A Review of Plant-Mediated and Fertilization-Induced Shifts in Ammonia Oxidizers: Implications for Nitrogen Cycling in Agroecosystems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The presented work deals with (synthesize current knowledge on how plant-mediated factors and fertilization practices influence ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities, and to explore their implications for nitrogen cycling and nitrogen use efficiency (NUE) in agroecosystems. It focuses on the ecological niches of AOA and AOB and their responses to plant community composition, root exudates, and allelopathic compounds. The review also examines the effects of different fertilization regimes, including inorganic nitrogen inputs and biological nitrification inhibition (BNI), on microbial adaptation and ammonia tolerance. The article aims to inform integrated nitrogen management approaches that enhance NUE and reduce nitrogen losses by highlighting the interactions between plants, microbes, and fertilization strategies. Furthermore, it emphasizes the need for future research utilizing metagenomic and isotopic techniques to unravel the mechanistic pathways governing AOA and AOB activity to develop more sustainable agricultural systems.

The manuscript is generally (good written) however, I observed some minor grammar and syntax errors, as well as capitalization and punctuation errors throughout the manuscript text

In the following contains some of in-depth comments, criticisms, worries, and recommendations that should be taken into account before a final judgment on the document is made. In light of the criticisms I've provided below, I predict that the rewritten document will produce a considerably different form from the one it is in now. As a result, I advise resubmitting this work because it typically yields some intriguing results.

The main criticism points are:

• There are many grammatical errors in the whole article.. check and correct, please …. Occasional awkward phrasing or grammatical slips need correction for example :

Ensure consistency in tense and formatting of citations (e.g., sometimes "[14,29,30]", sometimes "[54]").

Line 109–110: “yet their relative abundance and nitrification roles differ markedly” → consider simplifying: “yet they differ markedly in abundance and nitrification roles…”

Line 113: “since each plant species…” → rephrase to “as each plant species…”

Line 116–119: Consider combining sentences for better flow: "Multiple studies indicate that plant species can influence microbial populations through variations in root exudates and rhizosphere processes, although the exact mechanisms remain under investigation."

Line 125: "which may, in turn, results in" → should be "which may, in turn, result in"

Line 135: “where sequences from cluster 3b… disappear under forbs and grasses, while 3a… was absent” → use consistent tense: “while 3a was also absent…”

Line 138: “Additionally, no direct interaction (facilitation or antagonism)…” → could simplify to “No facilitative or antagonistic interactions were observed…”

Line 146: “this significantly influences…” → “these significantly influence…”

Line 149–151: The list is long. Break into two sentences: "...in the form of exudates. These include small molecules (e.g., carbohydrates, amino acids, organic acids) and complex ones (e.g., proteins, mucilage)..."

Line 152: “Different antimicrobial metabolites include…” → consider: “Antimicrobial metabolites such as phenolic compounds (e.g., flavonoids, tannins) and amino sugars play…”

Line 157: “thus reducing nitrate availability for competing plants” → nicely worded.

 And so on …..check whole article …….

• The introduction provides a comprehensive background, but it lacks a clearly defined statement of the review’s specific objectives or research questions. End the introduction with a concise paragraph stating the aim of the review, emphasizing its focus on AOA/AOB responses to plant and fertilizer influences, and outlining the review structure.
• The introduction sometimes veers into general background on nitrogen cycling and microbial mechanisms without consistently tying it back to the core focus of the review—plant-mediated and fertilization-induced shifts in ammonia oxidizers. Streamline background content to reduce redundancy and focus more directly on ammonia oxidizers and their agroecosystem interactions.
• Some facts, such as the role of AOA and AOB in ammonia oxidation and the anthropogenic impact on nitrogen cycling, are mentioned multiple times without adding new context.
• Though the title focuses on plant-mediated effects, the introduction emphasizes microbial biochemistry and fertilizer-driven impacts more heavily, underrepresenting plant influence (e.g., root exudates, allelopathy, rhizosphere dynamics).Strengthen the discussion of plant-mediated mechanisms, including root exudation patterns, rhizosphere pH shifts, and allelopathic influences, to align better with the title
• Use transitional phrases and topic sentences to create a clearer, more cohesive flow between sections (e.g., from global nitrogen trends to microbial pathways to plant–soil interactions).
• Briefly mention the need for further research (e.g., using metagenomics or isotopic tracing) to set the stage for recommendations presented later in the review.
• The review is rich with literature references and detailed mechanisms. good structural organization using sub-sections, covers a broad range of plant traits (exudates, photosynthetic pathways, allelopathy, etc.). But some repetition and slightly verbose passages could be condensed for better readability.
• The review predominantly summarizes qualitative findings without a meta-analysis or systematic quantification of trends (e.g., effect sizes of fertilization on AOA/AOB ratios). Incorporate meta-analytical techniques to statistically aggregate results from cited studies, enhancing the robustness of conclusions.
• Expand the discussion on Comammox, comparing their niches, enzymatic pathways, and responses to fertilization with AOA/AOB.
• The environmental impacts of fertilization (e.g., N₂O emissions, leaching) are fragmented rather than synthesized into a coherent framework. Dedicate a section to trade-offs between agronomic benefits (yield, NUE) and ecological costs (GHG emissions, soil acidification).
• Many cited experiments are lab-based microcosms, which may not reflect real-world agroecosystem dynamics. Highlight field studies to validate lab findings and discuss the scalability of interventions (e.g., nitrification inhibitors).
• Strategies like biological nitrification inhibition (BNI) lack discussion on barriers to adoption (e.g., crop compatibility, economic viability). Could you address practical challenges (farmer education, cost-effectiveness) and propose solutions (policy incentives, breeding programs)?
• The review does not systematically explore how climate variables (e.g., drought, warming) interact with nitrogen cycling and microbial communities. Include a subsection on climate-microbe-plant feedbacks, citing studies on temperature/pH shifts affecting nitrification.
• Plant-microbe signaling (e.g., VOCs, root exudates) is described superficially without linking molecular mechanisms to ecosystem outcomes. Use diagrams or tables to map specific exudate compounds to their effects on AOA/AOB activity and nitrogen retention.

Author Response

Please find attched the responses . We have included individual responses to reviewers in which we address each comment the reviewers made. In our response to reviewers, reviewers’ comments are in plain text and our responses are in blue text. Please kindly note that the line numbers highlighted in the responses
are for the “Revised Manuscript with track changes". 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well-structured and logically coherent, comprehensively addressing the impacts of plant-mediated processes and fertilization management on ammonia-oxidizing microorganisms (AOA and AOB) and their implications for agricultural nitrogen cycling. From the introduction to the conclusion, the sections are naturally connected, with a clear thematic focus. However, the following aspects require further refinement.

Lack of Visual Aids: The manuscript lacks figures or tables (e.g., mechanistic diagrams, comparative data charts, microbial community dynamic models), resulting in insufficient visual support for key conclusions (e.g., AOA/AOB niche differentiation, fertilization effects on soil pH). The authors should supplement figures and tables to enhance information delivery efficiency.

Lines 261-277: While the manuscript extensively cites classical studies (e.g., discussions on AOA/AOB niches), recent critical advances (e.g., the discovery of comammox bacteria and their role in nitrification) are inadequately incorporated. It is recommended to update the literature to reflect the diversity of nitrification pathways.

Lines 488-517: Heterogeneity in AOA/AOB responses to fertilization across studies (e.g., AOA dominance in acidic soils vs. AOB activity under high pH) is noted, but the manuscript lacks in-depth exploration of underlying mechanisms (e.g., soil type, organic matter content, microbial interactions). A systematic analysis of this heterogeneity is needed.

Lines 592-598: Although 15 N isotope tracing and metagenomic techniques are mentioned, the manuscript does not specify how these methods are applied in practical research designs (e.g., experimental conditions, sample processing, data analysis). The authors should supplement examples or case studies illustrating technical workflows.

Lines 616-624: While nitrification inhibitors (NIs) are reported to be more effective against AOB, their practical efficacy in complex soil environments (e.g., effects of organic matter content or moisture on inhibitor stability) is insufficiently discussed. Additional field trial data should be cited to validate the feasibility of proposed strategies.

Lines 680-684: Biological nitrification inhibition (BNI) is highlighted as an eco-friendly strategy, but its real-world application faces challenges (e.g., specificity of plant-microbe interactions, compound stability). The authors should supplement field validation cases of BNIs in major crops (e.g., wheat, rice).

Author Response

Please find attched the responses . We have included individual responses to reviewers in which we address each comment the reviewers made. In our response to reviewers, reviewers’ comments are in plain text and our responses are in blue text. Please kindly note that the line numbers highlighted in the responses
are for the “Revised Manuscript with track changes". 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

accept

Reviewer 2 Report

Comments and Suggestions for Authors

The authors carefully revised the manuscript, and the revised version is now acceptable.