented? |
( ) |
(x) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
(x) |
( ) |
( ) |
Comments and Suggestions for Authors
Dear authors,
My comments are as follows.
Abstract: No problem.
Answer: Thank you
Introduction:
- Line 44: add a citation.
Answer: Added
- Line 54: besides reference 8 and 9, suggest to find more update reference for support.
Answer: Added
- Line 68: Suggest to find a proper citation to replace this No. 22 citation.
Answer: Replaced with “22. Weeds of Australia, 2017. Biosecurity Queensland Edition. Fact Sheet Index http://keyserver.lucidcentral.org/weeds/data/media/Html/index.htmAndersen, M.; Jordheim M. Anthocyanins. Encyclope-dia of Life Sciences (ELS). 2010, 1-12.
- GRIIS, 2017. Global Register of Introduced and Invasive Species., http://www.griis.org/
- Line 80: need one citation to support this sentence.
Answer: One citation was added “Paton, A.; Mwanyambo, M. & Culham, A. (2018). "Phylogenetic study of Plectranthus, Coleus and allies (Lamiaceae): Taxonomy, distribution and medicinal use". Botanical Journal of the Linnean Society. 188 (4): 355–376.).
- Suggest exchange the two paragraph order (Line 67 and Line 80).
Answer: The two paragraphs were changed as suggested.
Results:
First, 2.3.2: You need to do major revised for this ITS phylogeny. NJ method without BP is not accepted here. You should use parsimony, ML or Bayesian to establish this phylogenetic tree, including bootstrap.
Answer: The ITS phylogeny was reestablished using the Maximum Parsimony method as requested.
Second: Table 3 format has to be edited.
Answer: Formatted
Discussion
Line 245: Need citations.
Answer: Added “Abada, K. A.; Mostafa, S. H.; Hillal, M. R. 2008. Effect of some chemical salts on suppressing the infection by early blight disease of tomato. Egypt. J. Appl. Sci. 23(20): 47-58.
Ashour, A.M.A. 2009. A protocol suggested for managing tomato early blight. Egypt. J. Phytopathol. 37(1): 9-20.
Line 263-267: Need citations.
Answer: Added
Line 268-273: Could you find more recent citation for those?
Answer: We added more recent citations.
Line 284: Need citation.
Answer: Citations added
Line 286-289: Suggest to add some more recent reference.
Answer: More recent citations were added.
Line 290-Line 296: Suggest to add some more recent reference.
Answer: More recent citations were added.
Reference
No 43: format error.
Answer; Corrected
Please check other than the format error.
All the referees have been checked
Methods
Please Edit phylogenetics section. 4.4.
Answer: Edited.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Dear authors,
1. You should write down the details how to perform this phylogenetics tree. For example, bootstraps duplicate number in your methods. You can read other similar research paper to know how others describe their methods in this section.
The figure legend changed into as follows.
Figure 4. Phylogenetic tree of Alternaria solani isolates (Acc. numbers, MT279570, MT279571, 325
MT279572 and MT279573) obtained in this study compared with ITS sequences by Maximum parsimony. A. solani isolates 326
collected from GenBank (Acc. Numbers, GU395512, MT135014, KX452728, KF999007, HQ270456 327
and MT199327).
Author Response
open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
(x) |
( ) |
( ) |
( ) |
|
Are the methods adequately described? |
( ) |
(x) |
( ) |
( ) |
|
Are the results clearly presented? |
( ) |
(x) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
(x) |
( ) |
( ) |
( ) |
Comments and Suggestions for Authors
Dear authors,
- You should write down the details how to perform this phylogenetics tree. For example, bootstraps duplicate number in your methods. You can read other similar research paper to know how others describe their methods in this section.
Answer: The evolutionary history was inferred using the Maximum Parsimony method. The consensus tree inferred from 10 most parsimonious trees is shown. Branches corresponding to partitions reproduced in less than 50% trees are collapsed. The consistency index is 1.000000 (1.000000), the retention index is 1.000000 (1.000000), and the composite index is 1.000000 (1.000000) for all sites and parsimony-informative sites (in parentheses). The percentage of parsimonious trees in which the associated taxa clustered together are shown next to the branches. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm [51] with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 10 nucleotide sequences. Sequence gaps were treated as missing data. There were a total of 288 positions in the final dataset.
The figure legend changed into as follows.
Figure 4. Phylogenetic tree of Alternaria solani isolates (Acc. numbers, MT279570, MT279571, 325 MT279572 and MT279573) obtained in this study compared with ITS sequences by Maximum parsimony. A. solani isolates 326 collected from GenBank (Acc. Numbers, GU395512, MT135014, KX452728, KF999007, HQ270456 327 and MT199327).
Answer: We changed the figure legend
Thank you so much
Reviewer 3 Report
The chapter of methodology should be included after the chapter with literature review, before the research results.
The manuscript lacks a clearly described research hypothesis at the beginning and a response to such hypothesis in the conclusion.
The discussion is well written but the conclusions are simplistic rather.
Author Response
Rev. 3#
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
|
Yes |
Can be improved |
Must be improved |
Not applicable |
|
|
Does the introduction provide sufficient background and include all relevant references? |
(x) |
( ) |
( ) |
( ) |
|
Is the research design appropriate? |
( ) |
(x) |
( ) |
( ) |
|
Are the methods adequately described? |
(x) |
( ) |
( ) |
( ) |
|
Are the results clearly presented? |
(x) |
( ) |
( ) |
( ) |
|
Are the conclusions supported by the results? |
( ) |
(x) |
( ) |
( ) |
Comments and Suggestions for Authors
The chapter of methodology should be included after the chapter with literature review, before the research results.
Answer; We rearranged those chapters.
The manuscript lacks a clearly described research hypothesis at the beginning and a response to such hypothesis in the conclusion.
Answer:
- In the introduction section we showed the cleared the research hypothesis as “This study is designed and carried out for the documentation and evaluation the activity of two biogents Trichoderma viride and Pseudomonas fluorescens as well as natural extracts from P. neochilus and P. quinquefolia against the growth of molecularly identified Alternaria solani isolates, the causal pathogen of tomato early blight in vitro and in vivo. The obtained resulted were compared to those for chemical fungicide (chlorothalonil). Furthermore, and for the characterization of two main flavonoid compounds, rutin and myricetin, were identified by chromatographic analysis, HPLC.
- we rewrote the conclusion section as “In agriculture, there is an important need for alternate ecofriendly materials to control plant diseases. This study provides insights for the development of new phytosanitary products based on plant extracts of Plectranthus neochilus and Parthenocissus quinquefolia and on the bioagents Trichoderma viride and Pseudomonas fluorescens for the control of A. solani in tomato plants. The tested bioagents, plant extracts and the fungicide chlorothalonil were significantly reduced disease index of A. solani. T. viride and chlorothalonil were suggested to be superior in the reduction of disease severity of A. solani follwied by P. fluorescens extract. In vivo, and with spray application of the tested agents, T. viride, chlorothalonil followed by P. fluorescens extract were observed the most reduction in the disease index of early blight. This study suggested and recommended alternatives to chemical pesticides to achieve organic production.”
The discussion is well written but the conclusions are simplistic rather.
Answer: Thank you for the comment, and we rewrote the conclusions as shown in previous comment.
