Impact of Antibacterial Agents in Horticulture: Risks to Non-Target Organisms and Sustainable Alternatives

Reviewer Comments (Reviewer 1)

Response

The present review is a well-organized and extensive work that covers all aspects of antibacterial agents.

The authors are thankful for your positive comments

The tables and figures enhance the understanding of the text and provide effective summaries. After reading the full manuscript, I would suggest that the title should be improved. It does not fully reflect the content of the paper, which includes topics such as fruticulture, horticulture, and soil health. Either the content should be adjusted to better match the current title, or the title should be revised to better align with the scope of the paper.

The authors are thankful for your comments. The contents and title of the manuscript have been revised in the light of comments

The question addressed in the present work is compiling a general topic like the use of antibacterial agents in different crops, not only horticultural crops.
Description of antibacterial agents, present status of the state of the art and future of the topic looking for a reduction in its use. In general, is not a focused review but a general description of the topic. From the mode of action, the environmental impact and ways of reduce the use of them.

I would say that is original but not specific.  But with more than 200 references is a good base to understand the field.  Reading this review allow for a good base in the knowledge about the topic.

The authors are thankful for your comments. The data related to other crops have been removed from the manuscript, and now it is focused on the use of antibacterial agents in horticultural crops

As mentioned before the value of this review is the amount of references and the general overview of the topic. It is not a review that focus specifically in a special par of antibacterial agents. The conclusions are in line with the evidences and arguments. Also the conclusions are nothing considered as new. Also the information inside the review do not show any novelty but is true that compile a big amount of information in not so many pages.

The references are update and I would say that in some cases may be too many (229 references) for a review I would consider a bit too much in some cases but I have not that feeling in the present work.

The authors are thankful for your comments

The tables and figures are ok, but nothing outstanding: It could be improved, and the figures are quite focused in the "molecular" topic. May be adding one or two more figures as regards other points would be welcomed. Tables are well organised and informative

The authors are thankful for your comments. One of the figures related to molecular work has been updated.

The number of citations is extensive and up to date.

The authors are thankful for your positive comments

Some issues to address:

·         Line 75: "Chemistry" should be replaced with "chemical."

Chemistry is replaced with chemical. Please check L 95

·         Line 77: The cited text is outdated, so the estimation is no longer valid. It should be replaced with a more recent source.

The authors are thankful for your comments. Updated data is presented in the manuscript at the same point. Please see L 98

Line 88: The phrase "that should be met" is incorrect in context, "be" should be removed

The authors are thankful for your comments. The phrase has been revised in the revised version of the manuscript

Reviewer Comments (Reviewer 2)

Response

The manuscript presents a well-structured overview, first outlining the problems associated with using antimicrobial substances in agriculture, and then detailing antibacterial agents, their mechanisms of action, and their effects on non-target organisms and the environment. Also, the problem of antibacterial products with remanence or residual is approached from several points of view. In separate chapters, alternatives for antibacterial treatments are presented using biotechnological means, but also new and sustainable approaches by applying integrated and digitalised measures that belong to precision agriculture.

The authors are thankful for your positive comments

The review article contains informative tables and original explanatory figures, and the references consulted are in appreciable number, 229, covering a time interval between 1990 and 2025.

The authors are thankful for your positive comments

Minor observations

·         Technical editing mistakes: L 33, 373, 542

The authors are thankful for your positive comments. The desired technical editing mistakes have been corrected in the revised version.

·         Abbreviations not explained or defined at first use, see IPDM in L 437

The authors are thankful for your positive comments. Abbreviations used in the manuscript have been elaborated at first instance throughout the manuscript. For specific abbreviation please see L 512

·         In References to check the Latin names of microbial species

The authors are thankful for your positive comments. Latin names of microbial species have been checked in the references section and italicized.

·         Nitrification and nitrogen fixation are two different processes. The bacteria from L 71 are only nitrogen fixers. To avoid confusion, I suggest removing the word "nitrifying"

·         The authors are thankful for your positive comments. The desired word “nitrifying” has been removed in the revised version.

·         Please see L 91

·         Table 1 . The last row, Phytophthora capsici is not a bacterial agent!!

·          

The authors are thankful for your positive comments. The mentioned mistake has been rectified from Table 1. Please see the revised version of the manuscript L 181

·         Subchapter 2. 3, nanoparticles are presented in 2 sections: inorganic and organic. Unfortunately, they are mixed (for example, carbon nanotubes are included in the organic section). I suggest presenting the nano antibacterial agents in a common chapter, without detailing by sections

The authors are thankful for your positive comments. The sectioning of subsection 2.3 has been removed and details regarding nano antibacterial agents have been presented under common section as suggested. Please see subsection 2.3. from L 154

·         The references in the text do not match those in the References. There may be a number gap. For example, Information attributed to [67] is sourced from [65]; information from [108] is sourced from [106].

·          

The authors are thankful for your positive comments. Reference order has been corrected in the revised version of manuscript.

·         It is necessary to check and correct the references throughout the entire review article!!!

·          

The authors are thankful for your positive comments. References have been corrected throughout the manuscript in the revised version of the manuscript.

Reviewer Comments (Reviewer 3)

Response

Line 52:  Delete “it seems to reach” and change to:  ‘predicted to reach’ 10 billion……

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 72

Lines 67 to 68:   can delete some of the words   (‘some’    ‘effects’;  ‘have an’   ‘on’)

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 87-88

Line 151.   ‘Environmentally friendly’.  

The authors are thankful for your comment. The desired correction has been made in the revised version of the manuscript. Please see L 185

Line 358.    Change ‘waterbodies’ to….’bodies of water’…throughout text..  and

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 415

Then change… ‘Waterbodies situated’  to  …’Water systems’ situated near agricultural lands are……

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 415

Line 368 can delete ‘that they are facing in their’  to….Common diseases in horticultural crops are…

The authors are thankful for your comments. The sentence has been rephrased to make it clear.

Line 377.  Change…’is reducing with time”--  is “decreasing” due to emergence of resistance in bacterial strains  (157).

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 443

Line 389. Delete ‘due to which’,  change to  “resulting in” restriction of their use in some countries [162].

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 455-456

Line  407.  Change wording: …”of the same antibacterial agents within the same crops (i.e. oxytetracycline or streptomycin) against E. amylovora (fire blight in apples and pears) has resulted in resistance to these treatments [167]

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 474-476.

Line 408. ‘ Delete  ‘are applied’

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 474-476.

Lines 409-410. Delete  ‘It makes difficult to manage the disease’

The authors are thankful for your comments. The mentioned sentence has been deleted in the revised version of the manuscript. Please see L 479-480.

Line 410.   Change wording:  “Similarly Xanthomonas and Pseudomonas, have developed resistance to the most commonly used copper-based antibacterial chemicals, worldwide due to continuous overuse [157].

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 480-482.

Line 413.  Change wording:    “of new chemicals as antibacterial agents.”

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 482-483.

Line 417;  change wording:  …’the toxicity of soil and water [150].   Then Delete the following sentence:  “This can leach-----and contaminate it’

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 487-488.

Line 420. Change wording:    …of health concerns, like antibiotic resistance [132].

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 489-490.

Line 420 to 422:  Delete following sentence: from   ‘ They also---case of bacterial infection”

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 490-492.

Line 426:  Add word    “can affect many of the most “ important bacteria…………

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 497

Line 427. Delete:  ‘Same in case’ .  and change to:   ‘Likewise, Kasugamycin can target beneficial mycorrhizal fungi along with targeted pathogens.”

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 499-500.

Line 430:  Change wording:    ‘….and most rapid methods

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 502-503

Line 433:  Change:   ‘in humans’,    to   ‘to human pathogens’.  In text before FIG 3.

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 507.

Line 443:  Add in text:   “should be based’…

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 518

Line 604-605:  change text:   “Plant Biotechnology  offers alternative approaches including host expressed RNA interference (RNAi) [Need reference? Li et al, 2015] and CRISPR technologies [Doudna and Charpentier 2014] for long term solutions, with development of externally applied treatments of antibacterial or antiviral aptamers like antisense oligonucleotides (need Refs?  Darrah et al, 2023; Hunter and Larson 2025; Patel et al, 2017;  Sandoval-Mojica et al, 2020;2021)

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 694-696.

Line 725: conclusions.   Word changes:    “Sustainable use of antibacterial agents are essential to reduce environmental and health hazards”. 

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see L 847-848.

Reference citations are incomplete, and inconsistent in formatting. Book citations need to be of same format. Suggest to include all doi: addresses for cited references for readers use to locate cited references.

The authors are thankful for your comments. The desired correction has been made in the revised version of the manuscript. Please see references section L 913

Li H, Guan R, Guo H, Miao X (2015) New insights into an RNAi approach for plant defence against piercing-sucking and stem-borer insect pests. Plant Cell Environ 38:2277–2285 doi:10.1111/pce.12546

Dalakouras A, Wassenegger M, Dadami E, et al (2020) Genetically Modified Organism- Free RNA interference: exogenous application of RNA molecules in plants. Plant Physiol. 182:38–50

Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096

Darrah KE, Albright S, Kumbhare R, Tsang M, Chen JK, Deiters A (2023) Antisense Oligonucleotide Activation via Enzymatic Antibiotic Resistance Mechanism https://doi.org/10.1021/acschembio.3c00027

Hunter, W.B., Larson, N.R. (2025). Invention of RNAi Biopesticides to Psyllid Vectors and Bacterial Pathogens of Citrus: Future Technologies for Gene Targeting. In: Smagghe, G., Palli, S.R., Swevers, L. (eds) RNA interference in Agriculture: Basic Science to Applications. Springer, Cham. https://doi.org/10.1007/978-3-031-81549-2_17

Patel RR, Sundin GW, Yang C-H, Wang J, Huntley RB, Yuan X, Zeng Q (2017) Exploration of using antisense peptide nucleic acid (PNA)-cell penetrating peptide (CPP) as a novel bactericide against fire blight pathogen Erwinia amylovora. Front Microbiol 8:687

Sandoval-Mojica A.F, Altman S, Hunter W.B., Pelz-Stelinski K.S. Peptide conjugated morpholino’s for management of the huanglongbing pathosystem. Pest Manag. Sci. 2020; 10.1002/ps.5877. https://doi:10.1002/ps.5877.

Sandoval-Mojica AF, Hunter WB, Aishwarya V, Bonilla S, Pelz-Stelinski KS (2021) Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem. Nat Sci Rep 11:2760.  https://link.springer.com/articles/cas-redirect/1%3ACAS%3A528%3ADC%252BB3MXjtleisrs%253D

The authors are thankful for suggesting references. Most of them have been incorporated in the revised version of the manuscript. Please see ref 208; 204; 205; 193; 203; 194

Comments on the Quality of English Language

The manuscript "Sustainable use of antibacterial agents against biotic stresses of horticultural crops" is a focused review on the need for environmentally safer antimicrobials (bactericides, viricides, fungicides) and pesticides (Insects).  Overall, the authors present the case that dependence upon too few, and applied too often treatments are causing problems as treatment resistance occurs with no new products to replace currently used agents/chemistries. This topic is vast, so the topic is covered with a view examples (refs) of each point raised by the authors, but these are adequate to support statements.  The figures are well done and easy to understand, providing a visual of pertinent discussion points.  While it is difficult to cover the entire topic which goes beyond antibiotics, antimicrobials such as natural and synthetic chemicals,  microbicidal peptides and genetics - host expressed RNAi, aptamers, and CRISPR gene editing; as well as emerging approvals for externally applied sprays of antisense oligonucleotides, the authors present a fairly complete vision in support of the need for better microbicides for agricultural use.   Once the manuscript has been corrected grammatically and the references corrected to proper formatting, this manuscript would be ready for publication.

The authors are thankful for your comments. The grammatical mistakes have been corrected throughout the manuscript.