Potentiality of Formulated Bioagents from Lab to Field: A Sustainable Alternative for Minimizing the Use of Chemical Fungicide in Controlling Potato Late Blight

Round 1

Reviewer 1 Report

The article entitled “Potential of formulated bioagents from Lab to field: a sustainable alternative of minimizing the use of chemical fungicide in controlling potato late blight” Manuscript id: sustainability-1613569, appears very appealing and informative. Application of chemical fungicide must be reduced for several stated regions. Your article was nicely written and discussed with proper citation. However, I observed the possibility of little improvement in English language. I would also suggest to make a flow chart of overall experiment for clarity of readers.

Author Response

Response to Reviewer 1 Comments

 

 

Point 1: The article entitled “Potential of formulated bioagents from Lab to field: a sustainable alternative of minimizing the use of chemical fungicide in controlling potato late blight” Manuscript id: sustainability-1613569, appears very appealing and informative. Application of chemical fungicide must be reduced for several stated regions. Your article was nicely written and discussed with proper citation. However, I observed the possibility of little improvement in English language. I would also suggest to make a flow chart of overall experiment for clarity of readers.

 

Response 1 (corrected in last paragraph on page 8): Thank you for your suggestion regarding improvement of the manuscript.

We have added a schematic diagram of all methodological approaches at the end of the materials and method section, we believe it would be more clear and precise concept for the readers.

 

 

 

 

 

 

 

 

 

 

 

 

 

A flow chart depicting the entire experimental procedures

 

 

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

I would like to thank you for the opportunity to prepare the review for this paper. Unfortunately, I need to present critical remarks. I don't deny the validity of the methods and the quality of the paper, but my objections are to the novelty of the method and research assumptions. In addition, the paper seems more appropriate for different journals related to biocontrol, agriculture, phytoparasites, etc. Sustainability is a cross-disciplinary access journal of environmental, cultural, economic, and social sustainability of human beings.  The fact that the pathogen of potatoes is controlled with alternative methods with respect to chemical plant protection products, which is the main core of the paper, is not enough, in my opinion, to justify the publication in a journal with a broader view.

The authors include some aspects of the economic impact of the tested formulated bacterial and fungal bioagents against the late blight of potato caused by Phytophthora infestans. However, they should increase more data on the potential environmental impact of the use of these agents and the acceptance of stakeholders and consumers. These aspects can make the paper more multidisciplinary and appropriate for the journal Sustainability.

While the authors' intentions seem genuine, however, it is a rather simplistic paper with respect to the goals of the Journal. The economic part and the stakeholder's acceptability of the proposed methods should be assessed on a deeper level.

Author Response

Response to Reviewer 2 Comments

Point 1: I would like to thank you for the opportunity to prepare the review for this paper. Unfortunately, I need to present critical remarks. I don't deny the validity of the methods and the quality of the paper, but my objections are to the novelty of the method and research assumptions. In addition, the paper seems more appropriate for different journals related to biocontrol, agriculture, phytoparasites, etc. Sustainability is a cross-disciplinary access journal of environmental, cultural, economic, and social sustainability of human beings.  The fact that the pathogen of potatoes is controlled with alternative methods with respect to chemical plant protection products, which is the main core of the paper, is not enough, in my opinion, to justify the publication in a journal with a broader view.

The authors include some aspects of the economic impact of the tested formulated bacterial and fungal bioagents against the late blight of potato caused by Phytophthora infestans. However, they should increase more data on the potential environmental impact of the use of these agents and the acceptance of stakeholders and consumers. These aspects can make the paper more multidisciplinary and appropriate for the journal Sustainability.

While the authors' intentions seem genuine, however, it is a rather simplistic paper with respect to the goals of the Journal. The economic part and the stakeholder's acceptability of the proposed methods should be assessed on a deeper level

Response 1 : Thank you for your suggestion regarding improvement of the manuscript.

Justification and novelty (Corrected in last paragraph on page 4): Two new native fungal and bacterial isolates have been identified from potato phylloplane and rhizosphere. We have assessed their efficacy on controlling late blight of potato but P. infestans is a polycyclic pathogen which can hardly be completely under control with bioagents only. The effects of fungicide application have numerous hazards to mankind and environment and apart from that many fungicides are banned in developed countries due to their toxic effects to human being and animals. Thus, in this study, we focused on the use of both fungicides and native formulated bioagents considered as a novel approach in reducing the application frequency of chemical fungicide to minimize the impact of late blight severity on potato yield.

 

 

 

Economical and environmental aspect (Corrected in 2^nd paragraph on page 3): With respect to environmental aspect, we have focused on the negative impact of fungicides on human being in the introduction section.

 

A tremendous increase in the application of pesticides especially fungicides lead to a number of health problems including reproductive problems [24,25], genetic damage [26], neurological disorders [27], increases in bladder cancer [28] and even breast cancer [29] because farmers are directly and indirectly exposed to pesticides (Corrected in 2^nd paragraph on page 3).

 

In the meanwhile, EU countries have decided in 2020 to ban mancozeb, the last cheap contact fungicide of the dithiocarbamates family, for its reproductive toxicity and endocrine disruptive action (regulation (EU) 2020/2087). It was largely used in potato late blight control, and one of the two or three most common pesticides in use worldwide, with a history of 60 years since its introduction in 1962 (Corrected in 1^st paragraph on page 3).

 

 

 

Approaches	Total expenditure for fungicides used (million Tk)	Economic return (million Tk)	Percent increase of income/ha compared to conventional practices	Total Increase of return in the country (million Tk) compared to conventional practices	Increase of income per farm family (000’Tk)  compared to conventional practices
Farmers Conventional approach	6500	85282.5	0	0	0
Improved Management with Bioagents 1	3250	91417.5	7.19	6135	20.45   7217
Improved Management with Bioagents 2	3250	94644	10.98	9361.5	31.21     24893

In terms of the acceptance of the stakeholders and consumers, we have added a table regarding detail economic analysis in the result section and we hope that will be enough for the acceptanc e of using these agents.

 

Table 9. Detailed Economic analysis of the improved management using bioagents based on the BCR calculated during 2019-2020 and 2020-2021

 

 

 

 

Farmers conventional approach: T₁[(Eight sprays of Curzate M8 (Positive control)],Improved management of bioagents 1:  T₆[Eight sprays of formulated P.putida + B.subtilis(T₂) + four sprays of Curzate M8] _,  Improved management of bioagents 2: T₁₆[Eight sprays of formulated P.putida, B.subtilisandT.erinaceum(T₁₂)+ four sprays of Curzate M8]_.  Tk = Bangladeshi currency (taka)

Total expenditure for fungicides used (million BDT): Area (0.5 milion hectare) * 1625 * 8kg Total Expenditure for Improved management with bioagents: Area (0.5 milion hectare) * 1625* 4kg. Where fungicide cost 1625 taka(BDT) /kg

Economic return (million BDT): [(Total production cost+13000) * 0.5 million * BCR]/ 10 million.

Percent increase of income/ ha compared to conventional practices for improved management with bioagents: Economic return – Economic return of farmers conventional approach/ Economic return of farmers conventional approach* 100.

Total Increase of return in the country (million BDT) compared to conventional practices: Economic return of improved management with bioagents - Economic return of farmers conventional approach.

Increase of income per farm family (000’BDT)  compared to conventional practices: Total Increase of return in the country (million BDT) / 0.3 million * 10 Million.

 

3.1.9. Detailed Economic analysis of the improved management using bioagents based on the BCR calculated during 2019-2020 and 2020-2021

Bangladesh has been producing 9.7 million tonnes potato on 0.5 million hectaresof land as mentioned earlier. Farmers are spending 6500 million Tk for total expenditures for fungicides per year with conventional approaches [Eight sprays of Curzate M8 (Positive control)]. Conversely, if we could apply two improved management approaches with bioagents 1 [(T₂) + four sprays of Curzate M8]& 2 [(T₁₂)+ four sprays of Curzate M8] then the total expenditures for fungicides could be drastically reduced to 3250 million Tk. Among three approaches, improved management with bioagents 1 & 2 showed better economic returns compared to farmers approach. Cultivation of potato with improved management approaches with bioagents 1 & 2 were satisfactory because farmers benefited 7.19%and 10.98% increase in their income in one hectare of land, respectively. With regards to country’s economic impact within two years, 9361.5 million Tk was the total increase of the country’s return applying improved management with bioagents 2 and 6135 million dollars from improved management with bioagents 1. Approximately 0.3 million farm families are closely engaged with potato production. In our detailed analysis we observed that income of individual farm family was raised 31.21 thousands Tk when we applied improved management with bioagents 2 which indicated that the use of bioagents with chemical fungicide to minimize the late blight severity have tremendous economic and social impact of our country. Thus, farmers will be willing to accept this technology as several factors are closely associated with their income return from one hectare of potato land (Table 9). (Corrected in 3.1.9)

 

 

 

 

Considering detailed economic analysis, improved management with bioagents 1 and 2 performed better compared to farmers conventional approach in terms of economic return as well as income of per farm family was raised up to 31.21 thousands Tk which indicated that using these bioagents have a positive economic impact on the country. Farmers are also benefitted while using these improved management with bioagents which significantly focused the acceptability of these bioagents among stakeholders, consumers and farmers. (corrected in last paragraph on page 13)

   

 

 

 

 

 

 

 

 

 

 

 

 

Overall responses from the authors:

 

Thank you all reviewers for your nice comments regarding the modification of this manuscript, we have modified our manuscript icluding

1. a) Slight changes though out the manuscripts have been done under track change including spacing, grammatical issues and others.
2. b) We have replaced our previous Table 6 and 8 with a new Table 8 in the revised file with a view to represent the average data of those previous tables.
3. c) We have also added a new Table (Table 9) to show the detailed economic analysis of this study.
4. d) Some references have been included in the text and reference also.

 

 

 

 

 

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

I have one major question for authors to be clarified.

In the title/aim of research you stated that you want to prove that with application of given biofungicides you will be able to reduce application of chemical agents. But as we see from experimental design of your research in several cases you have used also commercial fungicide. In addition, experimental data have proven that the best results for potato late blight are those with the maximal application of chemical fungicide combined with biofungicide (T₁₁ and T₁₆). In both experiments you applied chemical fungicide for even 4 times. So, how many times you will apply it if you did not combine with biofungicides? It is not explained anywhere in the text. So, I do not understand what is improvement obtained in current study? Please clarify and make additional explanations and clarifications.

 

The rest of the comments are listed below:

1. Add countries for affiliations 1 and 2 in the title of Manuscript.
2. In abstract you must specified what mean abbreviations used such as T₁, T_k, T₆ and T₁₆.
3. The final part about economical analysis of given research is completely unclear with English hard to follow. Please rewrite to make it more clear.
4. On Page 2 (Line 2) I think it should be "cool temperature area/region"? Check/correct.
5. On Page 2 (Line 7) delete () before and after 20.6 t/ha. It is surplus here.
6. On Page 2 (Line 17) it should be "... structure of  P. Infestans". Correct.
7. On Page 2 (Line 19) word potato should not be written with capital letter P. Correct.
8. On Page 3 (the first paragraph) please provide more references for given statements about "... numerous research finding promising outcomes".
9. On Page 3 (Lines 3 and 5) term Bacillus should be written in Italic style. Correct.
10. This complete paragraph (As fungicides are not ... to highest level) is unclear. It is either incorrectly written or something is missing in given sentences. Please check/clarify/correct.
11. On Page 3 (subsection 2.1.) it should be "... were isolated from potato.."
12. In the next Line on Page 3 correct technical error- split words shere and identified.
13. In the penultimate Line correct as in comment 11.
14. On Page 4 is this " for shoet period of time" incorrectly written? Please check.
15. On Page 5 (Line 1) correct technical error and split words.
16. On Page 5 (Line 6) It should be "P. putida" with a capital letter P. Correct.
17. On Page 5 (Line 7) it should be abbreviated name as "B. subtilis". Correct.
18. On Page 5 provide reference(s) for given statement "... previous experiments on the performance of bacterial...".
19. On Page 5 (subsection 2.5.) correct several typos i.e. split numerical values from units here.
20. On Page 6 (Line 2) correct chemical formula for calcium carbonate.
21. On Page 6 (Line 6) it can not be "was given" but "was added" or some other synonym. Check/correct.
22. On Page 8 (subsection 3.1.2.) this "that" in front of "deformation" is surplus or some verb is missing. Please check/correct.
23. On page 8 (subsections 3.1.3 and 3.1.4.) there are several typos where words should be split "T₅ in", "T₁₀ and", "T₁₁ and".
24. On Page 8 (subsection 3.1.4.) I do not understand how given values (2.43%, 3.37%, 10.33% and 21.30%) are not statistically different? I mean what was value for standard deviation if 2.43 is not different from 21.30?? Please check/clarify.
25. On Page 10 correct two typos - "Figure 2" and "Table 6"
26. Line 425: How it can be "organic potato" if we used chemical agents?? Ok, in reduced amount (If your presumption is correct) but still it is obviously that it must be applied in some doses. And with this we will go back on my the first and the main question.

Kind regards.

Author Response

Response to Reviewer 3 Comments

 

Point 1: Add countries for affiliations 1 and 2 in the title of Manuscript

Response 1( corrected in page 1): ¹Plant Bacteriology and Biotechnology Laboratory, Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

²Upazilla Agriculture Officer (L.R.), Department of Agricultural Extension (DAE), Khamarbari, Dhaka-1215, Bangladesh

 

Point 2: In abstract you must specified what mean abbreviations used such as T₁, T_k, T₆ and T₁₆.

Response 2 (corrected in page 1): T₆ (Eight sprays of formulated P.putida + B.subtilis + four sprays of Curzate M8), (Eight sprays of formulated P.putida, B.subtilis and T.erinaceum+ four sprays of Curzate M8), T₁ [Eight sprays of Curzate M8 (Positive control)], Bangladeshi Currency (Taka)

 

Point 3: The final part about economical analysis of given research is completely unclear with English hard to follow. Please rewrite to make it more clear.

Response 3 (corrected in 3.1.8 on page 12):  During 2019-2020 and 2020-2021, average cost benefit analysis revealed that the highest (Tk. 395111.11/ha) gross return was obtained from the treatmentT₁₆ followed by T₆ (Tk. 390222.23/ha), T₁ (379166.67/ha), T₁₁ (Tk. 374888.89/ha). Thus, the highest BCR (0.88) was calculated from the treatments T₁₆ and T₆ (0.85) which performed better than T₁ (0.83)(Table6). BCR results indicated a return ranging fromTaka0.78 to 0.88 over the investment of Taka 1.00 in these treatments in those two years. In both years, treatments (T_16, T_6, T_11, T₁) performed better in the field conditions reducing the fungicide application frequency to mitigate late blight severity as those treatments also performed better in the cost benefit analysis (Table 8). 

 

 

Point 4: On Page 2 (Line 2) I think it should be "cool temperature area/region"? Check/correct.

Response 4 (corrected in section 1 on page 2): Late blight caused by P. infestans (Mont.) De Bary restricts the yield of potato notably in cool temperature regions globally

 

Point 5: On Page 2 (Line 7) delete () before and after 20.6 t/ha. It is surplus here

Response 5 (corrected in paragraph 1 on page 2): The yield of potatoes in Bangladesh in 2019 as calculated by the FAO [2] was 20.6 t/ha which is lower as compared to the potential yield and to the yield of other potato growing countries of the world

 

Point 6: On Page 2 (Line 17) it should be "... structure of  P. Infestans". Correct.

Response 6 (corrected in 1^st paragraph on page 2): Genome structure of P. infestans allows itself to adapt by fostering genetic diversity [8,9].

 

Point 7: On Page 2 (Line 19) word potato should not be written with capital letter P. Correct.

Response 7 (corrected in 2^nd paragraph on page 2): As potato late blight may cause large economic losses quickly; potato growers must apply synthetic fungicides to plant surfaces almost weekly before sporangia appear

 

Point 8: On Page 3 (the first paragraph) please provide more references for given statements about "... numerous research finding promising outcomes"

Response 8 (corrected in 2^nd paragraph on page 3): The use of bacteria as bio-control agents for the treatment of potato late blight has recently gained in popularity throughout recent years, with numerous research finding promising outcomes [41-46].

 

Point 9: On Page 3 (Lines 3 and 5) term Bacillus should be written in Italic style. Correct.

Response 9: (corrected in 2^nd paragraph on page 3): Among the bacterial antagonists, many belong to the genus Bacillus and there are several other major genera but are of smaller practical value than Bacillus

[31].

 

Point 10: This complete paragraph (As fungicides are not ... to highest level) is unclear. It is either incorrectly written or something is missing in given sentences. Please check/clarify/correct.

Response 10 (corrected in 2^nd paragraph on page 4): The effects of fungicide application have numerous hazards to mankind and environment and apart from that many fungicides are banned in developedcountries due to their toxic effects to human being and animals. On the other hand, sometimes the efficacy level of bioagents alone are not deliver its best performance activity against poly cyclic pathogens like P. infestans. Thus, in this study, we focused on the use of both fungicides and bioagents in reducing the application frequency of chemical fungicide to minimize the impact of late blight severity on potato yield.

 

Point 11: On Page 3 (subsection 2.1.) it should be "... were isolated from potato.."

Response 11 (corrected in subsection 2.1. on page 4): Two bacterial isolates viz. P. putida (BDISO64RanP), B. subtilis (BDISO36ThaR) were isolated from potato phylloplane

 

Point 12: In the next Line on Page 3 correct technical error- split words shere and identified.

Response 12 (corrected in subsection 2.1. on page 3): rhizosphere identified

 

Point 13: In the penultimate Line correct as in comment 11.

Response 13 : comment 11 has been corrected 

 

Point 14: On Page 4 is this " for shoet period of time" incorrectly written? Please check

Response 14 (corrected in subsection 2.2. on page 5): short period of time

 

Point 15: On Page 5 (Line 1) correct technical error and split words.

Response 15 : word has been splitted

 

Point 16: On Page 5 (Line 6) It should be "P. putida" with a capital letter P. Correct.

Response 16(corrected in subsection 2.4. on page 5): P. putida

 

Point 17: On Page 5 (Line 7) it should be abbreviated name as "B. subtilis". Correct

Response 17: (corrected in subsection 2.4. on page 5): B. subtilis

 

Point 18: On Page 5 provide reference(s) for given statement "... previous experiments on the performance of bacterial..."

Response 18: (corrected in 2^nd paragraph on page 6): According to the results of the previous experiments on the efficacy of formulated two bacterial and two fungal bioagents in reducing late blight severity of potato under growth chamber conditions and field conditions during 2018-2019, we selected two bacterial (viz; P. putida, B. subtilis)and one fungal (viz. T. erinaceum) bioagents that were found effective in total growth inhibition of late blight pathogen. The next step was to compare treatments A) exclusively based on the current number of sprays of chemical fungicide, B) based on the same number of sprays but applying single or mixed bioagents, and C) the same as B) but reinforced by 1 to 4 additional sprays with chemical fungicide.

(previous experiments mean our experiment which we have done in the year of 2018-2019 depicting Table 1 and Table 2)

Point 19: On Page 5 (subsection 2.5.) correct several typos i.e. split numerical values from units here

Response 19: (corrected in subsection 2.6 on page 6): split numerical value has been done.

 

Point 20: On Page 6 (Line 2) correct chemical formula for calcium carbonate.

Response 20: (corrected in subsection 2.6. on page 6): CaCO₃ 

 

Point 21: On Page 6 (Line 6) it can not be "was given" but "was added" or some other synonym. Check/correct.

Response 21: (corrected in subsection 2.6. on page 6): was added

 

Point 22: On Page 8 (subsection 3.1.2.) this "that" in front of "deformation" is surplus or some verb is missing. Please check/correct.

Response 22: (corrected in subsection 3.1.2. on page 9): we observed the deformation of mycelial structures

 

Point 23:On page 8 (subsections 3.1.3 and 3.1.4.) there are several typos where words should be split "T₅ in", "T₁₀ and", "T₁₁ and".

Response 23: (corrected in page 9):words has been splitted

 

Point 24:On Page 8 (subsection 3.1.4.) I do not understand how given values (2.43%, 3.37%, 10.33% and 21.30%) are not statistically different? I mean what was value for standard deviation if 2.43 is not different from 21.30?? Please check/clarify.

Response 24: (corrected in subsection 3.1.4. on page 9 and 10): At 59DAP, minimal severity was recorded in T₁₁ (2.43%) followed by T₁₀ (3.37%) showing statistically identical data. These treatments performed better compared to all other treatments. In case of 71 DAP, the minimum (10.33%) severity was recorded in T₁₁ followed byT₁₀ (21.30%) which were statistically similar and performed better among all other treatments. Considering percent reduction of late blight severity over control, highest reduction was found when applied with T₁₁ (89.16%) followed by T₁₀ (77.50%), T₉ (27.07%), T₂ (16.71%) (Table 2).

 

Point 25:On Page 10 correct two typos - "Figure 2" and "Table 6"

Response 25: (corrected in subsection 3.1.8. on page 10): Two typos has been corrected

 

Point 25: Line 425: How it can be "organic potato" if we used chemical agents?? Ok, in reduced amount (If your presumption is correct) but still it is obviously that it must be applied in some doses. And with this we will go back on my the first and the main question.

Response 25 (corrected in last paragraph on page 2): At the same time, two counter-balancing factors have also grown: societal pressure for reducing pesticide use on crops and acreage of organically-grown food crops, potato and tomato included [14-16].  For many years, copper-based fungicides (e.g. Bordeaux combination, fixed-copper hydroxide, copper oxide, and copper oxychloride) were used to suppress late blight in organic potato and tomato cultivation. Organic fields in Brazil [15], USA [17] and Japan (Maff Notification No. 59, 2000) may employ these chemicals (www.maff.go.jp/soshiki/syokuhin/hinshitu/organic/eng yukihow.pd). Currently in the European Union only 6 kg of elemental copper per ha per year is allowed in organic production [16]. As soon as reliable alternatives to manage late blight are available, a complete ban of copper compounds should take place [18]

 

 

Response 25 (corrected in last paragraph on page 12): while generally all farmers of Bangladesh have been using at least eight sprays of chemical fungicides,which might be raised up to 16 sprays depending on the weather conditions,per hectareof land whether late blight severity is present or not, thus, we have standardized it (8 sprays)based on the field surveys in our experiment to evaluate the reduction of spray frequency of chemical fungicide with bioagent.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have made significant changes to improve the manuscript. The suggestions proposed were accepted and they explain in other cases well their reasons.

Reviewer 3 Report

I have no further comments.